@article{dye_muga_mwangi_hoyer_ly_rosado_sharpee_mware_wambugu_labadie_et al._2023, title={Cassava begomovirus species diversity changes during plant vegetative cycles}, volume={14}, ISSN={["1664-302X"]}, DOI={10.3389/fmicb.2023.1163566}, abstractNote={Cassava is a root crop important for global food security and the third biggest source of calories on the African continent. Cassava production is threatened by Cassava mosaic disease (CMD), which is caused by a complex of single-stranded DNA viruses (family: Geminiviridae , genus: Begomovirus ) that are transmitted by the sweet potato whitefly ( Bemisia tabaci ). Understanding the dynamics of different cassava mosaic begomovirus (CMB) species through time is important for contextualizing disease trends. Cassava plants with CMD symptoms were sampled in Lake Victoria and coastal regions of Kenya before transfer to a greenhouse setting and regular propagation. The field-collected and greenhouse samples were sequenced using Illumina short-read sequencing and analyzed on the Galaxy platform. In the field-collected samples, African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Kenya virus (EACMKV), and East African cassava mosaic virus-Uganda variant (EACMV-Ug) were detected in samples from the Lake Victoria region, while EACMV and East African mosaic Zanzibar virus (EACMZV) were found in the coastal region. Many of the field-collected samples had mixed infections of EACMV and another begomovirus. After 3 years of regrowth in the greenhouse, only EACMV-like viruses were detected in all samples. The results suggest that in these samples, EACMV becomes the dominant virus through vegetative propagation in a greenhouse. This differed from whitefly transmission results. Cassava plants were inoculated with ACMV and another EACMV-like virus, East African cassava mosaic Cameroon virus (EACMCV). Only ACMV was transmitted by whiteflies from these plants to recipient plants, as indicated by sequencing reads and copy number data. These results suggest that whitefly transmission and vegetative transmission lead to different outcomes for ACMV and EACMV-like viruses.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Dye, Anna E. E. and Muga, Brenda and Mwangi, Jenniffer and Hoyer, J. Steen and Ly, Vanessa and Rosado, Yamilex and Sharpee, William and Mware, Benard and Wambugu, Mary and Labadie, Paul and et al.}, year={2023}, month={May} }
 @article{kennedy_sharpee_jacobson_wambugu_mware_hanley-bowdoin_2023, title={Genome segment ratios change during whitefly transmission of two bipartite cassava mosaic begomoviruses}, volume={13}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-023-37278-8}, abstractNote={Cassava mosaic disease is caused by a complex of whitefly-transmitted begomoviruses, which often occur in co-infections. These viruses have bipartite genomes consisting of DNA-A and DNA-B that are encapsidated into separate virions. Individual viruses exist in plants and whitefly vectors as populations comprising both genome segments, which can occur at different frequencies. Both segments are required for infection, and must be transmitted for virus spread to occur. Cassava plants infected with African cassava mosaic virus (ACMV) and/or East African cassava mosaic Cameroon virus (EACMCV), in which the ratios of DNA-A:DNA-B titers differed between plants, were used to examine how titers of the segments in a plant relate to their respective probabilities of acquisition by whiteflies and to the titers of each segment acquired and subsequently transmitted by whiteflies. The probabilities of acquiring each segment of ACMV did not reflect their relative titers in the source plant but they did for EACMCV. However, for both viruses, DNA-A:DNA-B ratios acquired by whiteflies differed from those in the source plant and the ratios transmitted by the whitefly did not differ from one - the ratio at which the highest probability of transmitting both segments is expected.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Kennedy, George G. G. and Sharpee, William and Jacobson, Alana L. L. and Wambugu, Mary and Mware, Benard and Hanley-Bowdoin, Linda}, year={2023}, month={Jun} }
 @article{aimone_hoyer_dye_deppong_duffy_carbone_hanley-bowdoin_2022, title={An experimental strategy for preparing circular ssDNA virus genomes for next-generation sequencing}, volume={300}, ISSN={["1879-0984"]}, DOI={10.1016/j.jviromet.2021.114405}, abstractNote={The ability of begomoviruses to evolve rapidly threatens many crops and underscores the importance of detecting these viruses quickly and to understand their genome diversity. This study presents an improved protocol for the enhanced amplification and enrichment of begomovirus DNA for use in next generation sequencing of the viral genomes. An enhanced rolling circle amplification (RCA) method using EquiPhi29 polymerase was combined with size selection to generate a cost-effective, short-read sequencing method. This improved short-read sequencing produced at least 50 % of the reads mapping to the target viral reference genomes, African cassava mosaic virus and East African cassava mosaic virus. This study provided other insights into common misconceptions about RCA and lessons that could be learned from the sequencing of single-stranded DNA virus genomes. This protocol can be used to examine the viral DNA as it moves from host to vector, thus producing valuable information for viral DNA population studies, and would likely work well with other circular Rep-encoding ssDNA viruses (CRESS) DNA viruses.}, journal={JOURNAL OF VIROLOGICAL METHODS}, author={Aimone, Catherine D. and Hoyer, J. Steen and Dye, Anna E. and Deppong, David O. and Duffy, Siobain and Carbone, Ignazio and Hanley-Bowdoin, Linda}, year={2022}, month={Feb} }
 @article{peng_dallas_ascencio-ibanez_hoyer_legg_hanley-bowdoin_grieve_yin_2022, title={Early detection of plant virus infection using multispectral imaging and spatial-spectral machine learning}, volume={12}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-022-06372-8}, abstractNote={Cassava brown streak disease (CBSD) is an emerging viral disease that can greatly reduce cassava productivity, while causing only mild aerial symptoms that develop late in infection. Early detection of CBSD enables better crop management and intervention. Current techniques require laboratory equipment and are labour intensive and often inaccurate. We have developed a handheld active multispectral imaging (A-MSI) device combined with machine learning for early detection of CBSD in real-time. The principal benefits of A-MSI over passive MSI and conventional camera systems are improved spectral signal-to-noise ratio and temporal repeatability. Information fusion techniques further combine spectral and spatial information to reliably identify features that distinguish healthy cassava from plants with CBSD as early as 28 days post inoculation on a susceptible and a tolerant cultivar. Application of the device has the potential to increase farmers' access to healthy planting materials and reduce losses due to CBSD in Africa. It can also be adapted for sensing other biotic and abiotic stresses in real-world situations where plants are exposed to multiple pest, pathogen and environmental stresses.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Peng, Yao and Dallas, Mary M. and Ascencio-Ibanez, Jose T. and Hoyer, J. Steen and Legg, James and Hanley-Bowdoin, Linda and Grieve, Bruce and Yin, Hujun}, year={2022}, month={Feb} }
 @article{mclaughlin_hanley-bowdoin_kennedy_jacobson_2022, title={Vector acquisition and co-inoculation of two plant viruses influences transmission, infection, and replication in new hosts}, volume={12}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-022-24880-5}, abstractNote={This study investigated the role of vector acquisition and transmission on the propagation of single and co-infections of tomato yellow leaf curl virus (TYLCV,) and tomato mottle virus (ToMoV) (Family: Geminiviridae, Genus: Begomovirus) by the whitefly vector Bemisia tabaci MEAM1 (Gennadius) in tomato. The aim of this research was to determine if the manner in which viruses are co-acquired and co-transmitted changes the probability of acquisition, transmission and new host infections. Whiteflies acquired virus by feeding on singly infected plants, co-infected plants, or by sequential feeding on singly infected plants. Viral titers were also quantified by qPCR in vector cohorts, in artificial diet, and plants after exposure to viruliferous vectors. Differences in transmission, infection status of plants, and titers of TYLCV and ToMoV were observed among treatments. All vector cohorts acquired both viruses, but co-acquisition/co-inoculation generally reduced transmission of both viruses as single and mixed infections. Co-inoculation of viruses by the vector also altered virus accumulation in plants regardless of whether one or both viruses were propagated in new hosts. These findings highlight the complex nature of vector-virus-plant interactions that influence the spread and replication of viruses as single and co-infections.}, number={1}, journal={SCIENTIFIC REPORTS}, author={McLaughlin, Autumn A. and Hanley-Bowdoin, Linda and Kennedy, George G. and Jacobson, Alana L.}, year={2022}, month={Nov} }
 @article{aimone_de leon_dallas_ndunguru_ascencio-ibanez_hanley-bowdoin_2021, title={A New Type of Satellite Associated with Cassava Mosaic Begomoviruses}, volume={95}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.00432-21}, abstractNote={Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.}, number={21}, journal={JOURNAL OF VIROLOGY}, author={Aimone, Catherine D. and De Leon, Leandro and Dallas, Mary M. and Ndunguru, Joseph and Ascencio-Ibanez, Jose T. and Hanley-Bowdoin, Linda}, year={2021}, month={Nov} }
 @article{wang_gong_wu_huang_ismayil_zhang_li_gu_ludman_fatyol_et al._2021, title={A calmodulin-binding transcription factor links calcium signaling to antiviral RNAi defense in plants}, volume={29}, ISSN={["1934-6069"]}, DOI={10.1016/j.chom.2021.07.003}, abstractNote={RNA interference (RNAi) is an across-kingdom gene regulatory and defense mechanism. However, little is known about how organisms sense initial cues to mobilize RNAi. Here, we show that wounding to Nicotiana benthamiana cells during virus intrusion activates RNAi-related gene expression through calcium signaling. A rapid wound-induced elevation in calcium fluxes triggers calmodulin-dependent activation of calmodulin-binding transcription activator-3 (CAMTA3), which activates RNA-dependent RNA polymerase-6 and Bifunctional nuclease-2 (BN2) transcription. BN2 stabilizes mRNAs encoding key components of RNAi machinery, notably AGONAUTE1/2 and DICER-LIKE1, by degrading their cognate microRNAs. Consequently, multiple RNAi genes are primed for combating virus invasion. Calmodulin-, CAMTA3-, or BN2-knockdown/knockout plants show increased susceptibility to geminivirus, cucumovirus, and potyvirus. Notably, Geminivirus V2 protein can disrupt the calmodulin-CAMTA3 interaction to counteract RNAi defense. These findings link Ca2+ signaling to RNAi and reveal versatility of host antiviral defense and viral counter-defense.}, number={9}, journal={CELL HOST & MICROBE}, author={Wang, Yunjing and Gong, Qian and Wu, Yuyao and Huang, Fan and Ismayil, Asigul and Zhang, Danfeng and Li, Huangai and Gu, Hanqing and Ludman, Marta and Fatyol, Karoly and et al.}, year={2021}, month={Sep}, pages={1393-+} }
 @article{mickelson-young_wear_song_zynda_hanley-bowdoin_thompson_2021, title={A protocol for genome-wide analysis of DNA replication timing in intact root tips}, journal={Methods in Molecular Biology series}, author={Mickelson-Young, Leigh and Wear, Emily E. and Song, Jawon and Zynda, Gregory J. and Hanley-Bowdoin, Linda and Thompson, William F.}, year={2021} }
 @article{borges_donoghue_leblanc_wear_tanurdzic_berube_brooks_thompson_hanley-bowdoin_martienssen_2021, title={Loss of Small-RNA-Directed DNA Methylation in the Plant Cell Cycle Promotes Germline Reprogramming and Somaclonal Variation}, volume={31}, ISBN={1879-0445}, url={https://doi.org/10.1016/j.cub.2020.10.098}, DOI={10.1016/j.cub.2020.10.098}, abstractNote={5-methyl cytosine is widespread in plant genomes in both CG and non-CG contexts. During replication, hemi-methylation on parental DNA strands guides symmetric CG methylation on nascent strands, but non-CG methylation requires modified histones and small RNA guides. Here, we used immortalized Arabidopsis cell suspensions to sort replicating nuclei and determine genome-wide cytosine methylation dynamics during the plant cell cycle. We find that symmetric mCG and mCHG are selectively retained in actively dividing cells in culture, whereas mCHH is depleted. mCG becomes transiently asymmetric during S phase but is rapidly restored in G2, whereas mCHG remains asymmetric throughout the cell cycle. Hundreds of loci gain ectopic CHG methylation, as well as 24-nt small interfering RNAs (siRNAs) and histone H3 lysine dimethylation (H3K9me2), without gaining CHH methylation. This suggests that spontaneous epialleles that arise in plant cell cultures are stably maintained by siRNA and H3K9me2 independent of the canonical RNA-directed DNA methylation (RdDM) pathway. In contrast, loci that fail to produce siRNA may be targeted for demethylation when the cell cycle arrests. Comparative analysis with methylomes of various tissues and cell types suggests that loss of small-RNA-directed non-CG methylation during DNA replication promotes germline reprogramming and epigenetic variation in plants propagated as clones.}, number={3}, journal={CURRENT BIOLOGY}, publisher={Elsevier BV}, author={Borges, Filipe and Donoghue, Mark T. A. and LeBlanc, Chantal and Wear, Emily E. and Tanurdzic, Milos and Berube, Benjamin and Brooks, Ashley and Thompson, William F. and Hanley-Bowdoin, Linda and Martienssen, Robert A.}, year={2021}, pages={591-+} }
 @article{aimone_lavington_hoyer_deppong_mickelson-young_jacobson_kennedy_carbone_hanley-bowdoin_duffy_2021, title={Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation}, volume={102}, ISSN={["1465-2099"]}, DOI={10.1099/jgv.0.001622}, abstractNote={Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta , we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5′ intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.}, number={7}, journal={JOURNAL OF GENERAL VIROLOGY}, author={Aimone, Catherine D. and Lavington, Erik and Hoyer, J. Steen and Deppong, David O. and Mickelson-Young, Leigh and Jacobson, Alana and Kennedy, George G. and Carbone, Ignazio and Hanley-Bowdoin, Linda and Duffy, Siobain}, year={2021} }
 @article{shen_hanley-bowdoin_2021, title={SnRK1: a versatile plant protein kinase that limits geminivirus infection}, volume={47}, ISSN={["1879-6265"]}, DOI={10.1016/j.coviro.2020.12.002}, abstractNote={Geminiviruses are a family of single-stranded DNA viruses that infect many plant species and cause serious diseases in important crops. The plant protein kinase, SnRK1, has been implicated in host defenses against geminiviruses. Overexpression of SnRK1 makes plants more resistant to geminivirus infection, and knock-down of SnRK1 increases susceptibility to geminivirus infection. GRIK, the SnRK1 activating kinase, is upregulated by geminivirus infection, while the viral C2 protein inhibits the SnRK1 activity. SnRK1 also directly phosphorylates geminivirus proteins to reduce infection. These data suggest that SnRK1 is involved in the co-evolution of plant hosts and geminiviruses.}, journal={CURRENT OPINION IN VIROLOGY}, author={Shen, Wei and Hanley-Bowdoin, Linda}, year={2021}, month={Apr}, pages={18–24} }
 @article{he_wang_yin_fiallo-oliv_liu_hanley-bowdoin_wang_2020, title={A plant DNA virus replicates in the salivary glands of its insect vector via recruitment of host DNA synthesis machinery}, volume={117}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1820132117}, abstractNote={Whereas most of the arthropod-borne animal viruses replicate in their vectors, this is less common for plant viruses. So far, only some plant RNA viruses have been demonstrated to replicate in insect vectors and plant hosts. How plant viruses evolved to replicate in the animal kingdom remains largely unknown. Geminiviruses comprise a large family of plant-infecting, single-stranded DNA viruses that cause serious crop losses worldwide. Here, we report evidence and insight into the replication of the geminivirus tomato yellow leaf curl virus (TYLCV) in the whitefly (Bemisia tabaci) vector and that replication is mainly in the salivary glands. We found that TYLCV induces DNA synthesis machinery, proliferating cell nuclear antigen (PCNA) and DNA polymerase δ (Polδ), to establish a replication-competent environment in whiteflies. TYLCV replication-associated protein (Rep) interacts with whitefly PCNA, which recruits DNA Polδ for virus replication. In contrast, another geminivirus, papaya leaf curl China virus (PaLCuCNV), does not replicate in the whitefly vector. PaLCuCNV does not induce DNA-synthesis machinery, and the Rep does not interact with whitefly PCNA. Our findings reveal important mechanisms by which a plant DNA virus replicates across the kingdom barrier in an insect and may help to explain the global spread of this devastating pathogen.}, number={29}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={He, Ya-Zhou and Wang, Yu-Meng and Yin, Tian-Yan and Fiallo-Oliv, Elvira and Liu, Yin-Quan and Hanley-Bowdoin, Linda and Wang, Xiao-Wei}, year={2020}, month={Jul}, pages={16928–16937} }
 @article{wheeler_brooks_concia_vera_wear_leblanc_ramu_vaughn_bass_martienssen_et al._2020, title={Arabidopsis DNA Replication Initiates in Intergenic, AT-Rich Open Chromatin(1)([OPEN])}, volume={183}, ISSN={["1532-2548"]}, DOI={10.1104/pp.19.01520}, abstractNote={The selection and firing of DNA replication origins play key roles in ensuring that eukaryotes accurately replicate their genomes. This process is not well documented in plants due in large measure to difficulties in working with plant systems. We developed a new functional assay to label and map very early replicating loci that must, by definition, include at least a subset of replication origins. Arabidopsis (Arabidopsis thaliana) cells were briefly labeled with 5-ethynyl-2′-deoxy-uridine, and nuclei were subjected to two-parameter flow sorting. We identified more than 5500 loci as initiation regions (IRs), the first regions to replicate in very early S phase. These were classified as strong or weak IRs based on the strength of their replication signals. Strong initiation regions were evenly spaced along chromosomal arms and depleted in centromeres, while weak initiation regions were enriched in centromeric regions. IRs are AT-rich sequences flanked by more GC-rich regions and located predominantly in intergenic regions. Nuclease sensitivity assays indicated that IRs are associated with accessible chromatin. Based on these observations, initiation of plant DNA replication shows some similarity to, but is also distinct from, initiation in other well-studied eukaryotic systems.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Wheeler, Emily and Brooks, Ashley M. and Concia, Lorenzo and Vera, Daniel L. and Wear, Emily E. and LeBlanc, Chantal and Ramu, Umamaheswari and Vaughn, Matthew W. and Bass, Hank W. and Martienssen, Robert A. and et al.}, year={2020}, month={May}, pages={206–220} }
 @article{wear_song_zynda_mickelson-young_leblanc_lee_deppong_allen_martienssen_vaughn_et al._2020, title={Comparing DNA replication programs reveals large timing shifts at centromeres of endocycling cells in maize roots}, volume={16}, ISSN={["1553-7404"]}, DOI={10.1371/journal.pgen.1008623}, abstractNote={Plant cells undergo two types of cell cycles-the mitotic cycle in which DNA replication is coupled to mitosis, and the endocycle in which DNA replication occurs in the absence of cell division. To investigate DNA replication programs in these two types of cell cycles, we pulse labeled intact root tips of maize (Zea mays) with 5-ethynyl-2'-deoxyuridine (EdU) and used flow sorting of nuclei to examine DNA replication timing (RT) during the transition from a mitotic cycle to an endocycle. Comparison of the sequence-based RT profiles showed that most regions of the maize genome replicate at the same time during S phase in mitotic and endocycling cells, despite the need to replicate twice as much DNA in the endocycle and the fact that endocycling is typically associated with cell differentiation. However, regions collectively corresponding to 2% of the genome displayed significant changes in timing between the two types of cell cycles. The majority of these regions are small with a median size of 135 kb, shift to a later RT in the endocycle, and are enriched for genes expressed in the root tip. We found larger regions that shifted RT in centromeres of seven of the ten maize chromosomes. These regions covered the majority of the previously defined functional centromere, which ranged between 1 and 2 Mb in size in the reference genome. They replicate mainly during mid S phase in mitotic cells but primarily in late S phase of the endocycle. In contrast, the immediately adjacent pericentromere sequences are primarily late replicating in both cell cycles. Analysis of CENH3 enrichment levels in 8C vs 2C nuclei suggested that there is only a partial replacement of CENH3 nucleosomes after endocycle replication is complete. The shift to later replication of centromeres and possible reduction in CENH3 enrichment after endocycle replication is consistent with a hypothesis that centromeres are inactivated when their function is no longer needed.}, number={10}, journal={PLOS GENETICS}, author={Wear, Emily E. and Song, Jawon and Zynda, Gregory J. and Mickelson-Young, Leigh and LeBlanc, Chantal and Lee, Tae-Jin and Deppong, David O. and Allen, George C. and Martienssen, Robert A. and Vaughn, Matthew W. and et al.}, year={2020}, month={Oct} }
 @article{ismayil_yang_haxim_wang_li_han_wang_zheng_wei_nagalakshmi_et al._2020, title={Cotton leaf curl Multan virus beta C1 Protein Induces Autophagy by Disrupting the Interaction of Autophagy-Related Protein 3 with Glyceraldehyde-3-Phosphate Dehydrogenases([OPEN])}, volume={32}, ISBN={1532-298X}, DOI={10.1105/tpc.19.00759}, abstractNote={Abstract Autophagy plays an important role in plant–pathogen interactions. Several pathogens including viruses induce autophagy in plants, but the underpinning mechanism remains largely unclear. Furthermore, in virus–plant interactions, viral factor(s) that induce autophagy have yet to be identified. Here, we report that the βC1 protein of Cotton leaf curl Multan betasatellite (CLCuMuB) interacts with cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC), a negative autophagic regulator, to induce autophagy in Nicotiana benthamiana. CLCuMuB βC1 bound to GAPCs and disrupted the interaction between GAPCs and autophagy-related protein 3 (ATG3). A mutant βC1 protein (βC13A) in which I45, Y48, and I53 were all substituted with Ala (A), had a dramatically reduced binding capacity with GAPCs, failed to disrupt the GAPCs-ATG3 interactions and failed to induce autophagy. Furthermore, mutant virus carrying βC13A showed increased symptoms and viral DNA accumulation associated with decreased autophagy in plants. These results suggest that CLCuMuB βC1 activates autophagy by disrupting GAPCs–ATG3 interactions.}, number={4}, journal={PLANT CELL}, author={Ismayil, Asigul and Yang, Meng and Haxim, Yakupjan and Wang, Yunjing and Li, Jinlin and Han, Lu and Wang, Yan and Zheng, Xiyin and Wei, Xiang and Nagalakshmi, Ugrappa and et al.}, year={2020}, month={Apr}, pages={1124–1135} }
 @article{hoyer_fondong_dallas_aimone_deppong_duffy_hanley-bowdoin_2020, title={Deeply Sequenced Infectious Clones of Key Cassava Begomovirus Isolates from Cameroon}, volume={9}, ISSN={["2576-098X"]}, DOI={10.1128/MRA.00802-20}, abstractNote={We deeply sequenced two pairs of widely used infectious clones (4 plasmids) of the bipartite begomoviruses African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV). The ACMV clones were quite divergent from published sequences. Raw reads, consensus plasmid sequences, and the infectious clones themselves are all publicly available.}, number={46}, journal={MICROBIOLOGY RESOURCE ANNOUNCEMENTS}, author={Hoyer, J. Steen and Fondong, Vincent N. and Dallas, Mary M. and Aimone, Catherine Doyle and Deppong, David O. and Duffy, Siobain and Hanley-Bowdoin, Linda}, year={2020}, month={Nov} }
 @article{wang_wu_gong_ismayil_yuan_lian_jia_han_deng_hong_et al._2019, title={Geminiviral V2 Protein Suppresses Transcriptional Gene Silencing through Interaction with AGO4}, volume={93}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.01675-18}, abstractNote={In plants, RNA-directed DNA methylation (RdDM)-mediated transcriptional gene silencing (TGS) is a natural antiviral defense against geminiviruses. Several geminiviral proteins have been shown to target the enzymes related to the methyl cycle or histone modification; however, it remains largely unknown whether and by which mechanism geminiviruses directly inhibit RdDM-mediated TGS. In this study, we showed that Cotton leaf curl Multan virus (CLCuMuV) V2 directly interacts with Nicotiana benthamiana AGO4 (NbAGO4) and that the L76S mutation in V2 (V2L76S) abolishes such interaction. We further showed that V2, but not V2L76S, can suppresses RdDM and TGS. Silencing of NbAGO4 inhibits TGS, reduces the viral methylation level, and enhances CLCuMuV DNA accumulation. In contrast, the V2L76S substitution mutant attenuates CLCuMuV infection and enhances the viral methylation level. These findings reveal that CLCuMuV V2 contributes to viral infection by interaction with NbAGO4 to suppress RdDM-mediated TGS in plants.IMPORTANCE In plants, the RNA-directed DNA methylation (RdDM) pathway is a natural antiviral defense mechanism against geminiviruses. However, how geminiviruses counter RdDM-mediated defense is largely unknown. Our findings reveal that Cotton leaf curl Multan virus V2 contributes to viral infection by interaction with NbAGO4 to suppress RNA-directed DNA methylation-mediated transcriptional gene silencing in plants. Our work provides the first evidence that a geminiviral protein is able to directly target core RdDM components to counter RdDM-mediated TGS antiviral defense in plants, which extends our current understanding of viral counters to host antiviral defense.}, number={6}, journal={JOURNAL OF VIROLOGY}, author={Wang, Yunjing and Wu, Yuyao and Gong, Qian and Ismayil, Asigul and Yuan, Yuxiang and Lian, Bi and Jia, Qi and Han, Meng and Deng, Haiteng and Hong, Yiguo and et al.}, year={2019}, month={Mar} }
 @inbook{mbewe_hanley-bowdoin_ndunguru_duffy_2018, place={St. Paul, MN}, title={Cassava viruses – host jumps, virus recombination, spread in plant material}, booktitle={Emerging Plant Diseases and Global Food Security}, publisher={APS Press}, author={Mbewe, W. and Hanley-Bowdoin, L. and Ndunguru, J. and Duffy, S.}, editor={Records, A. and Ristaino, J.Editors}, year={2018} }
 @article{turpin_vera_savadel_lung_wear_mickelson-young_thompson_hanley-bowdoin_dennis_zhang_et al._2018, title={Chromatin structure profile data from DNS-seq: Differential nuclease sensitivity mapping of four reference tissues of B73 maize (Zea mays L)}, volume={20}, ISSN={["2352-3409"]}, DOI={10.1016/j.dib.2018.08.015}, abstractNote={Presented here are data from Next-Generation Sequencing of differential micrococcal nuclease digestions of formaldehyde-crosslinked chromatin in selected tissues of maize (Zea mays) inbred line B73. Supplemental materials include a wet-bench protocol for making DNS-seq libraries, the DNS-seq data processing pipeline for producing genome browser tracks. This report also includes the peak-calling pipeline using the iSeg algorithm to segment positive and negative peaks from the DNS-seq difference profiles. The data repository for the sequence data is the NCBI SRA, BioProject Accession PRJNA445708.}, journal={DATA IN BRIEF}, author={Turpin, Zachary M. and Vera, Daniel L. and Savadel, Savannah D. and Lung, Pei-Yau and Wear, Emily E. and Mickelson-Young, Leigh and Thompson, William F. and Hanley-Bowdoin, Linda and Dennis, Jonathan H. and Zhang, Jinfeng and et al.}, year={2018}, month={Oct}, pages={358–363} }
 @article{ismayil_haxim_wang_li_qian_han_chen_jia_yihao liu_zhu_et al._2018, title={Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase}, volume={14}, ISSN={1553-7374}, url={http://dx.doi.org/10.1371/journal.ppat.1007282}, DOI={10.1371/journal.ppat.1007282}, abstractNote={Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.}, number={8}, journal={PLOS Pathogens}, publisher={Public Library of Science (PLoS)}, author={Ismayil, Asigul and Haxim, Yakupjan and Wang, Yunjing and Li, Huangai and Qian, Lichao and Han, Ting and Chen, Tianyuan and Jia, Qi and Yihao Liu, Alexander and Zhu, Songbiao and et al.}, editor={Dinesh-Kumar, Savithramma P.Editor}, year={2018}, month={Aug}, pages={e1007282} }
 @article{caldo_shen_xu_hanley-bowdoin_chen_weselake_lemieux_2018, title={Diacylglycerol acyltransferase 1 is activated by phosphatidate and inhibited by SnRK1-catalyzed phosphorylation}, volume={96}, ISSN={["1365-313X"]}, DOI={10.1111/tpj.14029}, abstractNote={Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final and committed step in the Kennedy pathway for triacylglycerol (TAG) biosynthesis and, as such, elucidating its mode of regulation is critical to understand the fundamental aspects of carbon metabolism in oleaginous crops. In this study, purified Brassica napus diacylglycerol acyltransferase 1 (BnaDGAT1) in n-dodecyl-β-d-maltopyranoside micelles was lipidated to form mixed micelles and subjected to detailed biochemical analysis. The degree of mixed micelle fluidity appeared to influence acyltransferase activity. BnaDGAT1 exhibited a sigmoidal response and eventual substrate inhibition with respect to increasing concentrations of oleoyl-CoA. Phosphatidate (PA) was identified as a feed-forward activator of BnaDGAT1, enabling the final enzyme in the Kennedy pathway to adjust to the incoming flow of carbon leading to TAG. In the presence of PA, the oleoyl-CoA saturation plot became more hyperbolic and desensitized to substrate inhibition indicating that PA facilitates the transition of the enzyme into the more active state. PA may also relieve possible autoinhibition of BnaDGAT1 brought about by the N-terminal regulatory domain, which was shown to interact with PA. Indeed, PA is a key effector modulating lipid homeostasis, in addition to its well recognized role in lipid signaling. BnaDGAT1 was also shown to be a substrate of the sucrose non-fermenting-1-related kinase 1 (SnRK1), which catalyzed phosphorylation of the enzyme and converted it to a less active form. Thus, this known regulator of carbon metabolism directly influences TAG biosynthesis.}, number={2}, journal={PLANT JOURNAL}, author={Caldo, Kristian Mark P. and Shen, Wei and Xu, Yang and Hanley-Bowdoin, Linda and Chen, Guanqun and Weselake, Randall J. and Lemieux, M. Joanne}, year={2018}, month={Oct}, pages={287–299} }
 @article{concia_brooks_wheeler_zynda_wear_leblanc_song_lee_pascuzzi_martienssen_et al._2018, title={Genome-Wide Analysis of the Arabidopsis Replication Timing Program}, volume={176}, ISSN={["1532-2548"]}, url={http://europepmc.org/abstract/med/29301956}, DOI={10.1104/pp.17.01537}, abstractNote={Eukaryotes use a temporally regulated process, known as the replication timing program, to ensure that their genomes are fully and accurately duplicated during S phase. Replication timing programs are predictive of genomic features and activity and are considered to be functional readouts of chromatin organization. Although replication timing programs have been described for yeast and animal systems, much less is known about the temporal regulation of plant DNA replication or its relationship to genome sequence and chromatin structure. We used the thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to describe, at high-resolution, the genome-wide replication timing program for Arabidopsis (Arabidopsis thaliana) Col-0 suspension cells. We identified genomic regions that replicate predominantly during early, mid, and late S phase, and correlated these regions with genomic features and with data for chromatin state, accessibility, and long-distance interaction. Arabidopsis chromosome arms tend to replicate early while pericentromeric regions replicate late. Early and mid-replicating regions are gene-rich and predominantly euchromatic, while late regions are rich in transposable elements and primarily heterochromatic. However, the distribution of chromatin states across the different times is complex, with each replication time corresponding to a mixture of states. Early and mid-replicating sequences interact with each other and not with late sequences, but early regions are more accessible than mid regions. The replication timing program in Arabidopsis reflects a bipartite genomic organization with early/mid-replicating regions and late regions forming separate, noninteracting compartments. The temporal order of DNA replication within the early/mid compartment may be modulated largely by chromatin accessibility.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Concia, Lorenzo and Brooks, Ashley M. and Wheeler, Emily and Zynda, Gregory J. and Wear, Emily E. and LeBlanc, Chantal and Song, Jawon and Lee, Tae-Jin and Pascuzzi, Pete E. and Martienssen, Robert A. and et al.}, year={2018}, month={Mar}, pages={2166–2185} }
 @article{rajabu_kennedy_ndunguru_ateka_tairo_hanley-bowdoin_ascencio-ibanez_2018, title={Lanai: A small, fast growing tomato variety is an excellent model system for studying geminiviruses}, volume={256}, ISSN={["1879-0984"]}, DOI={10.1016/j.jviromet.2018.03.002}, abstractNote={Geminiviruses are devastating single-stranded DNA viruses that infect a wide variety of crops in tropical and subtropical areas of the world. Tomato, which is a host for more than 100 geminiviruses, is one of the most affected crops. Developing plant models to study geminivirus-host interaction is important for the design of virus management strategies. In this study, “Florida Lanai” tomato was broadly characterized using three begomoviruses (Tomato yellow leaf curl virus, TYLCV; Tomato mottle virus, ToMoV; Tomato golden mosaic virus, TGMV) and a curtovirus (Beet curly top virus, BCTV). Infection rates of 100% were achieved by agroinoculation of TYLCV, ToMoV or BCTV. Mechanical inoculation of ToMoV or TGMV using a microsprayer as well as whitefly transmission of TYLCV or ToMoV also resulted in 100% infection frequencies. Symptoms appeared as early as four days post inoculation when agroinoculation or bombardment was used. Symptoms were distinct for each virus and a range of features, including plant height, flower number, fruit number, fruit weight and ploidy, was characterized. Due to its small size, rapid growth, ease of characterization and maintenance, and distinct responses to different geminiviruses, “Florida Lanai” is an excellent choice for comparing geminivirus infection in a common host.}, journal={JOURNAL OF VIROLOGICAL METHODS}, author={Rajabu, C. A. and Kennedy, G. G. and Ndunguru, J. and Ateka, E. M. and Tairo, F. and Hanley-Bowdoin, L. and Ascencio-Ibanez, J. T.}, year={2018}, month={Jun}, pages={89–99} }
 @article{shen_bobay_greeley_reyes_rajabu_blackburn_dallas_goshe_ascencio-ibanez_hanley-bowdoin_2018, title={Sucrose Nonfermenting 1-Related Protein Kinase 1 Phosphorylates a Geminivirus Rep Protein to Impair Viral Replication and Infection}, volume={178}, ISSN={["1532-2548"]}, DOI={10.1104/pp.18.00268}, abstractNote={Geminiviruses are single-stranded DNA viruses that infect a wide variety of plants and cause severe crop losses worldwide. The geminivirus replication initiator protein (Rep) binds to the viral replication origin and catalyzes DNA cleavage and ligation to initiate rolling circle replication. In this study, we found that the Tomato golden mosaic virus (TGMV) Rep is phosphorylated at serine-97 by sucrose nonfermenting 1-related protein kinase 1 (SnRK1), a master regulator of plant energy homeostasis and metabolism. Phosphorylation of Rep or the phosphomimic S97D mutation impaired Rep binding to viral DNA. A TGMV DNA-A replicon containing the Rep S97D mutation replicated less efficiently in tobacco (Nicotiana tabacum) protoplasts than in wild-type or Rep phosphorylation-deficient replicons. The TGMV Rep-S97D mutant also was less infectious than the wild-type virus in Nicotiana benthamiana and was unable to infect tomato (Solanum lycopersicum). Nearly all geminivirus Rep proteins have a serine residue at the position equivalent to TGMV Rep serine-97. SnRK1 phosphorylated the equivalent serines in the Rep proteins of Tomato mottle virus and Tomato yellow leaf curl virus and reduced DNA binding, suggesting that SnRK1 plays a key role in combating geminivirus infection. These results established that SnRK1 phosphorylates Rep and interferes with geminivirus replication and infection, underscoring the emerging role for SnRK1 in the host defense response against plant pathogens.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Shen, Wei and Bobay, Benjamin G. and Greeley, Laura A. and Reyes, Maria I. and Rajabu, Cyprian A. and Blackburn, R. Kevin and Dallas, Mary Beth and Goshe, Michael B. and Ascencio-Ibanez, Jose T. and Hanley-Bowdoin, Linda}, year={2018}, month={Sep}, pages={372–389} }
 @article{reyes_flores-vergara_guerra-peraza_rajabu_desai_hiromoto-ruiz_ndunguru_hanley-bowdoin_kjemtrup_ascencio-ibanez_et al._2017, title={A VIGS screen identifies immunity in the Arabidopsis Pla-1 accession to viruses in two different genera of the Geminiviridae}, volume={92}, ISSN={["1365-313X"]}, DOI={10.1111/tpj.13716}, abstractNote={Summary Geminiviruses are DNA viruses that cause severe crop losses in different parts of the world, and there is a need for genetic sources of resistance to help combat them. Arabidopsis has been used as a source for virus‐resistant genes that derive from alterations in essential host factors. We used a virus‐induced gene silencing ( VIGS ) vector derived from the geminivirus Cabbage leaf curl virus (Ca LC uV) to assess natural variation in virus–host interactions in 190 Arabidopsis accessions. Silencing of CH ‐42 , encoding a protein needed to make chlorophyll, was used as a visible marker to discriminate asymptomatic accessions from those showing resistance. There was a wide range in symptom severity and extent of silencing in different accessions, but two correlations could be made. Lines with severe symptoms uniformly lacked extensive VIGS , and lines that showed attenuated symptoms over time (recovery) showed a concomitant increase in the extent of VIGS . One accession, Pla‐1, lacked both symptoms and silencing, and was immune to wild‐type infectious clones corresponding to Ca LC uV or Beet curly top virus ( BCTV ), which are classified in different genera in the Geminiviridae. It also showed resistance to the agronomically important Tomato yellow leaf curl virus ( TYLCV ). Quantitative trait locus mapping of a Pla‐1 X Col‐0 F 2 population was used to detect a major peak on chromosome 1, which is designated gip‐1 ( geminivirus immunity Pla‐1‐1 ). The recessive nature of resistance to Ca LC uV and the lack of obvious candidate genes near the gip‐1 locus suggest that a novel resistance gene(s) confers immunity.}, number={5}, journal={PLANT JOURNAL}, author={Reyes, Maria Ines and Flores-Vergara, Miguel A. and Guerra-Peraza, Orlene and Rajabu, Cyprian and Desai, Jigar and Hiromoto-Ruiz, Yokiko H. and Ndunguru, Joseph and Hanley-Bowdoin, Linda and Kjemtrup, Susanne and Ascencio-Ibanez, Jose T. and et al.}, year={2017}, month={Dec}, pages={796–807} }
 @article{michelmore_coaker_bart_beattie_bent_bruce_cameron_dangl_dinesh-kumar_edwards_et al._2017, title={Foundational and Translational Research Opportunities to Improve Plant Health}, volume={30}, ISSN={0894-0282}, url={http://dx.doi.org/10.1094/mpmi-01-17-0010-cr}, DOI={10.1094/mpmi-01-17-0010-cr}, abstractNote={The white paper reports the deliberations of a workshop focused on biotic challenges to plant health held in Washington, D.C. in September 2016. Ensuring health of food plants is critical to maintaining the quality and productivity of crops and for sustenance of the rapidly growing human population. There is a close linkage between food security and societal stability; however, global food security is threatened by the vulnerability of our agricultural systems to numerous pests, pathogens, weeds, and environmental stresses. These threats are aggravated by climate change, the globalization of agriculture, and an over-reliance on nonsustainable inputs. New analytical and computational technologies are providing unprecedented resolution at a variety of molecular, cellular, organismal, and population scales for crop plants as well as pathogens, pests, beneficial microbes, and weeds. It is now possible to both characterize useful or deleterious variation as well as precisely manipulate it. Data-driven, informed decisions based on knowledge of the variation of biotic challenges and of natural and synthetic variation in crop plants will enable deployment of durable interventions throughout the world. These should be integral, dynamic components of agricultural strategies for sustainable agriculture.}, number={7}, journal={Molecular Plant-Microbe Interactions}, publisher={Scientific Societies}, author={Michelmore, Richard and Coaker, Gitta and Bart, Rebecca and Beattie, Gwyn and Bent, Andrew and Bruce, Toby and Cameron, Duncan and Dangl, Jeffery and Dinesh-Kumar, Savithramma and Edwards, Rob and et al.}, year={2017}, month={Jul}, pages={515–516} }
 @article{wear_song_zynda_leblanc_lee_mickelson-young_concia_mulvaney_szymanski_allen_et al._2017, title={Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize (Zea mays) Root Tips}, volume={29}, ISSN={["1532-298X"]}, url={http://europepmc.org/abstract/med/28842533}, DOI={10.1105/tpc.17.00037}, abstractNote={All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize (Zea mays) that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle. Maize root tips have a complex replication timing program, including regions of distinct early, mid, and late S replication that each constitute between 20 and 24% of the genome, as well as other loci corresponding to ∼32% of the genome that exhibit replication activity in two different time windows. Analyses of genomic, transcriptional, and chromatin features of the euchromatic portion of the maize genome provide evidence for a gradient of early replicating, open chromatin that transitions gradually to less open and less transcriptionally active chromatin replicating in mid S phase. Our genomic level analysis also demonstrated that the centromere core replicates in mid S, before heavily compacted classical heterochromatin, including pericentromeres and knobs, which replicate during late S phase.}, number={9}, journal={PLANT CELL}, author={Wear, Emily E. and Song, Jawon and Zynda, Gregory J. and LeBlanc, Chantal and Lee, Tae-Jin and Mickelson-Young, Leigh and Concia, Lorenzo and Mulvaney, Patrick and Szymanski, Eric S. and Allen, George C. and et al.}, year={2017}, month={Sep}, pages={2126–2149} }
 @article{zynda_song_concia_wear_hanley-bowdoin_thompson_vaughn_2017, title={Repliscan: a tool for classifying replication timing regions}, volume={18}, ISSN={["1471-2105"]}, url={http://europepmc.org/abstract/med/28784090}, DOI={10.1186/s12859-017-1774-x}, abstractNote={Replication timing experiments that use label incorporation and high throughput sequencing produce peaked data similar to ChIP-Seq experiments. However, the differences in experimental design, coverage density, and possible results make traditional ChIP-Seq analysis methods inappropriate for use with replication timing. To accurately detect and classify regions of replication across the genome, we present Repliscan. Repliscan robustly normalizes, automatically removes outlying and uninformative data points, and classifies Repli-seq signals into discrete combinations of replication signatures. The quality control steps and self-fitting methods make Repliscan generally applicable and more robust than previous methods that classify regions based on thresholds. Repliscan is simple and effective to use on organisms with different genome sizes. Even with analysis window sizes as small as 1 kilobase, reliable profiles can be generated with as little as 2.4x coverage.}, journal={BMC BIOINFORMATICS}, author={Zynda, Gregory J. and Song, Jawon and Concia, Lorenzo and Wear, Emily E. and Hanley-Bowdoin, Linda and Thompson, William F. and Vaughn, Matthew W.}, year={2017}, month={Aug}, pages={1–14} }
 @article{mickelson-young_wear_mulvaney_lee_szymanski_allen_hanley-bowdoin_thompson_2016, title={A flow cytometric method for estimating S-phase duration in plants}, volume={67}, ISSN={["1460-2431"]}, url={http://europepmc.org/abstract/med/27697785}, DOI={10.1093/jxb/erw367}, abstractNote={The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry. The transition between G1 and G2 DNA contents can be followed by measuring the mean DNA content of EdU-labeled S-phase nuclei as a function of time after the labeling pulse. We applied this technique to intact root tips of maize (Zea mays L.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), and wheat (Triticum aestivum L.), and to actively dividing cell cultures of Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and rice. Estimates of S-phase duration in root tips were remarkably consistent, varying only by ~3-fold, although the genome sizes of the species analyzed varied >40-fold.}, number={21}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Mickelson-Young, Leigh and Wear, Emily and Mulvaney, Patrick and Lee, Tae-Jin and Szymanski, Eric S. and Allen, George and Hanley-Bowdoin, Linda and Thompson, William}, year={2016}, month={Nov}, pages={6077–6087} }
 @inbook{wear_concia_brooks_markham_lee_allen_thompson_hanley-bowdoin_2016, title={Isolation of Plant Nuclei at Defined Cell Cycle Stages Using EdU Labeling and Flow Cytometry}, ISBN={9781493931415 9781493931422}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-4939-3142-2_6}, DOI={10.1007/978-1-4939-3142-2_6}, abstractNote={5-Ethynyl-2'-deoxyuridine (EdU) is a nucleoside analog of thymidine that can be rapidly incorporated into replicating DNA in vivo and, subsequently, detected by using "click" chemistry to couple its terminal alkyne group to fluorescent azides such as Alexa Fluor 488. Recently, EdU incorporation followed by coupling with a fluorophore has been used to visualize newly synthesized DNA in a wide range of plant species. One particularly useful application is in flow cytometry, where two-parameter sorting can be employed to analyze different phases of the cell cycle, as defined both by total DNA content and the amount of EdU pulse-labeled DNA. This approach allows analysis of the cell cycle without the need for synchronous cell populations, which can be difficult to obtain in many plant systems. The approach presented here, which was developed for fixed, EdU-labeled nuclei, can be used to prepare analytical profiles as well as to make highly purified preparations of G1, S, or G2/M phase nuclei for molecular or biochemical analysis. We present protocols for EdU pulse labeling, tissue fixation and harvesting, nuclei preparation, and flow sorting. Although developed for Arabidopsis suspension cells and maize root tips, these protocols should be modifiable to many other plant systems.}, booktitle={Methods in Molecular Biology}, publisher={Springer New York}, author={Wear, Emily E. and Concia, Lorenzo and Brooks, Ashley M. and Markham, Emily A. and Lee, Tae-Jin and Allen, George C. and Thompson, William F. and Hanley-Bowdoin, Linda}, year={2016}, pages={69–86} }
 @article{ndunguru_de leon_doyle_sseruwagi_plata_legg_thompson_tohme_aveling_ascencio-ibanez_et al._2016, title={Two Novel DNAs That Enhance Symptoms and Overcome CMD2 Resistance to Cassava Mosaic Disease}, volume={90}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.02834-15}, abstractNote={ABSTRACT Cassava mosaic begomoviruses (CMBs) cause cassava mosaic disease (CMD) across Africa and the Indian subcontinent. Like all members of the geminivirus family, CMBs have small, circular single-stranded DNA genomes. We report here the discovery of two novel DNA sequences, designated SEGS-1 and SEGS-2 (for s equences e nhancing g eminivirus s ymptoms), that enhance symptoms and break resistance to CMD. The SEGS are characterized by GC-rich regions and the absence of long open reading frames. Both SEGS enhanced CMD symptoms in cassava ( Manihot esculenta Crantz) when coinoculated with African cassava mosaic virus (ACMV), East African cassava mosaic Cameroon virus (EACMCV), or East African cassava mosaic virus-Uganda (EACMV-UG). SEGS-1 also overcame resistance of a cassava landrace carrying the CMD2 resistance locus when coinoculated with EACMV-UG. Episomal forms of both SEGS were detected in CMB-infected cassava but not in healthy cassava. SEGS-2 episomes were also found in virions and whiteflies. SEGS-1 has no homology to geminiviruses or their associated satellites, but the cassava genome contains a sequence that is 99% identical to full-length SEGS-1. The cassava genome also includes three sequences with 84 to 89% identity to SEGS-2 that together encompass all of SEGS-2 except for a 52-bp region, which includes the episomal junction and a 26-bp sequence related to alphasatellite replication origins. These results suggest that SEGS-1 is derived from the cassava genome and facilitates CMB infection as an integrated copy and/or an episome, while SEGS-2 was originally from the cassava genome but now is encapsidated into virions and transmitted as an episome by whiteflies. IMPORTANCE Cassava is a major crop in the developing world, with its production in Africa being second only to maize. CMD is one of the most important diseases of cassava and a serious constraint to production across Africa. CMD2 is a major CMD resistance locus that has been deployed in many cassava cultivars through large-scale breeding programs. In recent years, severe, atypical CMD symptoms have been observed occasionally on resistant cultivars, some of which carry the CMD2 locus, in African fields. In this report, we identified and characterized two DNA sequences, SEGS-1 and SEGS-2, which produce similar symptoms when coinoculated with cassava mosaic begomoviruses onto a susceptible cultivar or a CMD2-resistant landrace. The ability of SEGS-1 to overcome CMD2 resistance and the transmission of SEGS-2 by whiteflies has major implications for the long-term durability of CMD2 resistance and underscore the need for alternative sources of resistance in cassava.}, number={8}, journal={JOURNAL OF VIROLOGY}, author={Ndunguru, Joseph and De Leon, Leandro and Doyle, Catherine D. and Sseruwagi, Peter and Plata, German and Legg, James P. and Thompson, Graham and Tohme, Joe and Aveling, Theresa and Ascencio-Ibanez, Jose T. and et al.}, year={2016}, month={Apr}, pages={4160–4173} }
 @article{bass_hoffman_lee_wear_joseph_allen_hanley-bowdoin_thompson_2015, title={Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei}, volume={89}, ISSN={["1573-5028"]}, DOI={10.1007/s11103-015-0364-4}, abstractNote={Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.}, number={4-5}, journal={PLANT MOLECULAR BIOLOGY}, author={Bass, Hank W. and Hoffman, Gregg G. and Lee, Tae-Jin and Wear, Emily E. and Joseph, Stacey R. and Allen, George C. and Hanley-Bowdoin, Linda and Thompson, William F.}, year={2015}, month={Nov}, pages={339–351} }
 @misc{bass_wear_lee_hoffman_gumber_allen_thompson_hanley-bowdoin_2014, title={A maize root tip system to study DNA replication programmes in somatic and endocycling nuclei during plant development}, volume={65}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/ert470}, abstractNote={The progress of nuclear DNA replication is complex in both time and space, and may reflect several levels of chromatin structure and 3-dimensional organization within the nucleus. To understand the relationship between DNA replication and developmental programmes, it is important to examine replication and nuclear substructure in different developmental contexts including natural cell-cycle progressions in situ. Plant meristems offer an ideal opportunity to analyse such processes in the context of normal growth of an organism. Our current understanding of large-scale chromosomal DNA replication has been limited by the lack of appropriate tools to visualize DNA replication with high resolution at defined points within S phase. In this perspective, we discuss a promising new system that can be used to visualize DNA replication in isolated maize (Zea mays L.) root tip nuclei after in planta pulse labelling with the thymidine analogue, 5-ethynyl-2'-deoxyuridine (EdU). Mixed populations of EdU-labelled nuclei are then separated by flow cytometry into sequential stages of S phase and examined directly using 3-dimensional deconvolution microscopy to characterize spatial patterns of plant DNA replication. Combining spatiotemporal analyses with studies of replication and epigenetic inheritance at the molecular level enables an integrated experimental approach to problems of mitotic inheritance and cellular differentiation.}, number={10}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Bass, Hank W. and Wear, Emily E. and Lee, Tae-Jin and Hoffman, Gregg G. and Gumber, Hardeep K. and Allen, George C. and Thompson, William F. and Hanley-Bowdoin, Linda}, year={2014}, month={Jun}, pages={2747–2756} }
 @article{pascuzzi_flores-vergara_lee_sosinski_vaughn_hanley-bowdoin_thompson_allen_2014, title={In Vivo Mapping of Arabidopsis Scaffold/Matrix Attachment Regions Reveals Link to Nucleosome-Disfavoring Poly(dA:dT) Tracts}, volume={26}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.113.121194}, abstractNote={Scaffold or matrix attachment regions (S/MARs) are found in all eukaryotes. The pattern of distribution and genomic context of S/MARs is thought to be important for processes such as chromatin organization and modulation of gene expression. Despite the importance of such processes, much is unknown about the large-scale distribution and sequence content of S/MARs in vivo. Here, we report the use of tiling microarrays to map 1358 S/MARs on Arabidopsis thaliana chromosome 4 (chr4). S/MARs occur throughout chr4, spaced much more closely than in the large plant and animal genomes that have been studied to date. Arabidopsis S/MARs can be divided into five clusters based on their association with other genomic features, suggesting a diversity of functions. While some Arabidopsis S/MARs may define structural domains, most occur near the transcription start sites of genes. Genes associated with these S/MARs have an increased probability of expression, which is particularly pronounced in the case of transcription factor genes. Analysis of sequence motifs and 6-mer enrichment patterns show that S/MARs are preferentially enriched in poly(dA:dT) tracts, sequences that resist nucleosome formation, and the majority of S/MARs contain at least one nucleosome-depleted region. This global view of S/MARs provides a framework to begin evaluating genome-scale models for S/MAR function.}, number={1}, journal={PLANT CELL}, author={Pascuzzi, Pete E. and Flores-Vergara, Miguel A. and Lee, Tae-Jin and Sosinski, Bryon and Vaughn, Matthew W. and Hanley-Bowdoin, Linda and Thompson, William F. and Allen, George C.}, year={2014}, month={Jan}, pages={102–120} }
 @article{shen_dallas_goshe_hanley-bowdoin_2014, title={SnRK1 Phosphorylation of AL2 Delays Cabbage Leaf Curl Virus Infection in Arabidopsis}, volume={88}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.00761-14}, abstractNote={ABSTRACT Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage Leaf Curl Virus (CaLCuV) and Tomato mottle virus . A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana , demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions. IMPORTANCE Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses carry genes that encode multifunctional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World and may have provided a selective advantage as begomoviruses adapted to a different environment and different plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host.}, number={18}, journal={JOURNAL OF VIROLOGY}, author={Shen, Wei and Dallas, Mary Beth and Goshe, Michael B. and Hanley-Bowdoin, Linda}, year={2014}, month={Sep}, pages={10598–10612} }
 @misc{hanley-bowdoin_bejarano_robertson_mansoor_2013, title={Geminiviruses: masters at redirecting and reprogramming plant processes}, volume={11}, ISSN={["1740-1534"]}, DOI={10.1038/nrmicro3117}, abstractNote={Geminiviruses are important plant pathogens that cause devastating crop losses worldwide. Here, Hanley-Bowdoin and colleagues review how viral proteins interact with cellular machineries and reprogramme cellular control pathways in their plant host to support viral DNA replication, gene expression and trafficking, and to interfere with host defences. The family Geminiviridae is one of the largest and most important families of plant viruses. The small, single-stranded DNA genomes of geminiviruses encode 5–7 proteins that redirect host machineries and processes to establish a productive infection. These interactions reprogramme plant cell cycle and transcriptional controls, inhibit cell death pathways, interfere with cell signalling and protein turnover, and suppress defence pathways. This Review describes our current knowledge of how geminiviruses interact with their plant hosts and the functional consequences of these interactions.}, number={11}, journal={NATURE REVIEWS MICROBIOLOGY}, author={Hanley-Bowdoin, Linda and Bejarano, Eduardo R. and Robertson, Dominique and Mansoor, Shahid}, year={2013}, month={Nov}, pages={777–788} }
 @article{reyes_nash_dallas_ascencio-ibanez_hanley-bowdoin_2013, title={Peptide Aptamers That Bind to Geminivirus Replication Proteins Confer a Resistance Phenotype to Tomato Yellow Leaf Curl Virus and Tomato Mottle Virus Infection in Tomato}, volume={87}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.01095-13}, abstractNote={ABSTRACT Geminiviruses constitute a large family of single-stranded DNA viruses that cause serious losses in important crops worldwide. They often exist in disease complexes and have high recombination and mutation rates, allowing them to adapt rapidly to new hosts and environments. Thus, an effective resistance strategy must be general in character and able to target multiple viruses. The geminivirus replication protein (Rep) is a good target for broad-based disease control because it is highly conserved and required for viral replication. In an earlier study, we identified a set of peptide aptamers that bind to Rep and reduce viral replication in cultured plant cells. In this study, we selected 16 of the peptide aptamers for further analysis in yeast two-hybrid assays. The results of these experiments showed that all 16 peptide aptamers interact with all or most of the Rep proteins from nine viruses representing the three major Geminiviridae genera and identified two peptide aptamers (A22 and A64) that interact strongly with different regions in the Rep N terminus. Transgenic tomato lines expressing A22 or A64 and inoculated with Tomato yellow leaf curl virus or Tomato mottle virus exhibited delayed viral DNA accumulation and often contained lower levels of viral DNA. Strikingly, the effect on symptoms was stronger, with many of the plants showing no symptoms or strongly attenuated symptoms. Together, these results established the efficacy of using Rep-binding peptide aptamers to develop crops that are resistant to diverse geminiviruses.}, number={17}, journal={JOURNAL OF VIROLOGY}, author={Reyes, Maria Ines and Nash, Tara E. and Dallas, Mary M. and Ascencio-Ibanez, J. Trinidad and Hanley-Bowdoin, Linda}, year={2013}, month={Sep}, pages={9691–9706} }
 @article{nash_dallas_reyes_buhrman_ascencio-ibanez_hanley-bowdoin_2011, title={Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins}, volume={85}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.02143-10}, abstractNote={ABSTRACT Members of the Geminiviridae have single-stranded DNA genomes that replicate in nuclei of infected plant cells. All geminiviruses encode a conserved protein (Rep) that catalyzes initiation of rolling-circle replication. Earlier studies showed that three conserved motifs—motifs I, II, and III—in the N termini of geminivirus Rep proteins are essential for function. In this study, we identified a fourth sequence, designated GRS ( g eminivirus R ep s equence), in the Rep N terminus that displays high amino acid sequence conservation across all geminivirus genera. Using the Rep protein of Tomato golden mosaic virus (TGMV AL1), we show that GRS mutants are not infectious in plants and do not support viral genome replication in tobacco protoplasts. GRS mutants are competent for protein-protein interactions and for both double- and single-stranded DNA binding, indicating that the mutations did not impair its global conformation. In contrast, GRS mutants are unable to specifically cleave single-stranded DNA, which is required to initiate rolling-circle replication. Interestingly, the Rep proteins of phytoplasmal and algal plasmids also contain GRS-related sequences. Modeling of the TGMV AL1 N terminus suggested that GRS mutations alter the relative positioning of motif II, which coordinates metal ions, and motif III, which contains the tyrosine involved in DNA cleavage. Together, these results established that the GRS is a conserved, essential motif characteristic of an ancient lineage of rolling-circle initiators and support the idea that geminiviruses may have evolved from plasmids associated with phytoplasma or algae.}, number={3}, journal={JOURNAL OF VIROLOGY}, author={Nash, Tara E. and Dallas, Mary B. and Reyes, Maria Ines and Buhrman, Gregory K. and Ascencio-Ibanez, J. Trinidad and Hanley-Bowdoin, Linda}, year={2011}, month={Feb}, pages={1182–1192} }
 @article{sanchez-duran_dallas_ascencio-ibanez_reyes_arroyo-mateos_ruiz-albert_hanley-bowdoin_bejarano_2011, title={Interaction between Geminivirus Replication Protein and the SUMO-Conjugating Enzyme Is Required for Viral Infection}, volume={85}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.02566-10}, abstractNote={ABSTRACT Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells by using plant DNA polymerases. These viruses encode a protein designated AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyzes the cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma-related protein (RBR), the DNA replication protein PCNA (proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO-conjugating enzyme [SCE1]). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85 to 114. Alanine mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed for other AL1 functions, such as oligomerization, DNA binding, DNA cleavage, and interaction with AL3 or RBR. The lysine mutations reduced or abolished virus infectivity in plants and viral DNA accumulation in transient-replication assays, suggesting that the AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected, indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection of plants and suggested that AL1 alters the sumoylation of selected host factors to create an environment suitable for viral infection.}, number={19}, journal={JOURNAL OF VIROLOGY}, author={Sanchez-Duran, Miguel A. and Dallas, Mary B. and Ascencio-Ibanez, Jose T. and Reyes, Maria Ines and Arroyo-Mateos, Manuel and Ruiz-Albert, Javier and Hanley-Bowdoin, Linda and Bejarano, Eduardo R.}, year={2011}, month={Oct}, pages={9789–9800} }
 @article{shen_liu_song_xie_hanley-bowdoin_zhou_2011, title={Tomato SlSnRK1 Protein Interacts with and Phosphorylates beta C1, a Pathogenesis Protein Encoded by a Geminivirus beta-Satellite}, volume={157}, ISSN={["0032-0889"]}, DOI={10.1104/pp.111.184648}, abstractNote={The βC1 protein of tomato yellow leaf curl China β-satellite functions as a pathogenicity determinant. To better understand the molecular basis of βC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using βC1 as bait. βC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that βC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with βC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the βC1 protein.}, number={3}, journal={PLANT PHYSIOLOGY}, author={Shen, Qingtang and Liu, Zhou and Song, Fengming and Xie, Qi and Hanley-Bowdoin, Linda and Zhou, Xueping}, year={2011}, month={Nov}, pages={1394–1406} }
 @article{lee_pascuzzi_settlage_shultz_tanurdzic_rabinowicz_menges_zheng_main_murray_et al._2010, title={Arabidopsis thaliana Chromosome 4 Replicates in Two Phases That Correlate with Chromatin State}, volume={6}, ISSN={1553-7404}, url={http://dx.doi.org/10.1371/journal.pgen.1000982}, DOI={10.1371/journal.pgen.1000982}, abstractNote={DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins.}, number={6}, journal={PLoS Genetics}, publisher={Public Library of Science (PLoS)}, author={Lee, Tae-Jin and Pascuzzi, Pete E. and Settlage, Sharon B. and Shultz, Randall W. and Tanurdzic, Milos and Rabinowicz, Pablo D. and Menges, Margit and Zheng, Ping and Main, Dorrie and Murray, James A. H. and et al.}, editor={Copenhaver, Gregory P.Editor}, year={2010}, month={Jun}, pages={e1000982} }
 @article{sozzani_maggio_giordo_umana_ascencio-ibañez_hanley-bowdoin_bergounioux_cella_albani_2010, title={The E2FD/DEL2 factor is a component of a regulatory network controlling cell proliferation and development in Arabidopsis}, volume={72}, ISSN={1573-5028}, DOI={10.1007/s11103-009-9577-8}, number={4-5}, journal={Plant Molecular Biology}, author={Sozzani, Rosangela and Maggio, Caterina and Giordo, Roberta and Umana, Elisabetta and Ascencio-Ibañez, Jose Trinidad and Hanley-Bowdoin, Linda and Bergounioux, Catherine and Cella, Rino and Albani, Diego}, year={2010}, month={Mar}, pages={381–395} }
 @article{shen_reyes_hanley-bowdoin_2009, title={Arabidopsis Protein Kinases GRIK1 and GRIK2 Specifically Activate SnRK1 by Phosphorylating Its Activation Loop}, volume={150}, ISSN={["1532-2548"]}, DOI={10.1104/pp.108.132787}, abstractNote={SNF1-related kinases (SnRK1s) play central roles in coordinating energy balance and nutrient metabolism in plants. SNF1 and AMPK, the SnRK1 homologs in budding yeast (Saccharomyces cerevisiae) and mammals, are activated by phosphorylation of conserved threonine residues in their activation loops. Arabidopsis (Arabidopsis thaliana) GRIK1 and GRIK2, which were first characterized as geminivirus Rep interacting kinases, are phylogenetically related to SNF1 and AMPK activating kinases. In this study, we used recombinant proteins produced in bacteria to show that both GRIKs specifically bind to the SnRK1 catalytic subunit and phosphorylate the equivalent threonine residue in its activation loop in vitro. GRIK-mediated phosphorylation increased SnRK1 kinase activity in autophosphorylation and peptide substrate assays. These data, together with earlier observations that GRIKs could complement yeast mutants lacking SNF1 activation activities, established that the GRIKs are SnRK1 activating kinases. Given that the GRIK proteins only accumulate in young tissues and geminivirus-infected mature leaves, the GRIK-SnRK1 cascade may function in a developmentally regulated fashion and coordinate the unique metabolic requirements of rapidly growing cells and geminivirus-infected cells that have been induced to reenter the cell cycle.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Shen, Wei and Reyes, Maria Ines and Hanley-Bowdoin, Linda}, year={2009}, month={Jun}, pages={996–1005} }
 @article{shultz_lee_allen_thompson_hanley-bowdoin_2009, title={Dynamic Localization of the DNA Replication Proteins MCM5 and MCM7 in Plants}, volume={150}, ISSN={["1532-2548"]}, DOI={10.1104/pp.109.136614}, abstractNote={Genome integrity in eukaryotes depends on licensing mechanisms that prevent loading of the minichromosome maintenance complex (MCM2-7) onto replicated DNA during S phase. Although the principle of licensing appears to be conserved across all eukaryotes, the mechanisms that control it vary, and it is not clear how licensing is regulated in plants. In this work, we demonstrate that subunits of the MCM2-7 complex are coordinately expressed during Arabidopsis (Arabidopsis thaliana) development and are abundant in proliferating and endocycling tissues, indicative of a role in DNA replication. We show that endogenous MCM5 and MCM7 proteins are localized in the nucleus during G1, S, and G2 phases of the cell cycle and are released into the cytoplasmic compartment during mitosis. We also show that MCM5 and MCM7 are topologically constrained on DNA and that the MCM complex is stable under high-salt conditions. Our results are consistent with a conserved replicative helicase function for the MCM complex in plants but not with the idea that plants resemble budding yeast by actively exporting the MCM complex from the nucleus to prevent unauthorized origin licensing and rereplication during S phase. Instead, our data show that, like other higher eukaryotes, the MCM complex in plants remains in the nucleus throughout most of the cell cycle and is only dispersed in mitotic cells.}, number={2}, journal={PLANT PHYSIOLOGY}, author={Shultz, Randall W. and Lee, Tae-Jin and Allen, George C. and Thompson, William F. and Hanley-Bowdoin, Linda}, year={2009}, month={Jun}, pages={658–669} }
 @inbook{lopez-ochoa_nash_ramirez-prado_hanley-bowdoin_2009, title={Isolation of Peptide Aptamers to Target Protein Function}, ISBN={9781934115893 9781597455572}, ISSN={1064-3745 1940-6029}, url={http://dx.doi.org/10.1007/978-1-59745-557-2_19}, DOI={10.1007/978-1-59745-557-2_19}, abstractNote={Peptide aptamers are small recombinant proteins typically inserted into a supportive protein scaffold. These short peptide domains can bind to their target proteins with high specificity and affinity, often resulting in an altered target protein. We describe high-throughput protocols that facilitate the selection and characterization of peptide aptamers from yeast dihybrid libraries. These protocols include the preparation and evaluation of the bait fusion and the peptide aptamer screen. They also include confirmation of interaction specificity as well as isolation and sequencing of peptide inserts. Once the amino acid sequence is determined, we describe a protocol for aligning and comparing short peptide sequences and assessing the statistical significance of the alignments.}, booktitle={Methods in Molecular Biology}, publisher={Humana Press}, author={Lopez-Ochoa, Luisa and Nash, Tara E. and Ramirez-Prado, Jorge and Hanley-Bowdoin, Linda}, year={2009}, pages={333–360} }
 @misc{ascencio-ibanez_sozzani_lee_chu_wolfinger_cella_hanley-bowdoin_2008, title={Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection}, volume={148}, number={1}, journal={Plant Physiology}, author={Ascencio-Ibanez, J. T. and Sozzani, R. and Lee, T. J. and Chu, T. M. and Wolfinger, R. D. and Cella, R. and Hanley-Bowdoin, L.}, year={2008}, pages={436–454} }
 @article{jordan_shen_hanley-bowdoin_robertson_2007, title={Geminivirus-induced gene silencing of the tobacco retinoblastoma-related gene results in cell death and altered development}, volume={65}, ISSN={["1573-5028"]}, DOI={10.1007/s11103-007-9206-3}, number={1-2}, journal={PLANT MOLECULAR BIOLOGY}, author={Jordan, Chad V. and Shen, Wei and Hanley-Bowdoin, Linda K. and Robertson, Dominique}, year={2007}, month={Sep}, pages={163–175} }
 @misc{shultz_tatineni_hanley-bowdoin_thompson_2007, title={Genome-wide analysis of the core DNA replication machinery in the higher plants Arabidopsis and rice(1[W][OA])}, volume={144}, number={4}, journal={Plant Physiology}, author={Shultz, R. W. and Tatineni, V. M. and Hanley-Bowdoin, L. and Thompson, W. F.}, year={2007}, pages={1697–1714} }
 @article{arguello-astorga_ascencio-ibanez_dallas_orozco_hanley-bowdoin_2007, title={High-frequency reversion of geminivirus replication protein mutants during infection}, volume={81}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.00925-07}, abstractNote={ABSTRACT The geminivirus replication protein AL1 interacts with retinoblastoma-related protein (RBR), a key regulator of the plant division cell cycle, to induce conditions permissive for viral DNA replication. Previous studies of tomato golden mosaic virus (TGMV) AL1 showed that amino acid L148 in the conserved helix 4 motif is critical for RBR binding. In this work, we examined the effect of an L148V mutation on TGMV replication in tobacco cells and during infection of Nicotiana benthamiana plants. The L148V mutant replicated 100 times less efficiently than wild-type TGMV in protoplasts but produced severe symptoms that were delayed compared to those of wild-type infection in plants. Analysis of progeny viruses revealed that the L148V mutation reverted at 100% frequency in planta to methionine, leucine, isoleucine, or a second-site mutation depending on the valine codon in the initial DNA sequence. Similar results were seen with another geminivirus, cabbage leaf curl virus (CaLCuV), carrying an L145A mutation in the equivalent residue. Valine was the predominant amino acid recovered from N. benthamiana plants inoculated with the CaLCuV L145A mutant, while threonine was the major residue in Arabidopsis thaliana plants. Together, these data demonstrated that there is strong selection for reversion of the TGMV L148V and CaLCuV L145A mutations but that the nature of the selected revertants is influenced by both the viral background and host components. These data also suggested that high mutation rates contribute to the rapid evolution of geminivirus genomes in plants.}, number={20}, journal={JOURNAL OF VIROLOGY}, author={Arguello-Astorga, Gerardo and Ascencio-Ibanez, J. Trinidad and Dallas, Mary Beth and Orozco, Beverly M. and Hanley-Bowdoin, Linda}, year={2007}, month={Oct}, pages={11005–11015} }
 @article{shen_hanley-bowdoin_2006, title={Geminivirus infection up-regulates the expression of two Arabidopsis protein kinases related to yeast SNF1-and mammalian AMPK-activating kinases}, volume={142}, ISSN={["1532-2548"]}, DOI={10.1104/pp.106.088476}, abstractNote={Geminivirus Rep-interacting kinase 1 (GRIK1) and GRIK2 constitute a small protein kinase family in Arabidopsis (Arabidopsis thaliana). An earlier study showed that a truncated version of GRIK1 binds to the geminivirus replication protein AL1. We show here both full-length GRIK1 and GRIK2 interact with AL1 in yeast two-hybrid studies. Using specific antibodies, we showed that both Arabidopsis kinases are elevated in infected leaves. Immunoblot analysis of healthy plants revealed that GRIK1 and GRIK2 are highest in young leaf and floral tissues and low or undetectable in mature tissues. Immunohistochemical staining showed that the kinases accumulate in the shoot apical meristem, leaf primordium, and emerging petiole. Unlike the protein patterns, GRIK1 and GRIK2 transcript levels only show a small increase during infection and do not change significantly during development. Treating healthy seedlings and infected leaves with the proteasome inhibitor MG132 resulted in higher GRIK1 and GRIK2 protein levels, whereas treatment with the translation inhibitor cycloheximide reduced both kinases, demonstrating that their accumulation is modulated by posttranscriptional processes. Phylogenetic comparisons indicated that GRIK1, GRIK2, and related kinases from Medicago truncatula and rice (Oryza sativa) are most similar to the yeast kinases PAK1, TOS3, and ELM1 and the mammalian kinase CaMKK, which activate the yeast kinase SNF1 and its mammalian homolog AMPK, respectively. Complementation studies using a PAK1/TOS3/ELM1 triple mutant showed that GRIK1 and GRIK2 can functionally replace the yeast kinases, suggesting that the Arabidopsis kinases mediate one or more processes during early plant development and geminivirus infection by activating SNF1-related kinases.}, number={4}, journal={PLANT PHYSIOLOGY}, author={Shen, Wei and Hanley-Bowdoin, Linda}, year={2006}, month={Dec}, pages={1642–1655} }
 @article{lopez-ochoa_ramirez-prado_hanley-bowdoin_2006, title={Peptide aptamers that bind to a geminivirus replication protein interfere with viral replication in plant cells}, volume={80}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.02698-05}, abstractNote={The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses.}, number={12}, journal={JOURNAL OF VIROLOGY}, author={Lopez-Ochoa, Luisa and Ramirez-Prado, Jorge and Hanley-Bowdoin, Linda}, year={2006}, month={Jun}, pages={5841–5853} }
 @article{shultz_settlage_hanley-bowdoin_thompson_2005, title={A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue}, volume={23}, ISSN={["0735-9640"]}, DOI={10.1007/BF02788888}, number={4}, journal={PLANT MOLECULAR BIOLOGY REPORTER}, author={Shultz, RW and Settlage, SB and Hanley-Bowdoin, L and Thompson, WF}, year={2005}, month={Dec}, pages={405–409} }
 @article{settlage_see_hanley-bowdoin_2005, title={Geminivirus C3 protein: Replication enhancement and protein interactions}, volume={79}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.79.15.9885-9895.2005}, abstractNote={ABSTRACT Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus , we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.}, number={15}, journal={JOURNAL OF VIROLOGY}, author={Settlage, SB and See, RG and Hanley-Bowdoin, L}, year={2005}, month={Aug}, pages={9885–9895} }
 @article{arguello-astorga_lopez-ochoa_kong_orozco_settlage_hanley-bowdoin_2004, title={A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein}, volume={78}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.78.9.4817-4826.2004}, abstractNote={ABSTRACT The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression. The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted α-helices designated 3 and 4. Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding. We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays. These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif. Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding. The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays. Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding. Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.}, number={9}, journal={JOURNAL OF VIROLOGY}, author={Arguello-Astorga, G and Lopez-Ochoa, L and Kong, LJ and Orozco, BM and Settlage, SB and Hanley-Bowdoin, L}, year={2004}, month={May}, pages={4817–4826} }
 @article{lee_shultz_hanley-bowdoin_thompson_2004, title={Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis}, volume={22}, ISSN={["1572-9818"]}, DOI={10.1007/BF02773136}, number={3}, journal={PLANT MOLECULAR BIOLOGY REPORTER}, author={Lee, TJ and Shultz, RW and Hanley-Bowdoin, L and Thompson, WF}, year={2004}, month={Sep}, pages={259–267} }
 @misc{hanley-bowdoin_orozco_kong_gruissem_2004, title={Geminivirus resistant transgenic plants}, volume={6,800,793}, number={2004 Oct. 5}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Hanley-Bowdoin, L. and Orozco, B. M. and Kong, L. J. and Gruissem, W.}, year={2004} }
 @misc{hanley-bowdoin_settlage_2004, title={Geminivirus resistant transgenic plants expressing a mutant geminivirus AL3/C3 coding sequence}, volume={6,747,188}, number={2004 June 8}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Hanley-Bowdoin, L. and Settlage, S.}, year={2004} }
 @article{castillo_kong_hanley-bowdoin_bejarano_2004, title={Interaction between a geminivirus replication protein and the plant sumoylation system}, volume={78}, ISSN={["1098-5514"]}, DOI={10.1128/JVI.78.6.2758-2769.2004}, abstractNote={Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells after accumulation of host replication machinery. Tomato golden mosaic virus (TGMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) encode a protein, RepAC1 (or Rep), that is essential for viral replication. Rep/RepAC1 is an oligomeric protein that binds to double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and is sufficient for host induction. It also interacts with several host proteins, including the cell cycle regulator, retinoblastoma, and essential components of the cell DNA replication machinery, like proliferating nuclear cell antigen (PCNA) and RFC-1. To identify other cellular proteins that interact with Rep/RepAC1 protein, a Nicotiana benthamiana cDNA library was screened with a yeast two-hybrid assay. The host cell sumoylation enzyme, NbSCE1 (N. benthamiana SUMO-conjugating enzyme, homolog to Saccharomyces cerevisiae UBC9), was found to interact specifically with RepAC1. Mapping studies localized the interaction to the N-terminal half of RepAC1. Effects on geminivirus replication were observed in transgenic plants with altered levels of SUMO, the substrate for UBC9.}, number={6}, journal={JOURNAL OF VIROLOGY}, author={Castillo, AG and Kong, LJ and Hanley-Bowdoin, L and Bejarano, ER}, year={2004}, month={Mar}, pages={2758–2769} }
 @misc{hanley-bowdoin_settlage_robertson_2004, title={Reprogramming plant gene expression: a prerequisite to geminivirus DNA replication}, volume={5}, ISSN={["1364-3703"]}, DOI={10.1111/J.1364-3703.2004.00214.X}, abstractNote={SUMMARY Geminiviruses constitute a large family of plant‐infecting viruses with small, single‐stranded DNA genomes that replicate through double‐stranded intermediates. Because of their limited coding capacity, geminiviruses supply only the factors required to initiate their replication and use plant nuclear DNA polymerases to amplify their genomes. Many geminiviruses replicate in differentiated cells that no longer contain detectable levels of host DNA polymerases and associated factors. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells by reprogramming host gene expression. The mammalian DNA tumour viruses activate host genes required for DNA replication by binding to the retinoblastoma protein, a negative regulator of cell cycle progression, and relieving repression through the E2F family of transcription factors. In this review, we discuss recent experiments showing that geminiviruses also modulate components of the retinoblastoma/E2F transcription regulatory network to induce quiescent plant cells to re‐enter the cell cycle and regain the capacity to support high levels of DNA replication. Regulation of the cell division cycle and its integration with developmental pathways is complex, with many factors, including hormones, sucrose and environmental signals, controlling re‐entry into the plant cell cycle. Geminivirus interactions with these regulatory networks are likely to determine if and where they can replicate their genomes in different plant tissues and hosts.}, number={2}, journal={MOLECULAR PLANT PATHOLOGY}, author={Hanley-Bowdoin, L and Settlage, SB and Robertson, D}, year={2004}, month={Mar}, pages={149–156} }
 @article{kong_hanley-bowdoin_2002, title={A geminivirus replication protein interacts with a protein kinase and a motor protein that display different expression patterns during plant development and infection}, volume={14}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.003681}, abstractNote={The geminivirus protein AL1 initiates viral DNA replication, regulates its own expression, and induces plant gene transcription. To better understand how AL1 interacts with host proteins during these processes, we used yeast two-hybrid library screening and a baculovirus protein interaction system to identify plant proteins that interact with AL1. These studies identified a Ser/Thr kinase, a kinesin, and histone H3 as AL1 partners. The kinase is autophosphorylated and can phosphorylate common kinase substrates in vitro. The kinesin is phosphorylated in insect cells by a cyclin-dependent kinase. Immunostaining of Nicotiana benthamiana and Arabidopsis showed that kinase protein levels and subcellular location are regulated during plant development and geminivirus infection. By contrast, the kinesin is ubiquitous even though it is associated with the spindle apparatus in mitotic cells. Together, our results establish that AL1 interacts with host proteins involved in plant cell division and development. Possible functions of these host factors in healthy and geminivirus-infected plants are discussed.}, number={8}, journal={PLANT CELL}, author={Kong, LJ and Hanley-Bowdoin, L}, year={2002}, month={Aug}, pages={1817–1832} }
 @article{nagara_hanley-bowdoin_robertson_2002, title={Host DNA replication is induced by geminivirus infection of differentiated plant cells}, volume={14}, ISSN={["1040-4651"]}, DOI={10.1105/tpc.005777}, abstractNote={The geminivirus Tomato golden mosaic virus (TGMV) replicates in differentiated plant cells using host DNA synthesis machinery. We used 5-bromo-2-deoxyuridine (BrdU) incorporation to examine DNA synthesis directly in infected Nicotiana benthamiana plants to determine if viral reprogramming of host replication controls had an impact on host DNA replication. Immunoblot analysis revealed that up to 17-fold more BrdU was incorporated into chromosomal DNA of TGMV-infected versus mock-infected, similarly treated healthy leaves. Colocalization studies of viral DNA and BrdU demonstrated that BrdU incorporation was specific to infected cells and was associated with both host and viral DNA. TGMV and host DNA synthesis were inhibited differentially by aphidicolin but were equally sensitive to hydroxyurea. Short BrdU labeling times resulted in some infected cells showing punctate foci associated with host DNA. Longer periods showed BrdU label uniformly throughout host DNA, some of which showed condensed chromatin, only in infected nuclei. By contrast, BrdU associated with viral DNA was centralized and showed uniform, compartmentalized labeling. Our results demonstrate that chromosomal DNA is replicated in TGMV-infected cells.}, number={12}, journal={PLANT CELL}, author={Nagara, S and Hanley-Bowdoin, L and Robertson, D}, year={2002}, month={Dec}, pages={2995–3007} }
 @article{egelkrout_mariconti_settlage_cella_robertson_hanley-bowdoin_2002, title={Two E2F elements regulate the proliferating cell nuclear antigen promoter differently during leaf development}, volume={14}, ISSN={["1040-4651"]}, DOI={10.1105/tpc.006403}, abstractNote={E2F transcription factors regulate genes expressed at the G1/S boundary of the cell division cycle in higher eukaryotes. Although animal E2F proteins and their target promoters have been studied extensively, little is known about how these factors regulate plant promoters. An earlier study identified two E2F consensus binding sites in the promoter of a Nicotiana benthamiana gene encoding proliferating cell nuclear antigen (PCNA) and showed that the proximal element (E2F2) is required for the full repression of PCNA expression in mature leaves. In this study, we examined the distal element (E2F1) and how it interacts with the E2F2 site to regulate the PCNA promoter. Gel shift assays using plant nuclear extracts or purified Arabidopsis E2F and DP proteins showed that different complexes bind to the two E2F sites. Mutation of the E2F1 site or both sites differentially altered PCNA promoter function in transgenic plants. As reported previously for the E2F2 mutation, the E2F1 and E2F1+2 mutations partially relieved the repression of the PCNA promoter in mature leaves. In young tissues, the E2F1 mutation resulted in a threefold reduction in PCNA promoter activity, whereas the E2F1+2 mutation had no detectable effect. The activity of E2F1+2 mutants was indistinguishable from that of E2F2 mutants. These results demonstrate that both E2F elements contribute to the repression of the PCNA promoter in mature leaves, whereas the E2F1 site counters the repression activity of the E2F2 element in young leaves.}, number={12}, journal={PLANT CELL}, author={Egelkrout, EM and Mariconti, L and Settlage, SB and Cella, R and Robertson, D and Hanley-Bowdoin, L}, year={2002}, month={Dec}, pages={3225–3236} }
 @article{settlage_miller_gruissem_hanley-bowdoin_2001, title={Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator}, volume={279}, ISSN={["0042-6822"]}, DOI={10.1006/viro.2000.0719}, abstractNote={Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.}, number={2}, journal={VIROLOGY}, author={Settlage, SB and Miller, AB and Gruissem, W and Hanley-Bowdoin, L}, year={2001}, month={Jan}, pages={570–576} }
 @article{egelkrout_robertson_hanley-bowdoin_2001, title={Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves}, volume={13}, ISSN={["1531-298X"]}, DOI={10.1105/tpc.13.6.1437}, abstractNote={The geminivirus tomato golden mosaic virus (TGMV) amplifies its DNA genome in differentiated plant cells that lack detectable levels of DNA replication enzymes. Earlier studies showed that TGMV induces the accumulation of proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase delta, in mature cells of Nicotiana benthamiana. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gel blot analysis detected an approximately 1200-nucleotide PCNA transcript in young leaves. The same RNA was found in mature leaves of infected but not healthy plants. Reporter gene analysis showed that a 633-bp promoter fragment of the N. benthamiana PCNA gene supports high levels of expression in cultured cells and in young but not mature leaves of healthy transgenic plants. In contrast, PCNA promoter activity was detected in both young and mature leaves of TGMV-infected plants. Developmental studies established a strong relationship between symptom severity, viral DNA accumulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in the PCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Unlike the wild-type PCNA promoter, TGMV infection had no detectable effect on the activity of the mutant E2F promoter. Together, these results demonstrate that geminivirus infection induces the accumulation of a host replication factor by activating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression.}, number={6}, journal={PLANT CELL}, author={Egelkrout, EM and Robertson, D and Hanley-Bowdoin, L}, year={2001}, month={Jun}, pages={1437–1452} }
 @article{peele_jordan_muangsan_turnage_egelkrout_eagle_hanley-bowdoin_robertson_2001, title={Silencing of a meristematic gene using geminivirus-derived vectors}, volume={27}, ISSN={["0960-7412"]}, DOI={10.1046/j.1365-313x.2001.01080.x}, abstractNote={Summary Geminiviruses are DNA viruses that replicate and transcribe their genes in plant nuclei. They are ideal vectors for understanding plant gene function because of their ability to cause systemic silencing in new growth and ease of inoculation. We previously demonstrated DNA episome‐mediated gene silencing from a bipartite geminivirus in Nicotiana benthamiana . Using an improved vector, we now show that extensive silencing of endogenous genes can be obtained using less than 100 bp of homologous sequence. Concomitant symptom development varied depending upon the target gene and insert size, with larger inserts producing milder symptoms. In situ hybridization of silenced tissue in attenuated infections demonstrated that silencing occurs in cells that lack detectable levels of viral DNA. A mutation confining the virus to vascular tissue produced extensive silencing in mesophyll tissue, further demonstrating that endogenous gene silencing can be separated from viral infection. We also show that two essential genes encoding a subunit of magnesium chelatase and proliferating cell nuclear antigen (PCNA) can be silenced simultaneously from different components of the same viral vector. Immunolocalization of silenced tissue showed that the PCNA protein was down‐regulated throughout meristematic tissues. Our results demonstrate that geminivirus‐derived vectors can be used to study genes involved in meristem function in intact plants.}, number={4}, journal={PLANT JOURNAL}, author={Peele, C and Jordan, CV and Muangsan, N and Turnage, M and Egelkrout, E and Eagle, P and Hanley-Bowdoin, L and Robertson, D}, year={2001}, month={Aug}, pages={357–366} }
 @article{kong_orozco_roe_nagar_ou_feiler_durfee_miller_gruissem_robertson_et al._2000, title={A geminivirus replication protein interacts with the retinoblastoma protein through a novel domain to determine symptoms and tissue specificity of infection in plants}, volume={19}, ISSN={["0261-4189"]}, DOI={10.1093/emboj/19.13.3485}, abstractNote={Geminiviruses replicate in nuclei of mature plant cells after inducing the accumulation of host DNA replication machinery. Earlier studies showed that the viral replication factor, AL1, is sufficient for host induction and interacts with the cell cycle regulator, retinoblastoma (pRb). Unlike other DNA virus proteins, AL1 does not contain the pRb binding consensus, LXCXE, and interacts with plant pRb homo logues (pRBR) through a novel amino acid sequence. We mapped the pRBR binding domain of AL1 between amino acids 101 and 180 and identified two mutants that are differentially impacted for AL1-pRBR interactions. Plants infected with the E-N140 mutant, which is wild-type for pRBR binding, developed wild-type symptoms and accumulated viral DNA and AL1 protein in epidermal, mesophyll and vascular cells of mature leaves. Plants inoculated with the KEE146 mutant, which retains 16% pRBR binding activity, only developed chlorosis along the veins, and viral DNA, AL1 protein and the host DNA synthesis factor, proliferating cell nuclear antigen, were localized to vascular tissue. These results established the importance of AL1-pRBR interactions during geminivirus infection of plants.}, number={13}, journal={EMBO JOURNAL}, author={Kong, LJ and Orozco, BM and Roe, JL and Nagar, S and Ou, S and Feiler, HS and Durfee, T and Miller, AB and Gruissem, W and Robertson, D and et al.}, year={2000}, month={Jul}, pages={3485–3495} }
 @article{bass_nagar_hanley-bowdoin_robertson_2000, title={Chromosome condensation induced by geminivirus infection of mature plant cells}, volume={113}, number={7}, journal={Journal of Cell Science}, author={Bass, H. W. and Nagar, S. and Hanley-Bowdoin, L. and Robertson, D.}, year={2000}, pages={1149–1160} }
 @article{hanley-bowdoin_settlage_orozco_nagar_robertson_2000, title={Geminiviruses - Models for plant DNA replication, transcription and cell cycle regulation ([correction to] vol 35, pg 105, 2000)}, volume={35}, number={4}, journal={Critical Reviews in Biochemistry and Molecular Biology}, author={Hanley-Bowdoin, L. and Settlage, S. B. and Orozco, B. M. and Nagar, S. and Robertson, D.}, year={2000}, pages={U4} }
 @article{hanley-bowdoin_settlage_orozco_nagar_robertson_2000, title={Geminiviruses: Models for plant DNA replication, transcription, and cell cycle regulation}, volume={35}, number={2}, journal={Critical Reviews in Biochemistry and Molecular Biology}, author={Hanley-Bowdoin, L. and Settlage, S. B. and Orozco, B. M. and Nagar, S. and Robertson, D.}, year={2000}, pages={105–140} }
 @article{orozco_kong_batts_elledge_hanley-bowdoin_2000, title={The multifunctional character of a geminivirus replication protein is reflected by its complex oligomerization properties}, volume={275}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.275.9.6114}, abstractNote={Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157–159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157–159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein. Tomato golden mosaic virus (TGMV), a member of the geminivirus family, encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its plant host. TGMV AL1 is an oligomeric protein that binds double-stranded DNA and catalyzes cleavage and ligation of single-stranded DNA. The oligomerization domain, which is required for DNA binding, maps to a region that displays strong sequence and structural homology to other geminivirus Rep proteins. To assess the importance of conserved residues, we generated a series of site-directed mutations and analyzed their impact on AL1 function in vitro and in vivo. Two-hybrid experiments revealed that mutation of amino acids 157–159 inhibited AL1-AL1 interactions, whereas mutations at nearby residues reduced complex stability. Changes at positions 157–159 also disrupted interaction between the full-length mutant protein and a glutathione S-transferase-AL1 oligomerization domain fusion in insect cells. The mutations had no detectable effect on oligomerization when both proteins contained full-length AL1 sequences, indicating that AL1 complexes can be stabilized by amino acids outside of the oligomerization domain. Nearly all of the oligomerization domain mutants were inhibited or severely attenuated in their ability to support AL1-directed viral DNA replication. In contrast, the same mutants were enhanced for AL1-mediated transcriptional repression. The replication-defective AL1 mutants also interfered with replication of a TGMV A DNA encoding wild type AL1. Full-length mutant AL1 was more effective in the interference assays than truncated proteins containing the oligomerization domain. Together, these results suggested that different AL1 complexes mediate viral replication and transcriptional regulation and that replication interference involves multiple domains of the AL1 protein. tomato golden mosaic virus activation domain glutathioneS-transferase maize streak virus Geminiviruses are a large family of plant viruses with circular, single-stranded DNA genomes that replicate in the nuclei of infected cells (reviewed in Ref. 1.Hanley-Bowdoin L. Settlage S.B. Orozco B.M. Nagar S. Robertson D. CRC Crit. Rev. Plant Sci. 1999; 18: 71-106Crossref Google Scholar). The single-stranded genome is converted to a double-stranded DNA that serves as the template for rolling circle replication (2.Saunders K. Lucy A. Stanley J. Nucleic Acids Res. 1991; 19: 2325-2330Crossref PubMed Scopus (167) Google Scholar, 3.Stenger D.C. Revington G.N. Stevenson M.C. Bisaro D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8029-8033Crossref PubMed Scopus (288) Google Scholar, 4.Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (88) Google Scholar) and transcription (5.Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 6.Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). Geminiviruses do not encode their own polymerases and, instead, rely on host enzymes for viral DNA and RNA synthesis. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms. The geminivirus, tomato golden mosaic virus (TGMV),1 has a bipartite genome that encodes seven open reading frames that are divergently transcribed. The 5′-intergenic region separating the transcription units is nearly identical between the two DNA components and includes the plus strand origin of replication (7.Lazarowitz S.G. Wu L.C. Rogers S.G. Elmer J.S. Plant Cell. 1992; 4: 799-809Crossref PubMed Scopus (116) Google Scholar, 8.Orozco B.M. Gladfelter H.J. Settlage S.B. Eagle P.A. Gentry R. Hanley-Bowdoin L. Virology. 1998; 242: 346-356Crossref PubMed Scopus (68) Google Scholar). The promoter for complementary sense transcription overlaps the replication origin (5.Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar,9.Hanley-Bowdoin L. Elmer J.S. Rogers S.G. Plant Cell. 1989; 1: 1057-1067PubMed Google Scholar) and shares some of the cis-elements involved in origin function (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). A directly repeated sequence, GGTAG, is required for origin recognition (11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar) and transcriptional repression of the complementary sense (AL1) promoter (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Similarly, the TATA-box and G-box transcription factor binding sites in the AL1 promoter act as replication enhancer elements (12.Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar). In contrast, three elements in the TGMV intergenic region are necessary for origin function but have little or no effect on AL1 promoter activity. A hairpin structure with a 9-base pair loop sequence that is conserved among all geminiviruses is essential for replication and contains the cleavage site for initiating plus strand DNA synthesis (4.Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (88) Google Scholar, 13.Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (259) Google Scholar, 14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). A conserved sequence between the AL1 binding site and the hairpin, the AG-motif, is also required for replication (8.Orozco B.M. Gladfelter H.J. Settlage S.B. Eagle P.A. Gentry R. Hanley-Bowdoin L. Virology. 1998; 242: 346-356Crossref PubMed Scopus (68) Google Scholar). The third element, the CA motif, is located outside of the minimal origin but its deletion reduced replication 20-fold (8.Orozco B.M. Gladfelter H.J. Settlage S.B. Eagle P.A. Gentry R. Hanley-Bowdoin L. Virology. 1998; 242: 346-356Crossref PubMed Scopus (68) Google Scholar). The role of the AG- and CA-motifs in TGMV origins is not known, but one possibility is that they bind host factors that facilitate initiation of plus strand DNA replication. TGMV encodes two proteins, AL1 and AL3, that are required for efficient viral replication. AL1 is necessary for replication, whereas AL3 enhances viral DNA accumulation by an unknown mechanism (15.Elmer J.S. Brand L. Sunter G. Gardiner W.E. Bisaro D.M. Rogers S.G. Nucleic Acids Res. 1988; 16: 7043-7060Crossref PubMed Scopus (212) Google Scholar, 16.Sunter G. Hartitz M.D. Hormuzdi S.G. Brough C.L. Bisaro D.M. Virology. 1990; 179: 69-77Crossref PubMed Scopus (167) Google Scholar) (AL1 homologues are also designated C1 or Rep.) AL1 is a multifunctional protein that mediates both virus-specific recognition of its cognate origin (17.Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar) and transcriptional repression by binding to the directly repeated sequence in the intergenic region (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar, 12.Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar). AL1 initiates and terminates plus strand replication (13.Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (259) Google Scholar, 14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar, 18.Heyraud-Nitschke F. Schumacher S. Laufs J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. 1995; 23: 910-916Crossref PubMed Scopus (142) Google Scholar) and induces the accumulation of a host replication factor, proliferating cell nuclear antigen, in infected cells (19.Nagar S. Pedersen T.J. Carrick K. Hanley-Bowdoin L. Robertson D. Plant Cell. 1995; 7: 705-719Crossref PubMed Scopus (156) Google Scholar). Recombinant AL1 specifically binds double-stranded DNA (11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 20.Fontes E.P.B. Luckow V.A. Hanley-Bowdoin L. Plant Cell. 1992; 4: 597-608Crossref PubMed Scopus (136) Google Scholar), cleaves and ligates single-stranded DNA in the invariant sequence of the hairpin loop (14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar, 21.Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar), and hydrolyzes ATP (22.Desbiez C. David C. Mettouchi A. Laufs J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5640-5644Crossref PubMed Scopus (106) Google Scholar, 23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TGMV AL1 also interacts with itself (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), the viral replication enhancer protein AL3 (24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar), and a maize homologue of the cell cycle regulatory protein, retinoblastoma (25.Ach R.A. Durfee T. Miller A.B. Taranto P. Hanley-Bowdoin L. Zambriski P.C. Gruissem W. Mol. Cell. Biol. 1997; 17: 5077-5086Crossref PubMed Scopus (202) Google Scholar). We previously mapped the TGMV AL1 domains for double-stranded DNA binding, single-stranded DNA cleavage and ligation, and AL1 oligomerization (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26.Orozco B.M. Hanley-Bowdoin L. J. Biol. Chem. 1998; 273: 24448-24456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The DNA cleavage/ligation domain was located to the first 120 amino acids, and the oligomerization domain was mapped between amino acids 120 and 181. DNA binding activity required amino acids 1–130 for protein-DNA contacts and the AL1 oligomerization domain. Because of its importance in AL1 function, we have further characterized the sequences required for oligomerization in vitro and assessed the impact of site-directed mutations in the domain in vivo. Constructs are listed in Table I. The plasmid pNSB148, which contains the AL1 coding sequence in a pUC118-background, was used as the template for site-directed mutagenesis (27.Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 482-492Google Scholar). The oligonucleotide primers and resulting clones are also listed in TableI. DNA fragments containing the mutations were verified by DNA sequence analysis. Plant expression cassettes for mutant AL1 proteins were generated by subcloningSalI/NcoI fragments (TGMV A position 2442 and 2059) from the mutant clones into the same sites in the wild type AL1 plant expression cassette pMON1549 (11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). In pMON1549, AL1 expression is under the control of the cauliflower mosaic virus 35S promoter with a duplicated enhancer region and the pea E9 rbcS 3′-end.Table IAL1 mutationsMutationOligonucleotideBaculovirus vectorYeast GAL4-ADPlant expressionWild typeN/ApMON1680pNSB809pMON1549FQ118CACTTCGACCGTCGACCGCGGCTTCTCCCCAN/ApNSB872pNSB866D120CACTTCGGCCGGCGACCGCGGCTTCTCCCCAN/ApNSB871pNSB865RS-R125GCAACCTCCTgcAGCggccgcACCGTCGACCTGGAN/ApNSB786pNSB695QT130CAGCGTCGTTgctaGcTgcGCAACCTCCTCTAGCAN/ApNSB788pNSB696ND133CTGCTGCAGCGgCcgcAGATGTTTGGCAApNSB603PNSB790pNSB670E–N140GGAAGAAGCAgcTAACGCggCcGCTGCAGCGTCGTpNSB604PNSB893pNSB640KEE146TCTGCAGGGCTgCggCcgcGGAAGAAGCATTTAApNSB605PNSB894pNSB641REK154TTCTGGGATTgcggCcgcAATTATCTGCAGGGpNSB606pNSB759pNSB671EKY159GAACTGAAATAAAgcggccgCTGGGATTTTCTCTCpNSB607pNSB760pNSB672Q-HN165GCTATTTAGAgcGgcGAACgcAAATAAATATTTTTCTGGGATpNSB608pNSB761pNSB698N-DR172ATCAAATATCgcAgCTAgcgcGCTATTTAGATTGTGpNSB609pNSB762pNSB707K–E179GAAGCCATGGcgCcGGAGTCgcATCAAATATCCpNSB610pNSB763pNSB697 Open table in a new tab Baculovirus vectors were generated for expression of mutant and truncated AL1 proteins in insect cells. Expression vectors coding for mutant AL1 proteins were made by subcloningBglII/BamHI inserts from the mutant plant expression cassettes into the BamHI site of pMON27025 (28.Luckow V.A. Lee S.C. Barry G.F. Olins P.O. J. Virol. 1993; 67: 4566-4579Crossref PubMed Google Scholar). Expression vectors for the truncated proteins, AL1119–352(pNSB516), AL11–120 (pNSB388), and AL11–180(pNSB517), have been described previously (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The N-terminal truncations, AL1134–352 and AL1147–352, were generated by inserting a double-stranded oligonucleotide containing anSphI site into the NotI site of pNSB593 and pNSB595 to create in-frame start codons. AL1160–352 was created by inserting an SphI linker with a start codon (New England Biolabs, Beverly, MA) into the SspI site of pMON1539 (29.Hanley-Bowdoin L. Elmer J.S. Rogers S.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1446-1450Crossref PubMed Scopus (84) Google Scholar). SphI/BamHI fragments from the resulting clones were inserted into the same sites of the baculovirus vector, pNSB448 (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), to give pNSB803 (AL1134–352), pNSB876 (AL1147–352), and pNSB633 (AL1160–352). The C-terminal truncation, AL11–158 (pNSB646), was generated by digesting pMON1539 with NdeI and SspI, repairing with Klenow, and subcloning into the filled BamHI site of pMON27025 to create an in-frame stop codon. The AL11–168 truncation, pNSB708, was created by inserting anXbaI linker into the repaired BssHII of pNSB609, generating an in-frame stop codon. Yeast expression cassettes were generated using the pAS1–1 and pACT2 vectors from CLONTECH (Palo Alto, CA). TheBamHI/NdeI fragment of pMON1539 was cloned into the same sites of pAS2–1 to give pNSB736, which contained the GAL4 DNA binding domain fused to wild type AL1 sequences. The ends of the sameBamHI/NdeI fragment were repaired with Klenow and cloned into the SmaI site of pACT2 to give pNSB735. TheAatII/BamHI fragment of pNSB735 was then replaced with the AatII/BamHI fragment from pMON1549. The resulting clone, pNSB809, contained the GAL4 activation domain (AD) fused to wild type AL1 sequences. Mutant AL1 yeast expression cassettes were created by replacing the wild typeAatII/BamHI fragment of pNSB735 with mutantAatII/BamHI fragments from the corresponding plant expression cassettes. Protoplasts were isolated from Nicotiana tabacum (BY-2) orNicoxiana benthamiana suspension cells, electroporated, and cultured according to published methods (11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). For replication assays, N. tabacum transfections included 15 μg each of replicon DNA containing a partial tandem copy of TGMV B (pTG1.4B, Ref. 17.Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar), wild type or mutant AL1 plant expression cassette, and an AL3 plant expression cassette (pNSB46, Ref. 11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). For the interference assays, 2 μg of replicon DNA containing a partial tandem copy of TGMV A (pMON1565, Ref. 14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar) was cotransfected with 40 μg of mutant AL1 expression cassette or the empty expression vector (pMON921, Ref. 11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Total DNA was extracted 3 days post-transfection and analyzed for double- and single-stranded viral DNA accumulation by DNA gel blot hybridization (11.Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). The viral DNA was quantified by phosphorimager analysis in a minimum of three independent experiments. For transcriptional repression assays, N. benthamianaprotoplasts were transfected with 15 μg of luc reporter construct (pNSB114), 15 μg of AL1 expression cassette, and 36 μg of sheared salmon sperm DNA (10.Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Luciferase activity in total soluble protein extracts was measured 36 h post-transfection and standardized for protein concentration. Repression was determined as the ratio of Luc activity in the absence versus the presence of AL1. Each expression cassette was assayed in triplicate in at least three independent experiments. Recombinant proteins were produced inSpodoptera frugiperda Sf9 cells using a baculovirus expression system according to published protocols (14.Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar, 24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). Protein extracts from cells co-expressing authentic and GST-AL1 fusion proteins were assayed for AL1 oligomerization by co-purification on glutathione-Sepharose (24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). Co-purification was monitored by SDS-polyacrylamide electrophoresis followed by transfer to nitrocellulose membrane (Schleicher and Schuell) and immunoblotting using the ECL detection system (Amersham Pharmacia Biotech). Primary antibodies were rabbit polyclonal anti-GST (Upstate Biotechnology Inc.) and anti-AL1 antisera (24.Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). The Saccharomyces cerevisiae strain Y187 (MATα,ura3–52, his3–200, ade 2–101,trp 1–901, leu 2–3, 112,gal4Δ, met −, gal80Δ,URA3::GAL1 UAS-GAL1 TATA-lacZ) was transformed using lithium acetate/polyethylene glycol (30.Geitz D. St. Jean A. Woods R.A. Schiesti R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2895) Google Scholar). The DNAs were pNSB736, which expresses the GAL4 binding domain-wild type AL1 fusion, and either pNSB809, which produces the GAL4 AD-wild type AL1 protein, or the equivalent cassettes corresponding to the GAL4 AD-AL1 mutants. For β-galactosidase assays, yeast transformants were grown to an A 600 of 0.5 in 3 ml of synthetic dropout medium lacking tryptophan and leucine (31.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar). Yeast were pelleted at 1000 × g for 5 min, rinsed with Z buffer (0.1m NaPO4, pH 7, 10 mm KCl, 1 mm MgSO4, 40 mmβ-mercaptoethanol) (31.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar), and resuspended in 300 μl of Z buffer. The cells were subjected to three freeze/thaw cycles in liquid nitrogen and centrifuged at 5000 × g for 2 min. The supernatant (150 μl) was assayed for β-galactosidase activity in a total reaction volume of 250 μl using the substrateo-nitrophenyl β-d-galactopyranoside, as described by CLONTECH. Accumulation of theo-nitrophenol product was measured atA 420 using a BioKinetics microplate reader (Bio-Tek Instrument Inc., Winooski, VT). Protein concentrations were measured by Bradford assays (Bio-Rad). The enzyme-specific activity (1 unit = 1.0 μm product/min at pH7.3 at 37 °C) resulting from interaction between two-hybrid cassettes carrying wild type AL1 sequences was determined using purified β-galactosidase (Sigma) as the standard. The relative activities of the mutants were normalized against the wild type AL1 interaction level, which was set to 100%. The β-galactosidase specific activity for wild type and mutant AL1 proteins was adjusted for background from pNSB736 alone. The different constructs were tested in a minimum of two experiments, each of which assayed four independent transformants for each construct. For immunoblot analysis, individual yeast transformants were grown in 5 ml of medium containing 1% yeast extract, 2% bacto-peptone, 2% glucose, pH 5.8 (31.Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar) to an A 600 of 1. An equal volume of crushed ice was added and the culture was centrifuged at 1000 × g for 5 min. The resulting pellet was washed once with ice cold water and resuspended in 80 μl of modified radioimmune precipitation buffer (150 mm NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 50 mmTris-HCl, pH 7.5 (32.Harlow E. Lane D. Antibodies, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY1988Google Scholar), containing 1% (w/v) SDS) and protease inhibitors (6 μg/ml pepstatin A, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 8 mm benzamidine, 1 mmphenylmethylsulfonyl fluoride). Glass beads (40 μl, 425–600 μm, Sigma) were added and the sample was vortexed at maximum speed for four 30-s intervals separated by 2-min intervals on ice. The sample was then centrifuged at 5000 × g for 2 min at 4 °C. The supernatant was recovered, and the protein concentration was determined using Bradford assays. Total protein (100 μg) was resolved on 12% polyacrylamide/SDS gels and analyzed by immunoblotting using a GAL4 AD monoclonal antibody at 0.4 μg/ml (CLONTECH). The domains for TGMV AL1 DNA binding and DNA cleavage/ligation activities are well defined, and key structural and sequence motifs have been identified for these functions (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26.Orozco B.M. Hanley-Bowdoin L. J. Biol. Chem. 1998; 273: 24448-24456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), whereas the AL1 oligomerization domain has only been broadly located to the center of the protein (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). In this paper, closely spaced N- and C-terminal truncations were generated to define the limits of the AL1 oligomerization domain (Fig.1 A). A GST fusion corresponding to full-length AL1 (GST-AL1) was co-expressed with the truncated AL1 proteins in baculovirus-infected insect cells, and protein complexes were purified on glutathione-Sepharose resin. Total extracts and purified proteins were resolved by SDS-polyacrylamide gel electrophoresis, and proteins were visualized by immunoblotting with AL1 and GST polyclonal antisera. As reported previously (23.Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), the C-terminal truncation AL11–180 (Fig. 1 B,lanes 3 and 6) copurified with full-length GST-AL1. Further deletion of the C terminus to amino acid 168 (lanes 2 and 5) or 158 (lanes 1 and4) abolished interactions with GST-AL1, demonstrating that the C-terminal limit of the oligomerization domain is between position 168 and 180. The N-terminal truncations to amino acids 134 (Fig.1 C, lanes 3 and 6), 147 (lanes 2 and 5), and 160 (lanes 1 and 4) showed a gradual disappearance of interaction with GST-AL1. AL1134–352 consistently displayed interactions with GST-AL1, whereas AL1147–352 and AL1160–352interactions varied between weak to background levels, making it more difficult to define the N-terminal limit. Contacts outside the oligomerization domain may contribute to complex stability or multimerization and account for the low level interactions observed with AL1147–352 and AL1160–352. To address this possibility, we asked whether full-length AL1 copurified when only the oligomerization domain was fused to GST (GST-AL1119–180). Full-length AL1 interacted with GST-AL1119–180 but not with GST alone (Fig.2 B, lanes 1 and2), demonstrating that amino acids 119–180 are sufficient for oligomerization. We then examined the abilities of the N-terminal AL1 truncations to bind GST-AL1119–180. In this assay, deletion to positions 119 (Fig. 1 D, lanes 1 and5) and 134 (lanes 2 and 6) did not affect oligomerization, whereas further deletion to positions 147 (lanes 3 and 7) and 160 (lanes 4 and8) abolished interactions with GST-AL1119–180. Together, these results showed that AL1 amino acids 134–180 contain the oligomerization domain and that sequences outside the domain contribute stabilizing contacts. Alanine substitutions were generated in conserved charged or hydrophobic residues within the oligomerization domain to identify key amino acids that contribute to AL1 interactions (Fig.3, the mutations are designated by the corresponding wild type sequence and the position of the last amino acid that was altered; dashes indicate amino acids that were not changed). Alanine was selected because it is structurally neutral and should not interfere with normal protein folding. The mutations are within a region that includes a pair of predicted α-helices (26.Orozco B.M. Hanley-Bowdoin L. J. Biol. Chem. 1998; 273: 24448-24456Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and downstream sequences required for oligomerization. All of the mutant AL1 proteins co-purified with GST-AL1 on glutathione resin when co-expressed in insect cells (Fig. 2 A), showing that they formed stable complexes with the wild type protein. Similar results were observed when both the test and GST fusion proteins carried the mutations (data not shown). In contrast, when interactions between the mutant AL1 proteins and GST-AL1119–180 were examined mutant EKY159 (Fig. 2 B, lane 6) was defective for AL1 interactions. Thus, sequences outside of the oligomerization domain in the full-length AL1 masked the effect of the EKY159 mutation in Fig.2 A, which is consistent with our previous conclusion that sequences outside the oligomerization domain stabilize AL1 interactions. The oligomerization mutant, EKY159, was assayed with truncated GST-AL1 proteins to locate the stabilizing region. As shown above, EKY159 bound full-length GST-AL1 (Fig. 2 C,lanes 1 and 5) but not GST-AL1119–180 (lanes 4 and 8). EKY159 also bound an N-terminal truncation, GST-AL1119–352(lanes 3 and 7), whereas no interaction was detected with a C-terminal truncation, GST-AL11–180(lanes 2 and 6), demonstrating that the AL1 C terminus contributes stabilizing contacts. The quantitative impact of each mutation on AL1 oligomerization was measured using a yeast two-hybrid system. Expression cassettes for wild type or mutant AL1 fused to the GAL4 AD were cotransformed into yeast with a cassette for wild type AL1 fused to the GAL4 DNA binding domain. Activation of a GAL4-responsive promoter driving a lacZreporter was assayed by measuring β-galactosidase activity in total soluble protein extracts. In extracts from yeast cotransfected with AD and binding domain cassettes carrying wild type AL1 sequences, 142 mu/mg total protei}, number={9}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Orozco, BM and Kong, LJ and Batts, LA and Elledge, S and Hanley-Bowdoin, L}, year={2000}, month={Mar}, pages={6114–6122} }
 @article{hanley-bowdoin_settlage_orozco_nagar_robertson_1999, title={Geminiviruses: Models for plant DNA replication, transcription, and cell cycle regulation {review}}, volume={18}, DOI={10.1080/07352689991309162}, abstractNote={Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.}, number={1}, journal={Critical Reviews in Plant Sciences}, author={Hanley-Bowdoin, L. and Settlage, S. B. and Orozco, B. M. and Nagar, S. and Robertson, D.}, year={1999}, pages={71–106} }
 @article{orozco_hanley-bowdoin_1998, title={Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein}, volume={273}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.273.38.24448}, abstractNote={Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1–120) and oligomerization (amino acids 134–181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitroexperiments demonstrated that a pair of predicted α-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1–120) and oligomerization (amino acids 134–181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitroexperiments demonstrated that a pair of predicted α-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions. tomato golden mosaic virus intergenic region rolling circle replication bean golden mosaic virus tomato yellow leaf curl virus glutathione S-transferase nucleotide(s). Tomato golden mosaic virus (TGMV)1 is a member of the geminivirus family of plant-infecting viruses characterized by twin icosahedral particles and small, single-stranded DNA genomes (reviewed in Refs. 1Timmermans M.C.P. Das O.P. Messing J. Annu. Rev. Plant Physiol. 1994; 45: 79-112Crossref Scopus (103) Google Scholar and 2Hanley-Bowdoin, L., Settlage, S. B., Orozco, B. M., Nagar, S., and Robertson, D. (1998) Crit. Rev. Plant Sci., in pressGoogle Scholar). The single-stranded DNA is converted to a double-stranded form in the nucleus of infected cells and then serves as a template for rolling circle replication (RCR; Refs. 3Saunders K. Lucy A. Stanley J. Nucleic Acids Res. 1991; 19: 2325-2330Crossref PubMed Scopus (167) Google Scholar, 4Stenger D.C. Revington G.N. Stevenson M.C. Bisaro D.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8029-8033Crossref PubMed Scopus (289) Google Scholar, 5Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar) and viral gene transcription (6Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 7Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). Geminiviruses encode only a few proteins for these processes and depend on host DNA and RNA polymerases as well as their accessory factors. These characteristics make geminiviruses excellent model systems for studying plant DNA replication and transcription mechanisms. The TGMV genome consists of two circular DNA molecules, designated as A and B (8Hamilton W.D.O. Bisaro D.M. Coutts R.H.A. Buck K.W. Nucleic Acids Res. 1983; 11: 7387-7396Crossref PubMed Scopus (92) Google Scholar). Both components have a conserved 5′ intergenic region (IR) that separates divergent open reading frames (9Bisaro D.M. Hamilton W.D.O. Coutts R.H.A. Buck K.W. Nucleic Acids Res. 1982; 10: 4913-4922Crossref PubMed Scopus (52) Google Scholar). The IR includes the plus-strand origin of replication (10Orozco B.M. Gladfelter H.J. Settlage S.B. Eagle P.A. Gentry R. Hanley-Bowdoin L. Virology. 1998; 242: 346-356Crossref PubMed Scopus (68) Google Scholar) and the promoters for leftward and rightward transcription (6Sunter G. Gardiner W.E. Bisaro D.M. Virology. 1989; 170: 243-250Crossref PubMed Scopus (53) Google Scholar, 7Sunter G. Bisaro D.M. Virology. 1989; 173: 647-655Crossref PubMed Scopus (47) Google Scholar). A directly repeated sequence in the TGMV IR is required for recognition of the plus-strand origin and negative regulation of the overlapping promoter for leftward transcription (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 12Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar). Related motifs are found in the genomes of most dicot-infecting geminiviruses (13Arguello-Astorga G.R. Guevara-Gonzalez R.G. Herrera-Estrella L.R. Rivera-Bustamante R.F. Virology. 1994; 203: 90-100Crossref PubMed Scopus (278) Google Scholar), and their roles in virus-specific replication have been confirmed for bean golden mosaic virus (BGMV) and beet curly top virus (14Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar, 15Choi I.R. Stenger D.C. Virology. 1995; 206: 904-912Crossref PubMed Scopus (64) Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar). The IR also contains a hairpin with a 9-base pair loop sequence conserved among all geminiviruses that is cleaved during initiation and termination of RCR (5Heyraud F. Matzeit V. Kammann M. Schaefer S. Schell J. Gronenborn B. EMBO J. 1993; 12: 4445-4452Crossref PubMed Scopus (90) Google Scholar, 17Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (260) Google Scholar, 18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Genetic experiments established that the hairpin structure is essential for TGMV replication (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). TGMV encodes two proteins, AL1 and AL3, that are required for efficient viral replication. AL1 is necessary for replication, whereas AL3 enhances viral DNA accumulation by an unknown mechanism (19Elmer J.S. Brand L. Sunter G. Gardiner W.E. Bisaro D.M. Rogers S.G. Nucleic Acids Res. 1988; 16: 7043-7060Crossref PubMed Scopus (212) Google Scholar, 20Sunter G. Hartitz M.D. Hormuzdi S.G. Brough C.L. Bisaro D.M. Virology. 1990; 179: 69-77Crossref PubMed Scopus (167) Google Scholar). AL1 is a multifunctional protein that confers virus-specific recognition to its cognate plus-strand origin (15Choi I.R. Stenger D.C. Virology. 1995; 206: 904-912Crossref PubMed Scopus (64) Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar, 21Jupin I. Hericourt F. Benz B. Gronenborn B. FEBS Lett. 1995; 362: 116-120Crossref PubMed Scopus (74) Google Scholar) and initiates RCR (17Laufs J. Traut W. Heyraud F. Matzeit V. Rogers S.G. Schell J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3879-3883Crossref PubMed Scopus (260) Google Scholar, 18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar,22Heyraud-Nitschke F. Schumacher S. Laufs J. Schaefer S. Schell J. Gronenborn B. Nucleic Acids Res. 1995; 23: 910-916Crossref PubMed Scopus (143) Google Scholar). It also actively represses its own transcription in a virus-specific manner (12Eagle P.A. Orozco B.M. Hanley-Bowdoin L. Plant Cell. 1994; 6: 1157-1170PubMed Google Scholar, 16Gladfelter H.J. Eagle P.A. Fontes E.P.B. Batts L.A. Hanley-Bowdoin L. Virology. 1997; 239: 186-197Crossref PubMed Scopus (47) Google Scholar, 23Sunter G. Hartitz M.D. Bisaro D.M. Virology. 1993; 195: 275-280Crossref PubMed Scopus (100) Google Scholar, 24Eagle P.A. Hanley-Bowdoin L. J. Virol. 1997; 71: 6947-6955Crossref PubMed Google Scholar) and induces the expression of a host DNA synthesis protein, proliferating cell nuclear antigen, in nondividing plant cells (25Nagar S. Pedersen T.J. Carrick K. Hanley-Bowdoin L. Robertson D. Plant Cell. 1995; 7: 705-719Crossref PubMed Scopus (156) Google Scholar). Several biochemical activities have been described for AL1 in vitro. TGMV AL1 binds to double-stranded DNA at the conserved repeated motif in the plus-strand origin (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar, 26Fontes E.P.B. Luckow V.A. Hanley-Bowdoin L. Plant Cell. 1992; 4: 597-608Crossref PubMed Scopus (136) Google Scholar) and cleaves single-stranded DNA in the invariant sequence of the hairpin loop (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Analysis of the C1 protein of tomato yellow leaf curl virus (TYLCV), a TGMV AL1 homologue, revealed that it covalently attaches to the 5′ end of the cleaved DNA by a phosphotyrosyl bond and catalyzes ligation of the cleavage products (27Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar). ATPase activity has also been demonstrated for the AL1/C1 proteins from TYLCV and TGMV (28Desbiez C. David C. Mettouchi A. Laufs J. Gronenborn B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5640-5644Crossref PubMed Scopus (106) Google Scholar, 29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TGMV AL1 is involved in several protein interactions. It forms large multimeric complexes (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), binds AL3 (30Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar), and interacts with a maize homologue of the animal cell cycle regulatory protein, retinoblastoma (31Ach R.A. Durfee T. Miller A.B. Taranto P. Hanley-Bowdoin L. Zambriski P.C. Gruissem W. Mol. Cell. Biol. 1997; 17: 5077-5086Crossref PubMed Scopus (202) Google Scholar). The C1 protein from wheat dwarf virus also interacts with retinoblastoma proteins from human and maize (32Xie Q. Suarezlopez P. Gutierrez C. EMBO J. 1995; 14: 4073-4082Crossref PubMed Scopus (156) Google Scholar, 33Collin S. Fernandez-Lobato M. Gooding P.S. Mullineaux P.M. Fenoll C. Virology. 1996; 219: 324-329Crossref PubMed Scopus (66) Google Scholar, 34Xie Q. Sanzburgos P. Hannon G.J. Gutierrez C. EMBO J. 1996; 15: 4900-4908Crossref PubMed Scopus (195) Google Scholar). In an earlier study, we used truncated proteins produced in a baculovirus expression system to map the regions of TGMV AL1 that are responsible for DNA cleavage, DNA binding and oligomerization (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These experiments showed that the DNA cleavage domain is located in the first 120 amino acids of AL1 whereas the oligomerization domain maps to the protein center between amino acids 121 and 181. DNA binding requires a larger region that fully overlaps the DNA cleavage and oligomerization domains. Based on these experiments, we proposed that additional amino acids between position 121 and 181 are necessary for AL1/DNA binding and/or that AL1 complex formation is required for DNA binding. In this study, we identified key sequence and structural motifs in the DNA binding and cleavage domains. A series of site-directed mutations in the AL1 N terminus were analyzed for their impact on function in vivo and in vitro. These studies focused on three amino acid motifs, which are conserved among all geminivirus AL1/C1 proteins and many initiator proteins from other RCR systems (35Koonin E.V. Ilyina T.V. J. Gen. Virol. 1992; 73: 2763-2766Crossref PubMed Scopus (132) Google Scholar, 36Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (508) Google Scholar), and on a predicted helix-loop-helix motif in the N termini of AL1/C1 proteins of dicot-infecting geminiviruses (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). We also used a heterologous protein interaction domain to assess the importance of oligomerization for AL1/DNA binding and coupled DNA cleavage/ligation. The plasmid pNSB148, which contains the AL1 coding sequence in a pUC118 background, was used as the template for site-directed mutagenesis (37Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 482-492Google Scholar). The oligonucleotide primers and resulting clones are listed in Table I. The sequences of fragments containing the mutations and used for subsequent cloning were verified by DNA sequence analysis. Plant expression cassettes with the mutant AL1 coding sequences were generated by subcloning NdeI/SalI fragments (AL1 amino acids 1–120) from the mutant clones into the same sites in a wild type AL1 plant expression cassette pMON1549 (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). In pMON1549, AL1 expression is under the control of the cauliflower mosaic virus 35 S promoter with a duplicated enhancer (38Kay R. Chan A. Daly M. McPherson J. Science. 1987; 236: 1299-1302Crossref PubMed Scopus (736) Google Scholar) and the 3′ end from the pea E9 rbcS gene (39Coruzzi G. Broglie R. Edwards C. Chua N.-H. EMBO J. 1984; 3: 1671-1679Crossref PubMed Scopus (208) Google Scholar). Baculovirus expression vectors coding for AL1 proteins fused to a glutathione S-transferase tag (GST-AL1) were generated by digesting the mutant plant expression cassettes with NdeI and BamHI and repairing the ends with Escherichia coli DNA polymerase I (Klenow). The fragments, which included complete AL1 coding regions, were inserted into the SmaI site of pNSB314. The pNSB314 vector contains the GST coding sequence, followed by a glycine linker, a thrombin cleavage site, and a multiple cloning site for generating in-frame fusion proteins (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). The mutant GST-AL1 expression cassettes are listed in Table I.Table IAL1 mutationsMotifMutationOligonucleotideBaculovirus vectorPlant expressionHelix 1H1–1GAGAAAGTGATgCggCcgcGGACAAGGAGCACpNSB627pNSB655H1–2GTAATTGAGAAAGTacTTCTTCTTTGGACpNSB434Helix 2H2–1CTTCATGAAGCgCTgcaCAGATTgcTATGAATTTTTTGTTAATCGGpNSB628pNSB656H2–2CTCTGCAGATcTTTccGAATTTTTTGTTAATCGGpNSB629pNSB657H2–3CATCTTCATGtcGCTCTtcGCAGAcTTTTATGAATTTTTTGTTAATCGGpNSB630pNSB658Motif 1M1–1GCACTGAGGAgcgGccgcAgcATAATTTTTGGCATpNSB649pNSB652M1–2CACCTCGAGGATAcGTAAGAgcATAATTTTTGGCATTpNSB685pNSB681M1–3GACATGAGGAgcgGTAAGAAAATAATTTTTGGGGCATTpNSB686pNSB682Motif 2M2–1GAATAAGCACGgcggccgcAGGTTGCCCATCpNSB650pNSB653Motif 3M3–1GAGTATCTCCGgCTTTaTCGATGgcCGTCTTGACGTCGpNSB651pNSB654M3–2CTTTGTCGATcgcCGTCTTGACGTCGpNSB687pNSB683M3–3GAGTATCTCCGgCTTTaTCGATGTACGpNSB688pNSB684M3–4TACAAGAGTAgCTCCGgCTTTcgCGATGTACGTCTTGACpNSB741pNSB747M3–5CTCCGTCTTTaTCGATGaACGTCTTGACpNSB781pNSB779M3–6GAGTATCTCCGTCTgcaTCGATGTACGpNSB782pNSB780 Open table in a new tab Wild type and mutant GST-AL1 fusion proteins were expressed in Spodoptera frugiperda (Sf9) cells using a Tn7-based baculovirus expression system (40Luckow V.A. Lee S.C. Barry G.F. Olins P.O. J. Virol. 1993; 67: 4566-4579Crossref PubMed Google Scholar) and purified from Sf9 cell cultures by glutathione affinity chromatography according to published protocols (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). Purified proteins were visualized by electrophoresis in 16% polyacrylamide-SDS gels and staining with Coomassie Brilliant Blue dye. Interactions between authentic AL1 and mutant GST-AL1 proteins were assayed by copurification on glutathione-Sepharose, followed by immunoblot analysis using AL1 polyclonal antisera (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 30Settlage S.B. Miller B. Hanley-Bowdoin L. J. Virol. 1996; 70: 6790-6795Crossref PubMed Google Scholar). DNA gel shift assays were performed as described previously (18Orozco B.M. Hanley-Bowdoin L. J. Virol. 1996; 270: 148-158Crossref Google Scholar). An 83-base pair EcoRI fragment containing the AL1 DNA binding motif (TGMV A positions 28–84) was isolated from pNSB378 and 3′ end-labeled using Klenow and [α-32P]dATP. The radiolabeled DNA was incubated with purified GST-AL1 fusion proteins for 1 h at room temperature at the concentrations indicated in the figure legends. The bound and free probe were resolved on 1% agarose gels, dried onto DE-81 paper, and analyzed by autoradiography. For DNA cleavage and ligation, oligonucleotides corresponding to sequences in the hairpin of the TGMV (+)-strand origin were 5′ end-labeled using polynucleotide kinase and [γ-32P]ATP. The oligonucleotides CR13 (5′-GTTTAATATTACCGGATGGCCGC) and CR33 (5′-GCGGCCATCCGTTTAATATT) were used for assays with full-length mutant and wild type AL1 proteins. For assays with truncated AL1 proteins, only one oligonucleotide, CR13 or CR34 (5′-GCGGCCATCCGTTTAATATTACCGGATGG) was radiolabeled and the cold oligonucleotide was titrated into the reactions as described in the figure legend. Approximately 5000 cpm of each labeled DNA was incubated with ∼100 ng of purified GST-AL1 fusion protein in 10 μl of cleavage buffer (25 mm Tris-HCl, pH 7.5, 75 mmNaCl, 5 mm MgCl2, 2.5 mm EDTA, 2.5 mm dithiothreitol, and 250 ng of poly(dI-dC)) at 37 °C for 30 min. The reactions were terminated by adding 6 μl of gel loading buffer (95% formamide, 20 mm EDTA, 0.05% bromphenol blue) and heating to 90 °C for 2 min. The reaction products were resolved on 15% polyacrylamide denaturing gels and analyzed by autoradiography. Protoplasts were isolated fromNicotiana tabacum NT-1 suspension cells, electroporated, and cultured according to published methods (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). The transfections contained 15 μg each of replicon DNA containing a partial tandem copy of TGMV B (pTG1.4B described in Ref. 14Fontes E.P.B. Gladfelter H.J. Schaffer R.L. Petty I.T.D. Hanley-Bowdoin L. Plant Cell. 1994; 6: 405-416Crossref PubMed Scopus (171) Google Scholar), wild type or mutant AL1 expression cassette, and an AL3 plant expression cassette (pNSB46 described in Ref. 11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Total DNA was extracted 3 days after transfection and analyzed for double-stranded viral DNA accumulation by DNA gel blot hybridization (11Fontes E.P.B. Eagle P.A. Sipe P.A. Luckow V.A. Hanley-Bowdoin L. J. Biol. Chem. 1994; 269: 8459-8465Abstract Full Text PDF PubMed Google Scholar). Two key steps in initiation of RCR are origin recognition and generation of a free 3′-OH for priming plus-strand DNA synthesis. TGMV AL1 mediates both of these processes by binding double-stranded DNA in a sequence-specific manner and by cleaving at a unique site in the origin. Earlier studies established that the AL1 N terminus is necessary for both activities (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This region of AL1 contains several conserved sequence and structural motifs (Fig. 1), including a highly predicted pair of α-helices between amino acids 25 and 52 (29Orozco B.M. Miller A.B. Settlage S.B. Hanley-Bowdoin L. J. Biol. Chem. 1997; 272: 9840-9846Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The sequences of both helices are strongly conserved among dicot-infecting geminiviruses, and the second helix is amphipathic in character. This conservation suggested that the predicted α-helices may be necessary for AL1 function. To test this hypothesis, we generated four site-directed mutants of TGMV AL1 that are modified in either helix 1 or helix 2 (Fig. 1 B) and compared their activities to the wild type protein in vivo and in vitro (Fig. 1 B). The helix mutants were first tested for the ability to direct replication of TGMV B DNA in tobacco protoplasts with wild type TGMV AL3 also supplied in trans. Helix 1-mutant 2 (H1–2), which contains a S25V change and resembles the AL1 protein of the closely related geminivirus BGMV, supported wild type levels of TGMV B replication (Fig. 2 A, cf. lanes 1 and 2). In contrast, H1–1, which has alanine substitutions at three conserved charged residues in helix 1, failed to support viral DNA replication (Fig. 2 A, lane 3). The mutations in helix 2 either converted three conserved charged residues to alanine (H2–1), included a I45G change (H2–2), or altered the helix to resemble BGMV AL1 (H2–3). All helix 2 mutations abolished transient replication (Fig. 2 A, lanes 3–5). The mutant phenotypes established that, in general, the amino acid sequences of the predicted helices are essential for AL1 activity in vivo. The negative effect of the I45G replacement in H2–2, which should disrupt helix 2, suggested that the structure is also required for AL1 function. The AL1 helix mutants defective in replication assays were expressed as GST-AL1 fusion proteins in insect cells, purified by glutathione affinity chromatography, and examined for various AL1 activitiesin vitro. H1–2 was not included in these experiments because of its wild type replication phenotype. The mutant proteins were tested for DNA binding activity in gel shift assays using a radiolabeled double-stranded DNA probe that includes the TGMV AL1 DNA binding site. None of the helix mutants bound DNA (Fig. 2 B, lanes 3–6), even though wild type AL1 efficiently shifted the probe in a parallel reaction (lane 2). The mutant AL1 proteins were also tested for DNA cleavage activity using a radiolabeled, single-stranded oligonucleotide corresponding to the loop and right side of the hairpin in the plus-strand origin. Wild type GST-AL1 cleaved the 23-nt substrate to give a 10-nt radiolabeled product (Fig. 2 C, lane 2). Mutants H1–1 (Fig. 2 C, lane 3) and H2–3 (lane 6) displayed severely attenuated DNA cleavage activity, whereas H2–1 (lane 4) and H2–2 (lane 5) had no detectable activity. These results demonstrated that the predicted helices 1 and 2 are required both for DNA binding and cleavage by TGMV AL1. Helices 1 and 2 are outside of the AL1 interaction domain, and their mutation should have no effect on oligomerization if the proteins are properly expressed and folded. To verify that the failure of the AL1 helix mutants to bind and cleave DNA was not due to global misfolding, the proteins were assayed for their abilities to form oligomers with authentic AL1. Wild type GST-AL1 (Fig. 3 A, lane 1) and GST fusions of H1–1 (lane 2), H2–1 (lane 3), H2–2 (lane 4), and H2–3 (lane 5) were coexpressed with authentic AL1 in insect cells, as determined by immunoblotting of total protein extracts. Like wild type GST-AL1 (Fig. 3 A, lane 6), all of the mutant proteins copurified with authentic AL1 on glutathione-Sepharose (lanes 7–10), indicating that the helix mutations did not impair AL1/AL1 interactions and, instead, specifically affected the DNA binding and cleavage activities of AL1. The N terminus of AL1 also includes three conserved sequence motifs that are found in many RCR initiator proteins (Fig. 1; Refs. 35Koonin E.V. Ilyina T.V. J. Gen. Virol. 1992; 73: 2763-2766Crossref PubMed Scopus (132) Google Scholar and 36Ilyina T.V. Koonin E.V. Nucleic Acids Res. 1992; 20: 3279-3285Crossref PubMed Scopus (508) Google Scholar). Laufset al. (27Laufs J. Schumacher S. Geisler N. Jupin I. Gronenborn B. FEBS Lett. 1995; 377: 258-262Crossref PubMed Scopus (67) Google Scholar) showed that motif 3 corresponds to the endonucleolytic active site, but the roles of motifs 1 and 2 in RCR have not been investigated. We specifically modified these motifs in TGMV AL1 and analyzed the impact of the mutations on protein function (Fig. 1 B). In M1–1, all four motif I residues (F16LTY19) were replaced with alanine. In M1–2 and M1–3, individual aromatic residues were altered to give F16A and Y19A, respectively. The motif 2 mutant contained alanine substitutions for the core HLH sequence. Transient replication assays revealed that none of the motif 1 or 2 mutants (Fig. 4 A, lanes 2–5) supported TGMV B amplification in tobacco protoplasts, establishing that both motifs are essential for AL1 activity in vivo. To gain insight into the biochemical basis of the mutant replication phenotypes, we purified GST-AL1 fusion proteins corresponding to the motif 1 and 2 mutants from insect cells and tested them for DNA binding and cleavage in vitro. All of the mutant proteins failed to bind double-stranded DNA (Fig. 4 B, lanes 2–5). Similarly, none of the mutants had detectable single-stranded DNA cleavage activity (Fig. 4 C, lanes 2–5). In parallel assays, wild type GST-AL1 (lane 1) efficiently bound (Fig. 4 B) and cleaved (Fig. 4 C) the respective DNA probes. Protein interaction experiments (Fig. 3 B) established that M1–1 (lanes 3 and 8) and M2–1 (lanes 4 and 9) can form oligomers with wild type AL1 and, thus, are not globally misfolded. Together, these results showed that motifs 1 and 2 of TGMV AL1 are required for both DNA binding and cleavage during initiation of RCR. Motif 3 includes several conserved amino acids that may contribute to AL1 function. We generated six TGMV AL1 mutants that modified one or more residues in motif 3 and analyzed their activities in vivo and in vitro. In M3–1, alanines were substituted for the catalytic Tyr-103 and conserved Asp-107 residues. As expected, M3–1 did not support TGMV B replication in tobacco protoplasts (Fig. 5 A, lane 2) and was defective for DNA cleavage (Fig. 5 C, lane 2). Gel shift assays with M3–1 revealed that double-stranded DNA binding was also attenuated over a range of protein concentrations (Fig. 5 B, lanes 5–7), even though wild type GST-AL1 readily formed protein/DNA complexes at the same concentrations (lanes 2–4). In some experiments, low levels of DNA binding were observed with the M3–1 protein (data not shown). Protein interaction experiments showing that M3–1 can oligomerize with wild type AL1 (Fig. 3 B, lanes 5 and 10) indicated that the motif 3 mutant is not globally misfolded. The DNA binding defect of M3–1 was unexpected because of the direct involvement of motif 3 in catalysis of DNA cleavage. To better characterize the role of motif 3 and specifically Tyr-103 in DNA binding, we generated two different mutations at this position. In M3–2, Tyr-103 was changed to alanine, while M3–5 contained a phenylalanine substitution. Like M3–1, neither M3–2 or M3–5 supported viral DNA replication (Fig. 5 A, lanes 3 and 6) or cleaved DNA (Fig. 5 C, lanes 3 and 6), confirming that Tyr-103 is necessary for these processes. However, only M3–2 was impaired for DNA binding over a range of protein concentrations (Fig. 5 B, lanes 5–7), whereas M3–5 displayed wild type DNA binding properties (lanes 17–19). These results demonstrated that Tyr-103 contributes to both the DNA bind}, number={38}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Orozco, BM and Hanley-Bowdoin, L}, year={1998}, month={Sep}, pages={24448–24456} }
 @article{orozco_gladfelter_settlage_eagle_gentry_hanley-bowdoin_1998, title={Multiple cis elements contribute to geminivirus origin function}, volume={242}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1997.9013}, abstractNote={The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules which are dissimilar in sequence except for a highly conserved 200-bp common region that includes the origin for rolling circle replication. To better characterize the plus-strand origin, we analyzed the capacities of various TGMV common region sequences to support episomal replication in tobacco protoplasts when the viral replication proteins AL1 and AL3 were supplied in trans. These experiments demonstrated that the minimal origin is located in 89-bp common region fragment that includes the known AL1 binding motif and a hairpin structure containing the DNA cleavage site. Analyses of mutant origin sequences identified two additional cis elements--one that is required for origin activity and a second that greatly enhances replication. In contrast, a conserved partial copy of the AL1 binding site did not contribute to origin function. Mutational analysis of the functional AL1 binding site showed that both spacing and sequence of this motif are important for replication in vivo and AL1/DNA binding in vitro. Spacing changes between the AL1 binding site and hairpin also negatively impacted TGMV origin function in a position-dependent manner. Together, these results demonstrated that the organization of TGMV plus-strand origin is complex, involving multiple cis elements that are likely to interact with each other during initiation of replication.}, number={2}, journal={VIROLOGY}, author={Orozco, BM and Gladfelter, HJ and Settlage, SB and Eagle, PA and Gentry, RN and Hanley-Bowdoin, L}, year={1998}, month={Mar}, pages={346–356} }
 @article{eagle_hanley-bowdoin_1997, title={CIS elements that contribute to geminivirus transcriptional regulation and the efficiency of DNA replication}, volume={71}, number={9}, journal={Journal of Virology}, author={Eagle, P. A. and Hanley-Bowdoin, L. K.}, year={1997}, pages={6947–6955} }
 @article{orozco_miller_settlage_hanley-bowdoin_1997, title={Functional domains of a geminivirus replication protein}, volume={272}, DOI={10.1074/jbc.272.15.9840}, abstractNote={Tomato golden mosaic virus, a member of the geminivirus family, has a single-stranded DNA genome that is replicated and transcribed in infected plant cells through the concerted action of viral and host factors. One viral protein, AL1, contributes to both processes by binding to a directly repeated, double-stranded DNA sequence located in the overlapping (+) strand origin of replication and AL1 promoter. The AL1 protein, which occurs as a multimeric complex in solution, also catalyzes DNA cleavage during initiation of rolling circle replication. To identify the tomato golden mosaic virus AL1 domains that mediate protein oligomerization, DNA binding, and DNA cleavage, a series of truncated AL1 proteins were produced in a baculovirus expression system and assayed for each activity. These experiments localized the AL1 oligomerization domain between amino acids 121 and 181, the DNA binding domain between amino acids 1 and 181, and the DNA cleavage domain between amino acids 1 and 120. Deletion of the first 29 amino acids of AL1 abolished DNA binding and DNA cleavage, demonstrating that an intact N terminus is required for both activities. The observation that the DNA binding domain includes the oligomerization domain suggested that AL1-AL1 protein interaction may be a prerequisite for DNA binding but not for DNA cleavage. The significance of these results for AL1 function during geminivirus replication and transcription is discussed. Tomato golden mosaic virus, a member of the geminivirus family, has a single-stranded DNA genome that is replicated and transcribed in infected plant cells through the concerted action of viral and host factors. One viral protein, AL1, contributes to both processes by binding to a directly repeated, double-stranded DNA sequence located in the overlapping (+) strand origin of replication and AL1 promoter. The AL1 protein, which occurs as a multimeric complex in solution, also catalyzes DNA cleavage during initiation of rolling circle replication. To identify the tomato golden mosaic virus AL1 domains that mediate protein oligomerization, DNA binding, and DNA cleavage, a series of truncated AL1 proteins were produced in a baculovirus expression system and assayed for each activity. These experiments localized the AL1 oligomerization domain between amino acids 121 and 181, the DNA binding domain between amino acids 1 and 181, and the DNA cleavage domain between amino acids 1 and 120. Deletion of the first 29 amino acids of AL1 abolished DNA binding and DNA cleavage, demonstrating that an intact N terminus is required for both activities. The observation that the DNA binding domain includes the oligomerization domain suggested that AL1-AL1 protein interaction may be a prerequisite for DNA binding but not for DNA cleavage. The significance of these results for AL1 function during geminivirus replication and transcription is discussed.}, number={15}, journal={Journal of Biological Chemistry}, author={Orozco, B. M. and Miller, A. B. and Settlage, S. B. and Hanley-Bowdoin, Linda}, year={1997}, pages={9840–9846} }
 @article{ach_durfee_miller_taranto_hanleybowdoin_zambryski_gruissem_1997, title={RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein}, volume={17}, ISSN={["0270-7306"]}, DOI={10.1128/MCB.17.9.5077}, abstractNote={Unlike mammalian and yeast cells, little is known about how plants regulate G1 progression and entry into the S phase of the cell cycle. In mammalian cells, a key regulator of this process is the retinoblastoma tumor suppressor protein (RB). In contrast, G1 control in Saccharomyces cerevisiae does not utilize an RB-like protein. We report here the cloning of cDNAs from two Zea mays genes, RRB1 and RRB2, that encode RB-related proteins. Further, RRB2 transcripts are alternatively spliced to yield two proteins with different C termini. At least one RRB gene is expressed in all the tissues examined, with the highest levels seen in the shoot apex. RRB1 is a 96-kDa nuclear protein that can physically interact with two mammalian DNA tumor virus oncoproteins, simian virus 40 large-T antigen and adenovirus E1A, and with a plant D-type cyclin. These associations are abolished by mutation of a conserved cysteine residue in RRB1 that is also essential for RB function. RRB1 binding potential is also sensitive to deletions in the conserved A and B domains, although differences exist in these effects compared to those of human RB. RRB1 can also bind to the AL1 protein from tomato golden mosaic virus (TGMV), a protein which is essential for TGMV DNA replication. These results suggest that G1 regulation in plant cells is controlled by a mechanism which is much more similar to that found in mammalian cells than that in yeast.}, number={9}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Ach, RA and Durfee, T and Miller, AB and Taranto, P and HanleyBowdoin, L and Zambryski, PC and Gruissem, W}, year={1997}, month={Sep}, pages={5077–5086} }
 @article{gladfelter_eagle_fontes_batts_hanley-bowdoin_1997, title={Two domains of the AL1 protein mediate geminivirus origin recognition}, volume={239}, ISSN={["0042-6822"]}, DOI={10.1006/viro.1997.8869}, abstractNote={The geminiviruses tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) have bipartite genomes. Their A and B DNA components containcis-acting sequences that function as origins of replication, while their A components encode thetrans-acting replication proteins—AL1 and AL3. Earlier experiments demonstrated that virus-specific interactions between thecis- andtrans-acting functions are required for TGMV and BGMV replication and that the AL1 proteins of the two viruses specifically bind their respective origins. In the current study, characterization of AL1 and AL3 proteins produced from plant expression cassettes in transient replication assays revealed that interaction between AL1 and the origin is responsible for virus-specific replication. The AL3 protein does not contribute to specificity but can be preferred by its cognate AL1 protein when replication is impaired. Analysis of chimeric proteins showed that two regions of AL1 act as specificity determinants during replication. The first domain is located between amino acids 1 and 116 and recognizes the AL1 origin binding site. The second region, which is between amino acids 121 and 209, is not dependent on the known AL1 DNA binding site. Analysis of wild type and chimeric proteins in transient transcription assays showed that AL1 also represses its own promoter in a virus-specific manner. Transcriptional specificity is conferred primarily by AL1 amino acids 1–93 with amino acids 121–209 making a smaller contribution. Together, these results demonstrated that the virus-specific interactions of AL1 during replication and transcription are complex, involving at least two discreet domains of the protein.}, number={1}, journal={VIROLOGY}, author={Gladfelter, HJ and Eagle, PA and Fontes, EPB and Batts, L and Hanley-Bowdoin, L}, year={1997}, month={Dec}, pages={186–197} }
 @article{orozco_hanley-bowdoin_1996, title={A DNA structure is required for geminivirus replication origin function.}, volume={70}, ISSN={0022-538X}, url={http://dx.doi.org/10.1128/jvi.70.1.148-158.1996}, DOI={10.1128/jvi.70.1.148-158.1996}, abstractNote={The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two single-stranded circular DNAs, A and B, that replicate through a rolling-circle mechanism in nuclei of infected plant cells. The TGMV origin of replication is located in a conserved 5' intergenic region and includes at least two functional elements: the origin recognition site of the essential viral replication protein, AL1, and a sequence motif with the potential to form a hairpin or cruciform structure. To address the role of the hairpin motif during TGMV replication, we constructed a series of B-component mutants that resolved sequence changes from structural alterations of the motif. Only those mutant B DNAs that retained the capacity to form the hairpin structure replicated to wild-type levels in tobacco protoplasts when the viral replication proteins were provided in trans from a plant expression cassette. In contrast, the same B DNAs replicated to significantly lower levels in transient assays that included replicating, wild-type TGMV A DNA. These data established that the hairpin structure is essential for TGMV replication, whereas its sequence affects the efficiency of replication. We also showed that TGMV AL1 functions as a site-specific endonuclease in vitro and mapped the cleavage site to the loop of the hairpin. In vitro cleavage analysis of two TGMV B mutants with different replication phenotypes indicated that there is a correlation between the two assays for origin activity. These results suggest that the in vivo replication results may reflect structural and sequence requirements for DNA cleavage during initiation of rolling-circle replication.}, number={1}, journal={Journal of virology}, publisher={American Society for Microbiology}, author={Orozco, B M and Hanley-Bowdoin, L}, year={1996}, pages={148–158} }
 @inbook{hanley-bowdoin_eagle_orozco_robertson_settlage_1996, place={St. Paul, MN}, title={Geminivirus Replication}, booktitle={Biology of Plant-Microbe Interactions}, publisher={International Society of Molecular Plant-Microbe Interactions}, author={Hanley-Bowdoin, L. and Eagle, P.A. and Orozco, B.M. and Robertson, D. and Settlage, S.B.}, editor={Stacey, G. and Mullin, B. and Gresshoff, P.M.Editors}, year={1996}, pages={287–292} }
 @article{settlage_miller_hanley-bowdoin_1996, title={Interactions between geminivirus replication proteins.}, volume={70}, ISSN={0022-538X}, url={http://dx.doi.org/10.1128/jvi.70.10.6790-6795.1996}, DOI={10.1128/jvi.70.10.6790-6795.1996}, abstractNote={Geminiviruses are small DNA viruses that replicate in the nuclei of infected plant cells. The closely related geminiviruses tomato golden mosaic virus and bean golden mosaic virus each encode a protein, AL1, that catalyzes the initiation of rolling-circle replication. Both viruses also specify a second replication protein, AL3, that greatly enhances the level of viral DNA accumulation. Using recombinant proteins produced in a baculovirus expression system, we showed that AL1 copurifies with a protein fusion of glutathione S-transferase (GST) and AL1, independent of the GST domain. Similarly, authentic AL3 cofractionates with a GST-AL3 fusion protein. These results demonstrated that both AL1 and AL3 form oligomers. Immunoprecipitation of protein extracts from insect cells expressing both AL1 and AL3 showed that the two proteins also complex with each other. None of the protein interactions displayed virus specificity; the tomato and bean golden mosaic virus proteins complexed with each other. The addition of heterologous replication proteins had no effect on the efficiency of geminivirus replication in transient-replication assays, suggesting that heteroprotein complexes might be functional. The significance of these protein interactions is discussed with respect to geminivirus replication in plant cells.}, number={10}, journal={Journal of virology}, publisher={American Society for Microbiology}, author={Settlage, S B and Miller, A B and Hanley-Bowdoin, L}, year={1996}, pages={6790–6795} }
 @article{nagar_pedersen_carrick_hanley-bowdoin_robertson_1995, title={A geminivirus induces expression of a host DNA synthesis protein in terminally differentiated plant cells.}, volume={7}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.7.6.705}, DOI={10.1105/tpc.7.6.705}, abstractNote={Geminiviruses are plant DNA viruses that replicate through DNA intermediates in plant nuclei. The viral components required for replication are known, but no host factors have yet been identified. We used immunolocalization to show that the replication proteins of the geminivirus tomato golden mosaic virus (TGMV) are located in nuclei of terminally differentiated cells that have left the cell cycle. In addition, TGMV infection resulted in a significant accumulation of the host DNA synthesis protein proliferating cell nuclear antigen (PCNA). PCNA, an accessory factor for DNA polymerase delta, was not present at detectable levels in healthy differentiated cells. The TGMV replication protein AL1 was sufficient to induce accumulation of PCNA in terminally differentiated cells of transgenic plants. Analysis of the mechanism(s) whereby AL1 induces the accumulation of host replication machinery in quiescent plant cells will provide a unique opportunity to study plant DNA synthesis.}, number={6}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Nagar, S and Pedersen, T J and Carrick, K M and Hanley-Bowdoin, L and Robertson, D}, year={1995}, month={Jun}, pages={705–719} }
 @article{eagle_orozco_hanley-bowdoin_1994, title={A DNA sequence required for geminivirus replication also mediates transcriptional regulation.}, volume={6}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.6.8.1157}, DOI={10.1105/tpc.6.8.1157}, abstractNote={Tomato golden mosaic virus (TGMV), a member of the geminivirus family, requires a single virus-encoded protein for DNA replication. We show that the TGMV replication protein, AL1, also acts during transcription to specifically repress the activity of its promoter. An earlier study established that AL1 binds to a 13-bp sequence (5'-GGTAGTAAGGTAG) that is essential for activity of the TGMV replication origin. Analysis of AL1 binding site mutants in transient expression assays demonstrated that the same site, which is located between the transcription start site and TATA box in the AL1 promoter, also mediates transcriptional repression. These experiments revealed that the repeated motifs in the AL1 binding site contribute differentially to repression, as has been observed previously for AL1-DNA binding and viral replication. Introduction of the AL1 binding site into the 35S promoter of the cauliflower mosaic virus was sufficient to confer AL1-mediated repression to the heterologous promoter. Analysis of a truncated AL1 promoter and of mutant AL1 proteins showed that repression does not require a replication-competent template or a replication-competent AL1 protein. Transient expression studies using two different Nicotiana cell lines revealed that, although the two lines replicate plasmids containing the TGMV origin similarly, they support very different levels of AL1-mediated repression. These results suggest that geminivirus transcriptional repression and replication may be independent processes.}, number={8}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Eagle, P A and Orozco, B M and Hanley-Bowdoin, L}, year={1994}, month={Aug}, pages={1157–1170} }
 @article{fontes_gladfelter_schaffer_petty_hanley-bowdoin_1994, title={Geminivirus replication origins have a modular organization.}, volume={6}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.6.3.405}, DOI={10.1105/tpc.6.3.405}, abstractNote={Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for replication, and their A components encode trans-acting factors are required for this process. We showed that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication in tobacco protoplasts. We also demonstrated that, similar to the essential TGMV AL1 replication protein, BGMV AL1 binds specifically to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificities of the B components were exchanged by site-directed mutagenesis, but the resulting mutants were not replicated by either A component. These results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Geminivirus genomes also contain a stem-loop sequence that is required for origin function. A BGMV B mutant with the TGMV stem-loop sequence was replicated by BGMV A, indicating that BGMV AL1 does not discriminate between the two sequences. A BGMV B double mutant, with the TGMV AL1 binding site and stem-loop sequences, was not replicated by either A component, indicating that an additional element in the TGMV origin is required for productive interaction with TGMV AL1. These results suggested that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is required for replication but does not contribute to virus-specific recognition of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.}, number={3}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Fontes, E P and Gladfelter, H J and Schaffer, R L and Petty, I T and Hanley-Bowdoin, L}, year={1994}, month={Mar}, pages={405–416} }
 @article{fontes_eagle_sipe_luckow_hanley-bowdoin_1994, title={Interaction between a geminivirus replication protein and origin DNA is essential for viral replication}, volume={269}, journal={Journal of Biological Chemistry}, author={Fontes, E.P.B. and Eagle, P.A. and Sipe, P. and Luckow, V.A. and Hanley-Bowdoin, L.}, year={1994}, pages={8459–8465} }
 @article{pedersen_hanley-bowdoin_1994, title={Molecular Characterization of the AL3 Protein Encoded by a Bipartite Geminivirus}, volume={202}, ISSN={0042-6822}, url={http://dx.doi.org/10.1006/viro.1994.1442}, DOI={10.1006/viro.1994.1442}, abstractNote={The genome of tomato golden mosaic virus (TGMV) is composed of two circular, single-stranded DNA molecules that together contain 6 open reading frames (ORFs). Three of these ORFs (designated AL1, AL2, and AL3) overlap and are specified by multiple polycistronic mRNAs. No RNA specifying the AL3 ORF alone has been detected, suggesting that the AL3 gene product is translated from an internal ORF. A recombinant histidine-tagged-AL3 fusion protein was purified from Escherichia coli and used to raise a polyclonal antiserum. Analysis of protein extracts from healthy plants and plants infected with TGMV by SDS-PAGE and immunoblotting showed that a protein corresponding to the predicted AL3 gene product is produced only in infected plants. This protein comprises approximately 0.05% of the cellular proteins and is present in the soluble and organelle fractions. These results are discussed with respect to the expression and role of the AL3 protein in the viral life cycle.}, number={2}, journal={Virology}, publisher={Elsevier BV}, author={Pedersen, Thomas J. and Hanley-Bowdoin, Linda}, year={1994}, month={Aug}, pages={1070–1075} }
 @article{fontes_luckow_hanley-bowdoin_1992, title={A geminivirus replication protein is a sequence-specific DNA binding protein.}, volume={4}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.4.5.597}, DOI={10.1105/tpc.4.5.597}, abstractNote={The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component encodes the only viral protein, AL1, that is required for viral replication. We showed that AL1 interacts specifically with TGMV A and B DNA by using an immunoprecipitation assay for AL1:DNA complex formation. In this assay, a monoclonal antibody against AL1 precipitated AL1:TGMV DNA complexes, whereas an unrelated antibody failed to precipitate the complexes. Competition assays with homologous and heterologous DNAs established the specificity of AL1:DNA binding. AL1 produced by transgenic tobacco plants and by baculovirus-infected insect cells exhibited similar DNA binding activity. The AL1 binding site maps to 52 bp on the left side of the common region, a 235-bp region that is highly conserved between the two TGMV genome components. The AL1:DNA binding site does not include the putative hairpin structure that is conserved in the common regions or the equivalent 5' intergenic regions of all geminiviruses. These studies demonstrate that a geminivirus replication protein is a sequence-specific DNA binding protein, and the studies have important implications for the role of this protein in virus replication.}, number={5}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Fontes, E P and Luckow, V A and Hanley-Bowdoin, L}, year={1992}, month={May}, pages={597–608} }
 @inbook{hanley bowdoin_hemenway_1992, title={Transgenic plants expressing viral genes}, booktitle={Genetic Engineering with Plant Viruses}, publisher={CRC Press, Inc}, author={Hanley Bowdoin, L. and Hemenway, C.}, editor={Wilson, T. Michael A. and Davies, Jeffrey W.Editors}, year={1992}, pages={251–295} }
 @article{hanley-bowdoin_elmer_rogers_1990, title={Expression of functional replication protein from tomato golden mosaic virus in transgenic tobacco plants.}, volume={87}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.87.4.1446}, DOI={10.1073/pnas.87.4.1446}, abstractNote={The A component of the bipartite genome of the geminivirus tomato golden mosaic virus (TGMV) encodes the viral protein (AL1) that is required for viral DNA replication. We have constructed transgenic Nicotiana benthamiana plants in which the AL1 open reading frame is transcribed under the control of the cauliflower mosaic virus 35S promoter. The transgenic plants, which were phenotypically normal, produced a single transcript from the 35S-AL1 construct and a 40-kDa protein that cross-reacted with a polyclonal antiserum raised against AL1 protein overproduced in Escherichia coli. Six of nine transgenic lines complemented a TGMV A variant with a mutation in AL1 when coinoculated with the B component of the TGMV genome. Single- and double-stranded forms of the B component were synthesized in leaf discs from a complementing, transgenic line in the absence of TGMV A. These results establish that the transgenic plants express functional AL1 protein and show that this viral protein is not only required, but sufficient, for single- and double-stranded replication of TGMV DNA in the presence of host proteins. These results also show that the AL1 protein is not by itself a determinant of disease or pathogenesis.}, number={4}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Hanley-Bowdoin, L. and Elmer, J. S. and Rogers, S. G.}, year={1990}, month={Feb}, pages={1446–1450} }
 @article{hanley-bowdoin_elmer_rogers_1989, title={Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobacco plants.}, volume={1}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.1.11.1057}, DOI={10.1105/tpc.1.11.1057}, abstractNote={The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. We have constructed Nicotiana benthamiana plants that are transgenic for the three overlapping open reading frames, AL1, AL2, and AL3, from the left side of TGMV A. In the transgenic plants, the AL open reading frames are under the control of the cauliflower mosaic virus (CaMV) 35S promoter. In TGMV infectivity assays, seven of 10 transgenic lines complemented TGMV A variants with mutations in AL1, AL2, or AL3 when co-inoculated with the B component. The 35S-AL construct was transcribed as a single RNA species in the transgenic plants, indicating that AL1, AL2, and AL3 were expressed from a polycistronic mRNA. This differs from the complex transcription pattern in TGMV-infected plants, which contains five AL transcripts. There was no quantitative correlation between the efficiency of complementation in the infectivity assay and the level of expression of transgenic AL RNA in the leaves of a transgenic line. One line that failed to complement defects in AL1, AL2, and AL3 in infectivity assays contained high levels of transgenic AL RNA and functional AL1 protein. These results provide evidence that chromosomal position can affect the cell- and tissue-specific transcription of the 35S promoter in transgenic plants. Comparison of the complementing plants and wild-type infected plants may provide insight into the TGMV infection process and the use of the CaMV 35S promoter for gene expression in transgenic plants.}, number={11}, journal={The Plant Cell}, publisher={American Society of Plant Biologists (ASPB)}, author={Hanley-Bowdoin, L and Elmer, J S and Rogers, S G}, year={1989}, month={Nov}, pages={1057–1067} }
 @article{hanley-bowdoin_chua_1989, title={Transcriptional interaction between the promoters of the maize chloroplast genes which encode the β subunit of ATP synthase and the large subunit of ribulose 1,5-bisphosphate carboxylase}, volume={215}, ISSN={0026-8925 1432-1874}, url={http://dx.doi.org/10.1007/bf00339720}, DOI={10.1007/bf00339720}, number={2}, journal={Molecular and General Genetics MGG}, publisher={Springer Science and Business Media LLC}, author={Hanley-Bowdoin, Linda and Chua, Nam-Hai}, year={1989}, month={Jan}, pages={217–224} }
 @article{lam_hanley bowdoin_chua_1988, title={Characterization of a chloroplast sequence-specific DNA binding factor}, volume={263}, journal={Journal of Biological Chemistry}, author={Lam, E. and Hanley Bowdoin, L. and Chua, N. H.}, year={1988}, pages={8288–8293} }
 @article{hanley-bowdoin_chua_1988, title={Transcription of the wheat chloroplast gene that encodes the 32 kd polypeptide}, volume={10}, ISSN={0167-4412 1573-5028}, url={http://dx.doi.org/10.1007/bf00029880}, DOI={10.1007/bf00029880}, number={4}, journal={Plant Molecular Biology}, publisher={Springer Science and Business Media LLC}, author={Hanley-Bowdoin, Linda and Chua, Nam-Hai}, year={1988}, pages={303–310} }
 @article{hanley-bowdoin_elmer_rogers_1988, title={Transient expression of heterologous RNAs using tomato golden mosaic virus}, volume={16}, ISSN={0305-1048 1362-4962}, url={http://dx.doi.org/10.1093/nar/16.22.10511}, DOI={10.1093/nar/16.22.10511}, abstractNote={The genome of the geminivirus tomato golden mosaic virus (TGMV) consists of two circular DNA molecules designated as components A and B. The A component contains the only virally-encoded function required for autonomous replication in infected plant cells. We used agroinoculation of petunia leaf discs with the A component to develop a transient expression system which permits direct examination of viral transcripts by S1 nuclease protection. The AR1 gene, which encodes the TGMV coat protein, was transcribed transiently in leaf discs after agroinoculation of TGMV A DNA. Synthesis of AR1 RNA was dependent on T-DNA transfer and TGMV DNA replication, demonstrating that it is a plant transcription product. The AL open reading frames of TGMV A were also expressed transiently in leaf discs. The ratio between AR1 RNA and the major leftward RNA was constant and was used to normalize AR1 transcription for viral DNA copy number. The bacterial genes encoding chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) were transiently expressed in leaf discs from the AR1 promoter in TGMV A. The levels of AR1 and GUS RNAs were similar in leaf discs after adjusting for viral DNA copy number, while CAT RNA was less abundant. The geminivirus transient expression system allows rapid analysis of RNAs transcribed from foreign genes and can serve as a preliminary screen in the construction of transgenic plants.}, number={22}, journal={Nucleic Acids Research}, publisher={Oxford University Press (OUP)}, author={Hanley-Bowdoin, L. and Elmer, J. S. and Rogers, S. G.}, year={1988}, month={Nov}, pages={10511–10528} }
 @article{hanley-bowdoin_chua_1987, title={Chloroplast promoters}, volume={12}, ISSN={0968-0004}, url={http://dx.doi.org/10.1016/0968-0004(87)90033-8}, DOI={10.1016/0968-0004(87)90033-8}, abstractNote={DNA sequences required for accurate initiation of chloroplast transcription have been characterized in higher plants using chloroplast in vitro transcription systems. Although chloroplast promoters resemble bacterial promoters, they also have some unique properties.}, journal={Trends in Biochemical Sciences}, publisher={Elsevier BV}, author={Hanley-Bowdoin, Linda and Chua, Nam-Hai}, year={1987}, month={Jan}, pages={67–70} }
 @inbook{orozco_mullet_hanley-bowdoin_chua_1986, title={In vitro transcription of chloroplast protein genes}, ISBN={9780121820183}, ISSN={0076-6879}, url={http://dx.doi.org/10.1016/0076-6879(86)18076-1}, DOI={10.1016/0076-6879(86)18076-1}, abstractNote={This chapter discusses the methods that are useful for the in vitro analysis of plastid gene expression. Many plastid genes have multiple transcripts in vivo that may be the result of transcription initiation and subsequent RNA processing events. Heterologous chloroplast transcription extracts may be useful for the analysis of plastid RNA processing. To characterize the potential regulatory nature of the plastid RNA and other plastid DNA sequences, the use of a homologous plastid in vitro transcription system is required. The criteria for such a transcription system are ease of preparation, accuracy of transcription initiation, and stability of enzyme activity. The optimal time and temperature of the in vitro transcription reaction varies with the gene transcribed and the extract employed. Part of this variance may be due to specific RNA processing and nonspecific degradation of in vitro transcripts by endogenous ribonucleases in the chloroplast extracts.}, booktitle={Methods in Enzymology}, publisher={Elsevier}, author={Orozco, Emil M., Jr. and Mullet, John E. and Hanley-Bowdoin, Linda and Chua, Nam-Hai}, year={1986}, pages={232–253} }
 @article{hanley-bowdoin_orozco_chua_1985, title={In vitro synthesis and processing of a maize chloroplast transcript encoded by the ribulose 1,5-bisphosphate carboxylase large subunit gene.}, volume={5}, ISSN={0270-7306 1098-5549}, url={http://dx.doi.org/10.1128/mcb.5.10.2733}, DOI={10.1128/mcb.5.10.2733}, abstractNote={The large subunit gene (rbcL) of ribulose 1,5-bisphosphate carboxylase was transcribed in vitro by using maize and pea chloroplast extracts and a cloned plastid DNA template containing 172 base pairs (bp) of the maize rbcL protein-coding region and 791 bp of upstream sequences. Three major in vitro RNA species were synthesized which correspond to in vivo maize rbcL RNAs with 5' termini positioned 300, 100 to 105, and 63 nucleotides upstream of the protein-coding region. A deletion of 109 bp, including the "-300" 5' end (the 5' end at position -300), depressed all rbcL transcription in vitro. A plasmid DNA containing this 109-bp fragment was sufficient to direct correct transcription initiation in vitro. A cloned template, containing 191 bp of plastid DNA which includes the -105 and -63 rbcL termini, did not support transcription in vitro. Exogenously added -300 RNA could be converted to the -63 transcript by maize chloroplast extract. These results established that the -300 RNA is the primary maize rbcL transcript, the -63 RNA is a processed form of the -300 transcript, and synthesis of the -105 RNA is dependent on the -300 region. The promoter for the maize rbcL gene is located within the 109 bp flanking the -300 site. Mutagenesis of the 109-bp chloroplast sequence 11 bp upstream of the -300 transcription initiation site reduced rbcL promoter activity in vitro.}, number={10}, journal={Molecular and Cellular Biology}, publisher={American Society for Microbiology}, author={Hanley-Bowdoin, L and Orozco, E M, Jr and Chua, N H}, year={1985}, month={Oct}, pages={2733–2745} }
 @inbook{hanley-bowdoin_orozco_chua_1985, title={Transcription of chloroplast genes by homo-logous and heterologous RNA polymerases}, booktitle={Molecular biology of the photosynthetic apparatus}, publisher={Cold Spring Harbor Press}, author={Hanley-Bowdoin, L. and Orozco, E.M. and Chua, N. H}, editor={Steinback, Katherine E. and Bonitz, Susan and Arntzen, Charles J. and Bogorad, LawrenceEditors}, year={1985}, pages={311–318} }
 @article{hanley-bowdoin_lane_1983, title={A novel protein programmed by the mRNA conserved in dry wheat embryos. The principal site of cysteine incorporation during early germination}, volume={135}, ISSN={0014-2956 1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1983.tb07611.x}, DOI={10.1111/j.1432-1033.1983.tb07611.x}, abstractNote={If bulk mRNA from dry wheat embryos (wheat germ) is used to direct cell-free incorporation of [35S]cysteine into proteins, a striking proportion of the total radioactivity is channeled into a single protein. During early postimbibition development, when protein synthesis is directed by the mRNA conserved in dry embryos, incorporation of cysteine is preponderantly (20-25%) directed into synthesis of this one protein: the 'early' cysteine-labeled protein (Ec). When conserved mRNA from the dry embryos has been fully degraded, as when cellular or cell-free protein synthesis is directed by the mRNA in germinated embryos, synthesis of Ec is not detected. Reliable detection of Ec requires prior alkylation of wheat embryo proteins, and it was especially interesting to find that when wheat embryo proteins are alkylated by iodo[14C]acetamide, two proteins co-dominate the distribution of radioalkylated products in dodecylsulphate/polyacrylamide gels: Ec and wheat germ agglutinin. Using co-electrophoresis with the isotopically labeled protein to detect a dye-staining counterpart, Ec has been purified by combined cation-exchange and gel-filtration chromatography of alkylated wheat germ proteins. The purified protein can be recovered in milligram quantity (5-10 mg/100 g wheat germ) and compositional analysis shows that it is unusually rich in cysteine (approx. 15%) and glycine (approx. 17%), as is wheat germ agglutinin.}, number={1}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={Hanley-Bowdoin, Linda and Lane, Byron G.}, year={1983}, month={Sep}, pages={9–15} }
 @article{grzelczak_sattolo_hanley-bowdoin_kennedy_lane_1982, title={Synthesis and turnover of proteins and mRNA in germinating wheat embryos}, volume={60}, ISSN={0008-4018}, url={http://dx.doi.org/10.1139/o82-046}, DOI={10.1139/o82-046}, abstractNote={The most prominent methionine-labeled protein made when cell-free systems are programmed with bulk mRNA from dry wheat embryos has been identified with what may be the most abundant protein in dry wheat embryos. The protein has been brought to purity and has a distinctive amino acid composition, Gly and Glx accounting for almost 40% of the total amino acids. Designated E because of its conspicuous association with early inhibition of dry wheat embryos, the protein and its mRNA are abundant during the "early" phase (0--1 h) of postimbibition development, and easily detected during "lag" phase (1--5 h), but they are almost totally degraded soon after entry into the "growth" phase of development, by about 10 h postimbibition. The most prominent methionine-labeled protein peculiar to the cell-free translational capacity of bulk mRNA from "growth" phase embryos is not detected as a product of in vivo synthesis. Its electrophoretic properties and its time course of emergence, after 5 h postimbibition development, suggest that this major product of cell-free synthesis may be an in vitro counterpart to a prominent methionine-labeled protein made only in vivo, by "growth" phase embryos. Designated G because of its conspicuous association with "growth" phase development, the cell-free product does not comigrate with any prominent dye-stained band in electrophoretic distributions of wheat proteins. The suspected cellular counterpart to G, also, does not comigrate with a prominent dye-stained wheat protein during electrophoresis, and although found in particulate as well as soluble fractions of wheat embryo homogenates it is not concentrated in either nuclei or mitochondria, as isolated.}, number={3}, journal={Canadian Journal of Biochemistry}, publisher={Canadian Science Publishing}, author={Grzelczak, Zbyszko F. and Sattolo, Mark H. and Hanley-Bowdoin, Linda K. and Kennedy, Theresa D. and Lane, Byron G.}, year={1982}, month={Mar}, pages={389–397} }
 @article{kennedy_hanley-bowdoin_lane_1981, title={Structural integrity of RNA and translational integrity of ribosomes in nuclease-treated cell-free protein synthesizing systems prepared from wheat germ and rabbit reticulocytes}, volume={256}, journal={Journal of Biological Chemistry}, author={Kennedy, T.D. and Hanley-Bowdoin, L. and Lane, B.G}, year={1981}, pages={5802–5809} }
 @article{bellvé_anderson_hanley-bowdoin_1975, title={Synthesis and amino acid composition of basic proteins in mammalian sperm nuclei}, volume={47}, ISSN={0012-1606}, url={http://dx.doi.org/10.1016/0012-1606(75)90289-4}, DOI={10.1016/0012-1606(75)90289-4}, abstractNote={The basic chromosomal proteins (SCP) of human, mouse, rabbit and guinea pig sperm nuclei were characterized by polyacrylamide gel electrophoresis and amino acid analysis. Spermatozoa were decapitated with 1% SDS and the nuclei recovered by density gradient centrifugation. Examination by Nomarski and electron microscopy revealed the nuclei to be intact and 99% pure. The basic proteins were extracted from nuclei, aminoethylated and purified by ion exchange chromatography and gel filtration chromatography. The SCP of human, rabbit and guinea pig gave single protein bands with similar mobilities when subjected to polyacrylamide gel electrophoresis. In contrast, aminoethylated mouse SCP consisted of two proteins, SCP·AE1 and SCP·AE2, which had different electrophoretic mobilities. The SCP of these mammalian species were characteristically rich in arginine (47–54.4%) and cysteine (7.7–12.2%). Major differences existed in the amino acid compositions of these proteins. Mouse and human SCP were rich in histidine (12.2 and 7.7%, respectively) and guinea pig was high in tyrosine (11.7%) and phenylalanine (3.5%). Valine was detected only in rabbit SCP and proline in human and guinea pig. Aspartic acid, methionine and tryptophan were not detected in all four species. Studies on the incorporation of [3H]arginine into mouse SCP demonstrated that these basic proteins are synthesized during the terminal stages of spermatogenesis and are subsequently conserved.}, number={2}, journal={Developmental Biology}, publisher={Elsevier BV}, author={Bellvé, Anthony R. and Anderson, Everett and Hanley-Bowdoin, Linda}, year={1975}, month={Dec}, pages={349–365} }