@misc{noar_thomas_daub_2022, title={Genetic Characteristics and Metabolic Interactions between Pseudocercospora fijiensis and Banana: Progress toward Controlling Black Sigatoka}, volume={11}, ISSN={["2223-7747"]}, url={https://www.mdpi.com/2223-7747/11/7/948}, DOI={10.3390/plants11070948}, abstractNote={The international importance of banana and severity of black Sigatoka disease have led to extensive investigations into the genetic characteristics and metabolic interactions between the Dothideomycete Pseudocercospora fijiensis and its banana host. P. fijiensis was shown to have a greatly expanded genome compared to other Dothideomycetes, due to the proliferation of retrotransposons. Genome analysis suggests the presence of dispensable chromosomes that may aid in fungal adaptation as well as pathogenicity. Genomic research has led to the characterization of genes and metabolic pathways involved in pathogenicity, including: secondary metabolism genes such as PKS10-2, genes for mitogen-activated protein kinases such as Fus3 and Slt2, and genes for cell wall proteins such as glucosyl phosphatidylinositol (GPI) and glycophospholipid surface (Gas) proteins. Studies conducted on resistance mechanisms in banana have documented the role of jasmonic acid and ethylene pathways. With the development of banana transformation protocols, strategies for engineering resistance include transgenes expressing antimicrobial peptides or hydrolytic enzymes as well as host-induced gene silencing (HIGS) targeting pathogenicity genes. Pseudocercospora fijiensis has been identified as having high evolutionary potential, given its large genome size, ability to reproduce both sexually and asexually, and long-distance spore dispersal. Thus, multiple control measures are needed for the sustainable control of black Sigatoka disease.}, number={7}, journal={PLANTS-BASEL}, author={Noar, Roslyn D. and Thomas, Elizabeth and Daub, Margaret E.}, year={2022}, month={Apr} }
 @article{thomas_noar_daub_2021, title={A polyketide synthase gene cluster required for pathogenicity of Pseudocercospora fijiensis on banana}, volume={16}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0258981}, DOI={10.1371/journal.pone.0258981}, abstractNote={Pseudocercospora fijiensis is the causal agent of the highly destructive black Sigatoka disease of banana. Previous research has focused on polyketide synthase gene clusters in the fungus, given the importance of polyketide pathways in related plant pathogenic fungi. A time course study of expression of the previously identified PKS7-1, PKS8-2, and PKS10-2 gene clusters showed high expression of all three PKS genes and the associated clustered genes in infected banana plants from 2 weeks post-inoculation through 9 weeks. Engineered transformants silenced for PKS8-2 and PKS10-2 were developed and tested for pathogenicity. Inoculation of banana plants with silencing transformants for PKS10-2 showed significant reduction in disease symptoms and severity that correlated with the degree of silencing in the conidia used for inoculation, supporting a critical role for PKS10-2 in disease development. Unlike PKS10-2, a clear role for PKS8-2 could not be determined. Two of four PKS8-2 silencing transformants showed reduced disease development, but disease did not correlate with the degree of PKS8-2 silencing in the transformants. Overall, the degree of silencing obtained for the PKS8-2 transformants was less than that obtained for the PKS10-2 transformants, which may have limited the utility of the silencing strategy to identify a role for PKS8-2 in disease. Orthologous PKS10-2 clusters had previously been identified in the related banana pathogens Pseudocercospora musae and Pseudocercospora eumusae. Genome analysis identified orthologous gene clusters to that of PKS10-2 in the newly sequenced genomes of Pseudocercospora fuligena and Pseudocercospora cruenta, pathogens of tomato and cowpea, respectively. Our results support an important role for the PKS10-2 polyketide pathway in pathogenicity of Pseudocercospora fijiensis, and suggest a possible role for this pathway in disease development by other Pseudocercospora species.}, number={10}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Thomas, Elizabeth and Noar, Roslyn D. and Daub, Margaret E.}, editor={Wilson, Richard A.Editor}, year={2021}, month={Oct}, pages={e0258981} }
 @article{thomas_herrero_eng_gomaa_gillikin_noar_beseli_daub_2020, title={Engineering Cercospora disease resistance via expression of Cercospora nicotianae cercosporin-resistance genes and silencing of cercosporin production in tobacco}, volume={15}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0230362}, DOI={10.1371/journal.pone.0230362}, abstractNote={Fungi in the genus Cercospora cause crop losses world-wide on many crop species. The wide host range and success of these pathogens has been attributed to the production of a photoactivated toxin, cercosporin. We engineered tobacco for resistance to Cercospora nicotianae utilizing two strategies: 1) transformation with cercosporin autoresistance genes isolated from the fungus, and 2) transformation with constructs to silence the production of cercosporin during disease development. Three C. nicotianae cercosporin autoresistance genes were tested: ATR1 and CFP, encoding an ABC and an MFS transporter, respectively, and 71cR, which encodes a hypothetical protein. Resistance to the pathogen was identified in transgenic lines expressing ATR1 and 71cR, but not in lines transformed with CFP. Silencing of the CTB1 polyketide synthase and to a lesser extent the CTB8 pathway regulator in the cercosporin biosynthetic pathway also led to the recovery of resistant lines. All lines tested expressed the transgenes, and a direct correlation between the level of transgene expression and disease resistance was not identified in any line. Resistance was also not correlated with the degree of silencing in the CTB1 and CTB8 silenced lines. We conclude that expression of fungal cercosporin autoresistance genes as well as silencing of the cercosporin pathway are both effective strategies for engineering resistance to Cercospora diseases where cercosporin plays a critical role.}, number={3}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Thomas, Elizabeth and Herrero, Sonia and Eng, Hayde and Gomaa, Nafisa and Gillikin, Jeff and Noar, Roslyn and Beseli, Aydin and Daub, Margaret E.}, editor={Wilson, Richard A.Editor}, year={2020}, month={Mar}, pages={e0230362} }
 @misc{swiderska-burek_daub_thomas_jaszek_pawlik_janusz_2020, title={Phytopathogenic Cercosporoid Fungi-From Taxonomy to Modern Biochemistry and Molecular Biology}, volume={21}, ISSN={["1422-0067"]}, DOI={10.3390/ijms21228555}, abstractNote={Phytopathogenic cercosporoid fungi have been investigated comprehensively due to their important role in causing plant diseases. A significant amount of research has been focused on the biology, morphology, systematics, and taxonomy of this group, with less of a focus on molecular or biochemical issues. Early and extensive research on these fungi focused on taxonomy and their classification based on in vivo features. Lately, investigations have mainly addressed a combination of characteristics such as morphological traits, host specificity, and molecular analyses initiated at the end of the 20th century. Some species that are important from an economic point of view have been more intensively investigated by means of genetic and biochemical methods to better understand the pathogenesis processes. Cercosporin, a photoactivated toxin playing an important role in Cercospora diseases, has been extensively studied. Understanding cercosporin toxicity in relation to reactive oxygen species (ROS) production facilitated the discovery and regulation of the cercosporin biosynthesis pathway, including the gene cluster encoding pathway enzymes. Furthermore, these fungi may be a source of other biotechnologically important compounds, e.g., industrially relevant enzymes. This paper reviews methods and important results of investigations of this group of fungi addressed at different levels over the years.}, number={22}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Swiderska-Burek, Urszula and Daub, Margaret E. and Thomas, Elizabeth and Jaszek, Magdalena and Pawlik, Anna and Janusz, Grzegorz}, year={2020}, month={Nov} }
 @article{noar_thomas_daub_2019, title={A novel polyketide synthase gene cluster in the plant pathogenic fungus Pseudocercospora fijiensis}, volume={14}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0212229}, DOI={10.1371/journal.pone.0212229}, abstractNote={Pseudocercospora fijiensis, causal agent of black Sigatoka of banana, produces polyketide synthase (PKS) pathways shown to be important in disease development by related Dothideomycete fungi. Genome analysis of the P. fijiensis PKS8-1 gene identified it as part of a gene cluster including genes encoding two transcription factors, a regulatory protein, a glyoxylase/beta-lactamase-like protein, an MFS transporter, a cytochrome P450, two aldo/keto reductases, a dehydrogenase, and a decarboxylase. Genome analysis of the related pathogens Pseudocercospora musae, Pseudocercospora eumusae, and Pseudocercospora pini-densiflorae, identified orthologous clusters containing a nearly identical combination of genes. Phylogenetic analysis of PKS8-1 identified homology to PKS proteins in the monodictyphenone and cladofulvin pathways in Aspergillus nidulans and Cladosporium fulvum, respectively. Analysis of clustered genes showed that the PKS8-1 cluster shares genes for enzymes involved in the production of the emodin intermediate in the monodictyphenone and cladofulvin pathways, but differs in many genes, suggesting production of a different metabolic product. Time course analysis of gene expression in infected banana showed up-regulation of PKS8-1 and four of eight clustered genes as early as 2 weeks post-inoculation and remaining high through 9 weeks. Overexpression of the pathway through constitutive expression of an aflR-like transcription factor gene in the cluster resulted in increased expression in culture of PKS8-1 as well as the four clustered genes that are up-regulated in infected plants. No differences were seen in timing or severity of disease symptoms with the overexpression strains relative to controls, however gene expression analysis showed no difference in expression in planta by an overexpression strain relative to controls. Thus constitutive expression of the aflR-like gene is not sufficient to upregulate the pathway above normal expression in planta. Pathway expression during all phases of disease development and conservation of the pathway in related Pseudocercospora species support a role for this pathway in disease.}, number={2}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Noar, Roslyn D. and Thomas, Elizabeth and Daub, Margaret E.}, editor={Lespinet, OlivierEditor}, year={2019}, month={Feb}, pages={e0212229} }
 @article{noar_thomas_xie_carter_ma_daub_2019, title={A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis}, volume={14}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0220319}, DOI={10.1371/journal.pone.0220319}, abstractNote={Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.}, number={7}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Noar, Roslyn D. and Thomas, Elizabeth and Xie, De-Yu and Carter, Morgan E. and Ma, Dongming and Daub, Margaret E.}, editor={Lespinet, OlivierEditor}, year={2019}, month={Jul}, pages={e0220319} }
 @article{noar_daub_2016, title={Bioinformatics Prediction of Polyketide Synthase Gene Clusters from Mycosphaerella fijiensis}, volume={11}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0158471}, abstractNote={Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode polyketides important in pathogenicity.}, number={7}, journal={PLOS ONE}, author={Noar, Roslyn D. and Daub, Margaret E.}, year={2016}, month={Jul} }
 @article{noar_daub_2016, title={Transcriptome sequencing of Mycosphaerella fijiensis during association with Musa acuminata reveals candidate pathogenicity genes}, volume={17}, ISSN={["1471-2164"]}, DOI={10.1186/s12864-016-3031-5}, abstractNote={Mycosphaerella fijiensis, causative agent of the black Sigatoka disease of banana, is considered the most economically damaging banana disease. Despite its importance, the genetics of pathogenicity are poorly understood. Previous studies have characterized polyketide pathways with possible roles in pathogenicity. To identify additional candidate pathogenicity genes, we compared the transcriptome of this fungus during the necrotrophic phase of infection with that during saprophytic growth in medium. Transcriptome analysis was conducted, and the functions of differentially expressed genes were predicted by identifying conserved domains, Gene Ontology (GO) annotation and GO enrichment analysis, Carbohydrate-Active EnZymes (CAZy) annotation, and identification of genes encoding effector-like proteins. The analysis showed that genes commonly involved in secondary metabolism have higher expression in infected leaf tissue, including genes encoding cytochrome P450s, short-chain dehydrogenases, and oxidoreductases in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Other pathogenicity-related genes with higher expression in infected leaf tissue include genes encoding salicylate hydroxylase-like proteins, hydrophobic surface binding proteins, CFEM domain-containing proteins, and genes encoding secreted cysteine-rich proteins characteristic of effectors. More genes encoding amino acid transporters, oligopeptide transporters, peptidases, proteases, proteinases, sugar transporters, and proteins containing Domain of Unknown Function (DUF) 3328 had higher expression in infected leaf tissue, while more genes encoding inhibitors of peptidases and proteinases had higher expression in medium. Sixteen gene clusters with higher expression in leaf tissue were identified including clusters for the synthesis of a non-ribosomal peptide. A cluster encoding a novel fusicoccane was also identified. Two putative dispensable scaffolds were identified with a large proportion of genes with higher expression in infected leaf tissue, suggesting that they may play a role in pathogenicity. For two other scaffolds, no transcripts were detected in either condition, and PCR assays support the hypothesis that at least one of these scaffolds corresponds to a dispensable chromosome that is not required for survival or pathogenicity. Our study revealed major changes in the transcriptome of Mycosphaerella fijiensis, when associating with its host compared to during saprophytic growth in medium. This analysis identified putative pathogenicity genes and also provides support for the existence of dispensable chromosomes in this fungus.}, journal={BMC GENOMICS}, author={Noar, Roslyn D. and Daub, Margaret E.}, year={2016}, month={Aug} }
 @article{beseli_noar_daub_2015, title={Characterization of Cercospora nicotianae Hypothetical Proteins in Cercosporin Resistance}, volume={10}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0140676}, abstractNote={The photoactivated toxin, cercosporin, produced by Cercospora species, plays an important role in pathogenesis of this fungus to host plants. Cercosporin has almost universal toxicity to cells due to its production of reactive oxygen species including singlet oxygen. For that reason, Cercospora species, which are highly resistant to their own toxin, are good candidates to identify genes for resistance to cercosporin and to the reactive oxygen species it produces. In previous research, the zinc cluster transcription factor CRG1 (cercosporin resistance gene 1) was found to be crucial for Cercospora species’ resistance against cercosporin, and subtractive hybridization analysis identified 185 genes differentially expressed between Cercospora nicotianae wild type (wt) and a crg1 mutant. The focus of this work was to identify and characterize the hypothetical proteins that were identified in the Cercospora nicotianae subtractive library as potential resistance factors. Quantitative RT-PCR analysis of the 20 genes encoding hypothetical proteins showed that two, 24cF and 71cR, were induced under conditions of cercosporin toxicity, suggesting a role in resistance. Transformation and expression of 24cF and 71cR in the cercosporin-sensitive fungus, Neurospora crassa, showed that 71cR provided increased resistance to cercosporin toxicity, whereas no significant increase was observed in 24cF transformants. Gene disruption was used to generate C. nicotianae 71cR mutants; these mutants did not differ from wt C. nicotianae in cercosporin resistance or production. Quantitative RT-PCR analysis showed induction of other resistance genes in the 71cR mutant that may compensate for the loss of 71cR. Analysis of 71cR conserved domains and secondary and tertiary structure identify the protein as having an NTF2-like superfamily DUF1348 domain with unknown function, to be intracellular and localized in the cytosol, and to have similarities to proteins in the steroid delta-isomerase family.}, number={10}, journal={PLOS ONE}, author={Beseli, Aydin and Noar, Roslyn and Daub, Margaret E.}, year={2015}, month={Oct} }
 @article{beseli_amnuaykanjanasin_herrero_thomas_daub_2015, title={Membrane transporters in self resistance of Cercospora nicotianae to the photoactivated toxin cercosporin}, volume={61}, ISSN={0172-8083 1432-0983}, url={http://dx.doi.org/10.1007/s00294-015-0486-x}, DOI={10.1007/s00294-015-0486-x}, abstractNote={The goal of this work is to characterize membrane transporter genes in Cercospora fungi required for autoresistance to the photoactivated, active-oxygen-generating toxin cercosporin they produce for infection of host plants. Previous studies implicated a role for diverse membrane transporters in cercosporin resistance. In this study, transporters identified in a subtractive cDNA library between a Cercospora nicotianae wild type and a cercosporin-sensitive mutant were characterized, including two ABC transporters (CnATR2, CnATR3), an MFS transporter (CnMFS2), a uracil transporter, and a zinc transport protein. Phylogenetic analysis showed that only CnATR3 clustered with transporters previously characterized to be involved in cercosporin resistance. Quantitative RT-PCR analysis of gene expression under conditions of cercosporin toxicity, however, showed that only CnATR2 was upregulated, thus this gene was selected for further characterization. Transformation and expression of CnATR2 in the cercosporin-sensitive fungus Neurospora crassa significantly increased cercosporin resistance. Targeted gene disruption of CnATR2 in the wild type C. nicotianae, however, did not decrease resistance. Expression analysis of other transporters in the cnatr2 mutant under conditions of cercosporin toxicity showed significant upregulation of the cercosporin facilitator protein gene (CFP), encoding an MFS transporter previously characterized as playing an important role in cercosporin autoresistance in Cercospora species. We conclude that cercosporin autoresistance in Cercospora is mediated by multiple genes, and that the fungus compensates for mutations by up-regulation of other resistance genes. CnATR2 may be a useful gene, alone or in addition to other known resistance genes, for engineering Cercospora resistance in crop plants.}, number={4}, journal={Current Genetics}, publisher={Springer Science and Business Media LLC}, author={Beseli, Aydin and Amnuaykanjanasin, Alongkorn and Herrero, Sonia and Thomas, Elizabeth and Daub, Margaret E.}, year={2015}, month={Apr}, pages={601–620} }
 @article{beseli_silva_daub_2015, title={The role of Cercospora zeae-maydis homologs of Rhodobacter sphaeroides O-1(2)-resistance genes in resistance to the photoactivated toxin cercosporin}, volume={362}, ISSN={["1574-6968"]}, DOI={10.1093/femsle/fnu036}, abstractNote={The photosynthetic bacterium Rhodobacter sphaeroides and plant pathogenic fungus Cercospora nicotianae have been used as models for understanding resistance to singlet oxygen ((1)O(2)), a highly toxic reactive oxygen species. In Rhodobacter and Cercospora, (1)O(2) is derived, respectively, from photosynthesis and from the (1)O(2)-generating toxin cercosporin which the fungus produces to parasitize plants. We identified common genes recovered in transcriptome studies of putative (1)O(2)-resistance genes in these two systems, suggesting common (1)O(2)-resistance mechanisms. To determine if the Cercospora homologs of R. sphaeroides (1)O(2)-resistance genes are involved in resistance to cercosporin, we expressed the genes in the cercosporin-sensitive fungus Neurospora crassa and assayed for increases in cercosporin resistance. Neurospora crassa transformants expressing genes encoding aldo/keto reductase, succinyl-CoA ligase, O-acetylhomoserine (thiol) lyase, peptide methionine sulphoxide reductase and glutathione S-transferase did not have elevated levels of cercosporin resistance. Several transformants expressing aldehyde dehydrogenase were significantly more resistant to cercosporin. Expression of the transgene and enzyme activity did not correlate with resistance, however. We conclude that although the genes tested in this study are important in (1)O(2) resistance in R. sphaeroides, their Cercospora homologs are not involved in resistance to (1)O(2) generated from cercosporin.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Beseli, Aydin and Silva, Marilia Goulart and Daub, Margaret E.}, year={2015}, month={Jan} }
 @article{coelho souza_herrero_maffia_daub_2014, title={Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein}, volume={139}, ISSN={["1573-8469"]}, DOI={10.1007/s10658-013-0301-9}, abstractNote={Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈108/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).}, number={2}, journal={EUROPEAN JOURNAL OF PLANT PATHOLOGY}, author={Coelho Souza, Andre Gomes and Herrero, Sonia and Maffia, Luiz Antonio and Daub, Margaret Elizabeth}, year={2014}, month={Jun}, pages={235–238} }
 @misc{daub_herrero_chung_2013, title={Reactive Oxygen Species in Plant Pathogenesis: The Role of Perylenequinone Photosensitizers}, volume={19}, ISSN={["1557-7716"]}, DOI={10.1089/ars.2012.5080}, abstractNote={SIGNIFICANCE
Reactive oxygen species (ROS) play multiple roles in interactions between plants and microbes, both as host defense mechanisms and as mediators of pathogenic and symbiotic associations. One source of ROS in these interactions are photoactivated, ROS-generating perylenequinone pigments produced via polyketide metabolic pathways in plant-associated fungi. These natural products, including cercosporin, elsinochromes, hypocrellins, and calphostin C, are being utilized as medicinal agents, enzyme inhibitors, and in tumor therapy, but in nature, they play a role in the establishment of pathogenic associations between fungi and their plant hosts.


RECENT ADVANCES
Photoactivated perylenequinones are photosensitizers that use light energy to form singlet oxygen (¹O₂) and free radical oxygen species which damage cellular components based on localization of the perylenequinone molecule. Production of perylenequinones during infection commonly results in lipid peroxidation and membrane damage, leading to leakage of nutrients from cells into the intercellular spaces colonized by the pathogen. Perylenequinones show almost universal toxicity against organisms, including plants, mice, bacteria, and most fungi. The producing fungi are resistant, however, and serve as models for understanding resistance mechanisms.


CRITICAL ISSUES
Studies of resistance mechanisms by perylenequinone-producing fungi such as Cercospora species are leading to an understanding of cellular resistance to ¹O₂ and oxidative stress. Recent studies show commonalities between resistance mechanisms in these fungi with extensive studies of ¹O₂ and oxidative stress responses in photosynthetic organisms.


FUTURE DIRECTIONS
Such studies hold promise both for improved medical use and for engineering crop plants for disease resistance.}, number={9}, journal={ANTIOXIDANTS & REDOX SIGNALING}, author={Daub, Margaret E. and Herrero, Sonia and Chung, Kuang-Ren}, year={2013}, month={Sep}, pages={970–989} }
 @article{rueschhoff_gillikin_sederoff_daub_2013, title={The SOS4 pyridoxal kinase is required for maintenance of vitamin B-6-mediated processes in chloroplasts}, volume={63}, ISSN={["0981-9428"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84872415352&partnerID=MN8TOARS}, DOI={10.1016/j.plaphy.2012.12.003}, abstractNote={Vitamin B(6) (pyridoxal 5'-phosphate and its vitamers) is an important cofactor in numerous enzymatic reactions. In spite of its importance, the consequences of altering vitamin B(6) content on plant growth and development are not well understood. This study compares two mutants for vitamin B(6)-metabolizing enzymes in Arabidopsis thaliana: a pdx1.3 mutant in the de novo synthesis pathway and a salvage pathway sos4 mutant that accumulates more vitamin B(6). We show that despite a difference in total B(6) content in leaf tissue, both mutants share similar phenotypes, including chlorosis, decreased size, altered chloroplast ultrastructure, and root sensitivity to sucrose. Assay of B(6) vitamer content from isolated chloroplasts showed that, despite differing B(6) vitamer content in whole leaf tissue, both mutants share a common deficiency in total and phosphorylated vitamers in chloroplasts. One of the splice variants of the SOS4 proteins was shown to be located in the chloroplast. Our data indicate that some of the phenotypic consequences shared between the pdx1.3 and sos4 mutants are due to B(6) deficiency in chloroplasts, and show that SOS4 is required for maintenance of phosphorylated B(6) vitamer concentrations in chloroplasts. Further, our data are consistent with a diffusion model for transport of vitamin B(6) into chloroplasts.}, journal={PLANT PHYSIOLOGY AND BIOCHEMISTRY}, author={Rueschhoff, Elizabeth E. and Gillikin, Jeffrey W. and Sederoff, Heike W. and Daub, Margaret E.}, year={2013}, month={Feb}, pages={281–291} }
 @article{herrero_gonzalez_gillikin_velez_daub_2011, title={Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis}, volume={76}, ISSN={["1573-5028"]}, DOI={10.1007/s11103-011-9777-x}, abstractNote={Vitamin B6 (pyridoxal phosphate) is an essential cofactor in enzymatic reactions involved in numerous cellular processes and also plays a role in oxidative stress responses. In plants, the pathway for de novo synthesis of pyridoxal phosphate has been well characterized, however only two enzymes, pyridoxal (pyridoxine, pyridoxamine) kinase (SOS4) and pyridoxamine (pyridoxine) 5' phosphate oxidase (PDX3), have been identified in the salvage pathway that interconverts between the six vitamin B6 vitamers. A putative pyridoxal reductase (PLR1) was identified in Arabidopsis based on sequence homology with the protein in yeast. Cloning and expression of the AtPLR1 coding region in a yeast mutant deficient for pyridoxal reductase confirmed that the enzyme catalyzes the NADPH-mediated reduction of pyridoxal to pyridoxine. Two Arabidopsis T-DNA insertion mutant lines with insertions in the promoter sequences of AtPLR1 were established and characterized. Quantitative RT-PCR analysis of the plr1 mutants showed little change in expression of the vitamin B6 de novo pathway genes, but significant increases in expression of the known salvage pathway genes, PDX3 and SOS4. In addition, AtPLR1 was also upregulated in pdx3 and sos4 mutants. Analysis of vitamer levels by HPLC showed that both plr1 mutants had lower levels of total vitamin B6, with significantly decreased levels of pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, and pyridoxamine 5'-phosphate. By contrast, there was no consistent significant change in pyridoxine and pyridoxine 5'-phosphate levels. The plr1 mutants had normal root growth, but were significantly smaller than wild type plants. When assayed for abiotic stress resistance, plr1 mutants did not differ from wild type in their response to chilling and high light, but showed greater inhibition when grown on NaCl or mannitol, suggesting a role in osmotic stress resistance. This is the first report of a pyridoxal reductase in the vitamin B6 salvage pathway in plants.}, number={1-2}, journal={PLANT MOLECULAR BIOLOGY}, author={Herrero, Sonia and Gonzalez, Eugenia and Gillikin, Jeffrey W. and Velez, Heriberto and Daub, Margaret E.}, year={2011}, month={May}, pages={157–169} }
 @article{amnuaykanjanasin_daub_2009, title={The ABC transporter ATR1 is necessary for efflux of the toxin cercosporin in the fungus Cercospora nicotianae}, volume={46}, ISSN={["1096-0937"]}, DOI={10.1016/j.fgb.2008.11.007}, abstractNote={The Cercospora nicotianae mutant deficient for the CRG1 transcription factor has marked reductions in both resistance and biosynthesis of the toxin cercosporin. We cloned and sequenced full-length copies of two genes, ATR1 and CnCFP, previously identified from a subtractive library between the wild type (WT) and a crg1 mutant. ATR1 is an ABC transporter gene and has an open reading frame (ORF) of 4368 bp with one intron. CnCFP encodes a MFS transporter with homology to Cercospora kikuchii CFP, previously implicated in cercosporin export, and has an ORF of 1975 bp with three introns. Disruption of ATR1 indicated atr1-null mutants had dramatic reductions in cercosporin production (25% and 20% of WT levels) in solid and liquid cultures, respectively. The ATR1 disruptants also showed moderately higher sensitivity to cercosporin. Constitutive expression of ATR1 in the crg1 mutant restored cercosporin biosynthesis and moderately increased resistance. In contrast, CnCFP overexpression in the mutant did not restore toxin production, however, it moderately enhanced toxin resistance. The results together indicate ATR1 acts as a cercosporin efflux pump in this fungus and plays a partial role in resistance.}, number={2}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Amnuaykanjanasin, Alongkom and Daub, Margaret E.}, year={2009}, month={Feb}, pages={146–158} }
 @article{veleaz_glassbrook_daub_2008, title={Mannitol biosynthesis is required for plant pathogenicity by Alternaria alternata}, volume={285}, ISSN={["0378-1097"]}, DOI={10.1111/j.1574-6968.2008.01224.x}, abstractNote={Mannitol has been hypothesized to play a role in antioxidant defense. In previous work, we confirmed the presence of the two mannitol biosynthetic enzymes, mannitol dehydrogenase (MtDH) and mannitol 1-phosphate 5-dehydrogenase (MPDH), in the fungus Alternaria alternata and created disruption mutants for both enzymes. These mutants were used to investigate the role of mannitol in pathogenicity of A. alternata on its host, tobacco. Conidia of all mutants were viable and germinated normally. GC-MS analysis demonstrated elevated levels of trehalose in the mutants, suggesting that trehalose may substitute for mannitol as a storage compound for germination. Tobacco inoculation showed no reduction in lesion severity caused by the MtDH mutant as compared with wild type; however, the MPDH mutant and a mutant in both enzymes caused significantly less disease. Microscopy analysis indicated that the double mutant was unaffected in the ability to germinate and produce appressoria on tobacco leaves and elicited a defense response from the host, indicating that it was able to penetrate and infect the host. We conclude that mannitol biosynthesis is required for pathogenesis of A. alternata on tobacco, but is not required for spore germination either in vitro or in planta or for initial infection.}, number={1}, journal={FEMS MICROBIOLOGY LETTERS}, author={Veleaz, Heriberto and Glassbrook, Norman J. and Daub, Margaret E.}, year={2008}, month={Aug}, pages={122–129} }
 @article{herrero_daub_2007, title={Genetic manipulation of Vitamin B-6 biosynthesis in tobacco and fungi uncovers limitations to up-regulation of the pathway}, volume={172}, ISSN={["0168-9452"]}, DOI={10.1016/j.plantsci.2006.11.011}, abstractNote={Transgenic expression of vitamin biosynthetic genes has been investigated for over-production of these dietary supplements in microorganisms and plants. In plants, successful efforts have been reported with Vitamins A, C, E and B-9, however information is lacking for other vitamins. Vitamin B-6 is an essential cofactor for numerous enzymatic reactions, and has also been shown to be a potent antioxidant involved in protecting phytopathogenic Cercospora fungi from their own toxin, cercosporin. In this report, we transformed and expressed two Vitamin B-6 biosynthetic genes (PDX1 and PDX2) isolated from Cercospora nicotianae in cercosporin-sensitive organisms, tobacco and the fungal species Aspergillus flavus and Neurospora crassa. Our goal was to determine if Vitamin B-6 levels could be increased by constitutive expression of these genes, and if over-production confers resistance to oxidative stresses induced by cercosporin and salinity stress. Elevated Vitamin B-6 levels were observed in one tobacco line. For other lines evaluated in this work, expression of PDX1 and PDX2 in transgenic organisms did not result in a significant increase in Vitamin B-6 content over controls. Analysis of gene expression in tobacco indicated that the lack of elevated B-6 content was not due to lack of enzymatic activity, but to down-regulation of the endogenous tobacco genes compounded with limited transgene expression. The single line with elevated B-6 levels had higher expression of both the PDX1 and PDX2 transgenes compared to the other lines, and the observed increase on Vitamin B-6 was correlated with higher enzyme activity. Consistent with our inability to elevate cellular B-6 levels, only small changes were observed in the response to either cercosporin or high salt, and most transgenic individuals were as susceptible as controls. Compared to tobacco lines transformed to express either PDX1 or PDX2 alone, half of the transgenic tobacco lines expressing both genes were impaired in seed germination and initial growth. However no correlation was observed between the observed phenotype and Vitamin B-6 levels in seeds. This is the first report on genetic engineering to manipulate the Vitamin B-6 pathway in plants. Our results suggest that genetic manipulation of the Vitamin B-6 biosynthetic pathway is possible but is limited by regulation of endogenous genes.}, number={3}, journal={PLANT SCIENCE}, author={Herrero, Sonia and Daub, Margaret E.}, year={2007}, month={Mar}, pages={609–620} }
 @article{herrero_amnuaykanjanasin_daub_2007, title={Identification of genes differentially expressed in the phytopathogenic fungus Cercospora nicotianae between cercosporin toxin-resistant and -susceptible strains}, volume={275}, ISSN={["1574-6968"]}, DOI={10.1111/j.1574-6968.2007.00903.x}, abstractNote={Plant pathogens from the genus Cercospora produce cercosporin, a photoactivated fungal toxin that generates toxic reactive oxygen species. Mechanisms governing toxin auto-resistance in Cercospora spp. are poorly understood. In this work, suppressive subtractive hybridization was used to identify genes differentially expressed between the cercosporin-resistant wild-type (WT) Cercospora nicotianae and a sensitive strain lacking a transcription factor (CRG1) that regulates resistance. Out of 338 sequences recovered, 185 unique expressed sequence tags (ESTs) were obtained and classified into functional categories. The majority of genes showed predicted expression differences, and 38.5% were differentially expressed at least twofold between the WT and mutant strain. ESTs were recovered with homology to genes involved in detoxification of noxious compounds, multidrug membrane transporters and antioxidant and polyketide biosynthetic enzymes as well as to ATPases and ATP synthases. The findings suggest that CRG1 regulates genes involved in pH responses in addition to those involved in toxin resistance and biosynthesis.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Herrero, Sonia and Amnuaykanjanasin, Alongkorn and Daub, Margaret E.}, year={2007}, month={Oct}, pages={326–337} }
 @article{velez_glassbrook_daub_2007, title={Mannitol metabolism in the phytopathogenic fungus Alternaria alternata}, volume={44}, ISSN={["1096-0937"]}, DOI={10.1016/j.fgb.2006.09.008}, abstractNote={Mannitol metabolism in fungi is thought to occur through a mannitol cycle first described in 1978. In this cycle, mannitol 1-phosphate 5-dehydrogenase (EC 1.1.1.17) was proposed to reduce fructose 6-phosphate into mannitol 1-phosphate, followed by dephosphorylation by a mannitol 1-phosphatase (EC 3.1.3.22) resulting in inorganic phosphate and mannitol. Mannitol would be converted back to fructose by the enzyme mannitol dehydrogenase (EC 1.1.1.138). Although mannitol 1-phosphate 5-dehydrogenase was proposed as the major biosynthetic enzyme and mannitol dehydrogenase as a degradative enzyme, both enzymes catalyze their respective reverse reactions. To date the cycle has not been confirmed through genetic analysis. We conducted enzyme assays that confirmed the presence of these enzymes in a tobacco isolate of Alternaria alternata. Using a degenerate primer strategy, we isolated the genes encoding the enzymes and used targeted gene disruption to create mutants deficient in mannitol 1-phosphate 5-dehydrogenase, mannitol dehydrogenase, or both. PCR analysis confirmed gene disruption in the mutants, and enzyme assays demonstrated a lack of enzymatic activity for each enzyme. GC–MS experiments showed that a mutant deficient in both enzymes did not produce mannitol. Mutants deficient in mannitol 1-phosphate 5-dehydrogenase or mannitol dehydrogenase alone produced 11.5 and 65.7 %, respectively, of wild type levels. All mutants grew on mannitol as a sole carbon source, however, the double mutant and mutant deficient in mannitol 1-phosphate 5-dehydrogenase grew poorly. Our data demonstrate that mannitol 1-phosphate 5-dehydrogenase and mannitol dehydrogenase are essential enzymes in mannitol metabolism in A. alternata, but do not support mannitol metabolism operating as a cycle.}, number={4}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Velez, Heriberto and Glassbrook, Norman J. and Daub, Margaret E.}, year={2007}, month={Apr}, pages={258–268} }
 @article{chen_lee_daub_chung_2007, title={Molecular analysis of the cercosporin biosynthetic gene cluster in Cercospora nicotianae}, volume={64}, ISSN={["1365-2958"]}, DOI={10.1111/j.1365-2958.2007.05689.x}, abstractNote={Summary}, number={3}, journal={MOLECULAR MICROBIOLOGY}, author={Chen, Huiqin and Lee, Miin-Huey and Daub, Margret E. and Chung, Kuang-Ren}, year={2007}, month={May}, pages={755–770} }
 @article{densiow_rueschhoff_daub_2007, title={Regulation of the Arabidopsis thaliana vitamin B-6 biosynthesis genes by abiotic stress}, volume={45}, ISSN={["0981-9428"]}, DOI={10.1016/j.plaphy.2007.01.007}, abstractNote={Vitamin B6 (pyridoxine and its vitamers) plays an essential role as a co-factor for enzymatic reactions and has also recently been implicated in defense against cellular oxidative stress. The biosynthetic pathway was thoroughly characterized in Escherichia coli, however most organisms, including plants, utilize an alternate pathway involving two genes, PDX1 and PDX2. Arabidopsis thaliana contains one copy of PDX2, but three full-length copies of PDX1, one each on chromosomes 2, 3, and 5 (referred to as PDX1.1, PDX1.2, and PDX1.3, respectively). Phylogenetic analysis of the PDX1 homologues in A. thaliana showed that PDX1.1 and PDX1.3 clustered with the homologues from the other dicots, whereas PDX1.2 was more divergent, and did not cluster with either the dicots or monocots. Expression analysis using quantitative PCR showed that PDX1.1 and PDX1.3 were highly expressed in A. thaliana rosettes, while PDX1.2 showed only low level expression. All three PDX1 genes and PDX2 were responsive to abiotic stressors including high light, chilling, drought, and ozone, however, the response of PDX1.2 was disparate from that of the other PDX genes, showing a lessened response to high light, chilling, and drought, but an increased response to ozone. Green fluorescent protein fusion studies demonstrated that PDX2 localizes in the nucleus and membranes of cells, consistent with recent published data for PDX1. Insight into regulation of the biosynthetic genes during abiotic stress could have important applications in the development of stress-tolerant crops.}, number={2}, journal={PLANT PHYSIOLOGY AND BIOCHEMISTRY}, author={Densiow, Sheri A. and Rueschhoff, Elizabeth E. and Daub, Margaret E.}, year={2007}, month={Feb}, pages={152–161} }
 @article{gonzalez_danehower_daub_2007, title={Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5 '-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B-6 salvage pathway}, volume={145}, ISSN={["1532-2548"]}, DOI={10.1104/pp.107.105189}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Gonzalez, Eugenia and Danehower, David and Daub, Margaret E.}, year={2007}, month={Nov}, pages={985–996} }
 @article{taylor_mitchell_daub_2006, title={An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae}, volume={72}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.00483-06}, abstractNote={ABSTRACT}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Taylor, Tanya V. and Mitchell, Thomas K. and Daub, Margaret E.}, year={2006}, month={Sep}, pages={6070–6078} }
 @misc{daub_herrero_chung_2005, title={Photoactivated perylenequinone toxins in fungal pathogenesis of plants}, volume={252}, ISSN={["0378-1097"]}, DOI={10.1016/j.femsle.2005.08.033}, abstractNote={Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Daub, ME and Herrero, S and Chung, KR}, year={2005}, month={Nov}, pages={197–206} }
 @article{denslow_walls_daub_2005, title={Regulation of biosynthetic genes and antioxidant properties of vitamin B-6 vitamers during plant defense responses}, volume={66}, ISSN={["0885-5765"]}, DOI={10.1016/j.pmpp.2005.09.004}, abstractNote={Vitamin B6, an essential cofactor in enzymatic reactions, has only recently been linked to cellular oxidative stress. We investigated the role of this vitamin as an antioxidant in oxidative responses linked to plant defense. B6 vitamers effectively quenched superoxide and had antioxidant activity when assayed in vitro. The de novo B6 biosynthetic genes (PDX1 and PDX2) were identified in Nicotiana tabacum cv. ‘Burley 21’ and their transcript abundance was assayed during defense responses. PDX1 and PDX2 transcript levels decreased following inoculation with the incompatible pathogen Pseudomonas syringae pv. phaseolicola and transiently increased in response to salicylic acid and methyl jasmonate. Excess vitamin B6 in tobacco leaves interfered with the development of a hypersensitive response caused by P. syringae pv. phaseolicola and increased disease severity caused by the compatible bacterium P. syringae pv. tabaci. Our findings indicate that during plant defense responses, vitamin B6 functions and its synthesis is regulated in a manner consistent with this vitamin's activity as an antioxidant and modulator of active oxygen species in vivo.}, number={6}, journal={PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY}, author={Denslow, SA and Walls, AA and Daub, ME}, year={2005}, month={Jun}, pages={244–255} }
 @article{choquer_dekkers_chen_cao_ueng_daub_chung_2005, title={The CTB1 gene encoding a fungal polyketide synthase is required for cercosporin biosynthesis and fungal virulence of Cercospora nicotianae}, volume={18}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI-18-0468}, abstractNote={ Cercosporin is a light-activated, non-host-selective toxin produced by many Cercospora fungal species. In this study, a polyketide synthase gene (CTB1) was functionally identified and molecularly characterized to play a key role in cercosporin biosynthesis by Cercospora nicotianae. We also provide conclusive evidence to confirm the crucial role of cercosporin in fungal pathogenesis. CTB1 encoded a polypeptide with a deduced length of 2,196 amino acids containing a keto synthase (KS), an acyltransferase (AT), a thioesterase/claisen cyclase (TE/CYC), and two acyl carrier protein (ACP) domains, and had high levels of similarity to many fungal type I polyketide synthases. Expression of a 6.8-kb CTB1 transcript was highly regulated by light and medium composition, consistent with the conditions required for cercosporin biosynthesis in cultures. Targeted disruption of CTB1 resulted in the loss of both CTB1 transcript and cercosporin biosynthesis in C. nicotianae. The ctb1-null mutants incited fewer necrotic lesions on inoculated tobacco leaves compared with the wild type. Complementation of ctb1-null mutants with a full-length CTB1 clone restored wild-type levels of cercosporin production as well as the ability to induce lesions on tobacco. Thus, we have demonstrated conclusively that cercosporin is synthesized via a polyketide pathway, and cercosporin is an important virulence factor in C. nicotianae. The results also suggest that strategies that avoid the toxicity of cercosporin will be useful in reduction of disease incidence caused by Cercospora spp. }, number={5}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Choquer, M and Dekkers, KL and Chen, HQ and Cao, LH and Ueng, PP and Daub, ME and Chung, KR}, year={2005}, month={May}, pages={468–476} }
 @article{wetzel_ehrenshaft_denslow_daub_2004, title={Functional complementation between the PDX1 vitamin B-6 biosynthetic gene of Cercospora nicotianae and pdxJ of Escherichia coli}, volume={564}, ISSN={["1873-3468"]}, DOI={10.1016/S0014-5793(04)00329-1}, abstractNote={The pathway for de novo vitamin B6 biosynthesis has been characterized in Escherichia coli, however plants, fungi, archaebacteria, and most bacteria utilize an alternative pathway. Two unique genes of the alternative pathway, PDX1 and PDX2, have been described. PDX2 encodes a glutaminase, however the enzymatic function of the product encoded by PDX1 is not known. We conducted reciprocal transformation experiments to determine if there was functional homology between the E. coli pdxA and pdxJ genes and PDX1 of Cercospora nicotianae. Although expression of pdxJ and pdxA in C. nicotianae pdx1 mutants, either separately or together, failed to complement the pyridoxine mutation in this fungus, expression of PDX1 restored pyridoxine prototrophy to the E. coli pdxJ mutant. Expression of PDX1 in the E. coli pdxA mutant restored very limited ability to grow on medium lacking pyridoxine. We conclude that the PDX1 gene of the alternative B6 pathway encodes a protein responsible for synthesis of the pyridoxine ring.}, number={1-2}, journal={FEBS LETTERS}, author={Wetzel, DK and Ehrenshaft, M and Denslow, SA and Daub, ME}, year={2004}, month={Apr}, pages={143–146} }
 @article{chung_ehrenshaft_wetzel_daub_2003, title={Cercosporin-deficient mutants by plasmid tagging in the asexual fungus Cercospora nicotianae}, volume={270}, ISSN={["1617-4623"]}, DOI={10.1007/s00438-003-0902-7}, abstractNote={We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.}, number={2}, journal={MOLECULAR GENETICS AND GENOMICS}, author={Chung, KR and Ehrenshaft, M and Wetzel, DK and Daub, ME}, year={2003}, month={Oct}, pages={103–113} }
 @article{chung_daub_ehrenshaft_2003, title={Expression of the cercosporin toxin resistance gene (CRG1) as a dicistronic mRNA in the filamentous fungus Cercospora nicotianae}, volume={43}, ISSN={["1432-0983"]}, DOI={10.1007/s00294-003-0414-3}, abstractNote={The CRG1 gene in Cercospora nicotianae encodes a transcription factor and is required for cercosporin toxin resistance and production. Cloning and sequencing of the downstream region of the CRG1 gene led to the discovery of an adjacent gene ( PUT1) encoding a putative uracil transporter. Expression of CRG1 and PUT1 as assessed by Northern analysis indicated that, in addition to the expected monocistronic mRNAs (2.6 kb and 2.0 kb, respectively), a common 4.5-kb mRNA could be identified, using either a CRG1 or a PUT1 gene probe. The 2.6-kb transcript identified only by the CRG1 probe was expressed constitutively, whereas the 2.0-kb transcript identified only by the PUT1 probe was differentially expressed in various media. Four cDNA clones containing CRG1, PUT1, and the CRG1- PUT1 intergenic region were identified as part of the products from the 4.5-kb transcript. Both the 4.5-kb and 2.6-kb transcripts were not detectable in three crg1-disrupted mutants, using the CRG1 probe. The 2.0-kb transcript, but not the 4.5-kb one was detected using the PUT1 probe in the three crg1-disrupted mutants. Taken together, we conclude that the 4.5-kb transcript is a dicistronic mRNA of both CRG1 and PUT1 in the fungus C. nicotianae. This is the first example of a dicistronic mRNA identified in filamentous fungi.}, number={6}, journal={CURRENT GENETICS}, author={Chung, KR and Daub, ME and Ehrenshaft, M}, year={2003}, month={Sep}, pages={415–424} }
 @article{chung_daub_kuchler_schuller_2003, title={The CRG1 gene required for resistance to the singlet oxygen-generating cercosporin toxin in Cercospora nicotianae encodes a putative fungal transcription factor}, volume={302}, ISSN={["1090-2104"]}, DOI={10.1016/S0006-291X(03)00171-2}, abstractNote={The Cercospora nicotianae CRG1 gene is involved in cellular resistance to the perylenequinone toxin, cercosporin, that generates highly toxic singlet oxygen upon exposure to light. The entire open reading frame (ORF) of CRG1 was isolated and sequenced. The gene contains an ORF of 1950 bp including a 65-bp intron. The predicted 650 amino acid CRG1 protein contains a Cys6Zn2 binuclear cluster DNA-binding motif with homology to various fungal regulatory proteins, indicating that CRG1 may act functionally as a transcription activator. Targeted gene disruption of CRG1 resulted in mutants that are partially sensitive to cercosporin and reduced in cercosporin production. Genetic complementation revealed that CRG1 fully restored cercosporin resistance, but only slightly restored cercosporin production in a UV-derived mutant (CS10) containing a single nucleotide substitution in crg1. Complementation of a crg1-null mutant, however, yielded strains that are similar to the wild-type in both phenotypes. These results indicate that the transcription regulator CRG1 is involved in the activation of genes associated with cercosporin resistance and production in the fungus Cercospora nicotianae.}, number={2}, journal={BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, author={Chung, KR and Daub, ME and Kuchler, K and Schuller, C}, year={2003}, month={Mar}, pages={302–310} }
 @article{mitchell_alejos-gonzalez_gracz_danehower_daub_chilton_2003, title={Xanosporic acid, an intermediate in bacterial degradation of the fungal phototoxin cercosporin}, volume={62}, ISSN={["0031-9422"]}, DOI={10.1016/S0031-9422(02)00517-4}, abstractNote={The red fungal perylenequinone phototoxin cercosporin is oxidized by Xanthomonas campestris pv zinniae to a non-toxic, unstable green metabolite xanosporic acid, identified via its lactone as 1,12-bis(2'R-hydroxypropyl)-4,9-dihydroxy-6,7-methylenedioxy-11-methoxy-3-oxaperylen-10H-10-one-2-carboxylic acid. Xanosporolactone was isolated in approximately 2:1 ratio of M:P atropisomers.}, number={5}, journal={PHYTOCHEMISTRY}, author={Mitchell, TK and Alejos-Gonzalez, F and Gracz, HS and Danehower, DA and Daub, ME and Chilton, WS}, year={2003}, month={Mar}, pages={723–732} }
 @article{mitchell_chilton_daub_2002, title={Biodegradation of the polyketide toxin cercosporin}, volume={68}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.68.9.4173-4181.2002}, abstractNote={ABSTRACT}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Mitchell, TK and Chilton, WS and Daub, ME}, year={2002}, month={Sep}, pages={4173–4181} }
 @article{jennings_daub_pharr_williamson_2002, title={Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata}, volume={32}, ISSN={["0960-7412"]}, DOI={10.1046/j.1365-313X.2001.01399.x}, abstractNote={Summary}, number={1}, journal={PLANT JOURNAL}, author={Jennings, DB and Daub, ME and Pharr, DM and Williamson, JD}, year={2002}, month={Oct}, pages={41–49} }
 @article{chung_ehrenshaft_daub_2002, title={Functional expression and cellular localization of cercosporin-resistance proteins fused with the GFP in Cercospora nicotianae}, volume={41}, ISSN={["0172-8083"]}, DOI={10.1007/s00294-002-0289-8}, abstractNote={The Cercospora nicotianae pdx1 and crg1 genes were previously identified as genes required for resistance to the singlet oxygen ((1)O(2))-generating toxin cercosporin. The pdx1 gene has subsequently been shown to be required for pyridoxine biosynthesis, but both the precise biochemical function of the PDX1 protein and the function of the CRG1 protein remain undefined, as both sequences lack defined enzymatic domains or cofactor-binding sites. The gfp gene encoding green fluorescent protein was translationally fused with pdx1 and crg1. Transformation of these constructs into strains mutant in these respective genes resulted in green-fluorescent transformants complemented for the mutant phenotype. Microscopic studies revealed that in transformants transformed with gfp alone, fluorescence was distributed evenly throughout the cytoplasm and excluded from the vacuoles. Expression of PDX1::GFP either under the constitutive Aspergillus nidulans gpdA promoter or its own native promoter was visualized as distinct fluorescent circular structures in the cytoplasm, suggesting that PDX1::GFP was probably localized in the intracellular vesicles. Expression of CRG1 fused with GFP at either its N- or C-terminus resulted in low green fluorescence, compared with that of GFP alone or PDX1::GFP. The green fluorescence of either of the CRG1::GFP fusion proteins was barely observable in transformants and was generally seen as a few scattered regions of fluorescence in the hyphae. Southern blot analysis indicated multiple copies of the constructs were integrated into the fungal genome. Northern analysis revealed that pdx1:: gfp and crg1:: gfp were each expressed as an intact transcriptional unit. Cell fractionation followed by immunoblotting against a GFP antibody showed that GFP alone and PDX1::GFP were detected exclusively in the cytoplasmic fraction. The two CRG1::GFP proteins were barely detected in the cytoplasmic fraction and not at all from the membrane fraction, a result inconsistent with microscopic observation and computer sequence analysis, which suggests that CRG1 is a membrane protein.}, number={3}, journal={CURRENT GENETICS}, author={Chung, KR and Ehrenshaft, M and Daub, ME}, year={2002}, month={Jun}, pages={159–167} }
 @article{mccarthy_wehner_xie_daub_2001, title={Improving culture efficiency of Cucumis metuliferus protoplasts}, ISBN={1064-5594}, number={24}, journal={Report (Cucurbit Genetics Cooperative)}, publisher={Cucurbit Genetics Cooperative}, author={McCarthy, W. H. and Wehner, T. C. and Xie, J. H. and Daub, M. E.}, year={2001}, pages={97} }
 @article{mccarthy_wehner_xie_daub_2001, title={Isolation and callus production from cotyledon protoplasts of Cucumis metuliferus}, ISBN={1064-5594}, number={24}, journal={Report (Cucurbit Genetics Cooperative)}, publisher={Cucurbit Genetics Cooperative}, author={McCarthy, W. H. and Wehner, T. C. and Xie, J. H. and Daub, M. E.}, year={2001}, pages={102} }
 @article{ehrenshaft_daub_2001, title={Isolation of PDX2, a second novel gene in the pyridoxine biosynthesis pathway of eukaryotes, archaebacteria, and a subset of eubacteria}, volume={183}, ISSN={["0021-9193"]}, DOI={10.1128/JB.183.11.3383-3390.2001}, abstractNote={ABSTRACT}, number={11}, journal={JOURNAL OF BACTERIOLOGY}, author={Ehrenshaft, M and Daub, ME}, year={2001}, month={Jun}, pages={3383–3390} }
 @article{herrero_rufty_daub_2001, title={Molecular determinants influencing the inheritance of transgenic virus resistance in segregating tobacco families transformed with the nucleocapsid gene of tomato spotted wilt virus}, volume={7}, ISSN={["1572-9788"]}, DOI={10.1023/A:1011381412397}, number={2}, journal={MOLECULAR BREEDING}, author={Herrero, S and Rufty, RC and Daub, ME}, year={2001}, pages={131–139} }
 @article{daub_li_bilski_chignell_2000, title={Dihydrocercosporin singlet oxygen production and subcellular localization: A possible defense against cercosporin phototoxicity in Cercospora}, volume={71}, ISSN={["0031-8655"]}, DOI={10.1562/0031-8655(2000)071<0135:SIPDSO>2.0.CO;2}, abstractNote={Abstract Fungi in the genus Cercospora produce cercosporin, a potent singlet oxygen (1O2)-generating photosensitizer that plays a critical role in the ability of these fungi to parasitize plants. Although plants, mice, bacteria and many fungi are sensitive to cercosporin, Cercospora species are resistant to its toxicity. The cellular resistance of these fungi to cercosporin has been correlated with fungal cell surface reducing ability and the ability to maintain cercosporin in a chemically reduced state. As a model for reduced cercosporin we employed a reduced, acetylated derivative (hexaacetyl-dihydrocercosporin, HAC) that we tested for 1O2 production in a range of solvents. We found that as a 1O2 photosensitizer, HAC was only moderately effective in organic solvents (ϕSO = 0.14–0.18) and very poor in water (ϕSO = 0.02–0.04). By contrast, the 1O2 quantum yield of cercosporin itself was unaffected by solvent (ϕSO = 0.84–0.97). To investigate the localization of reduced cercosporin in fungal cells, we developed a fluorescence assay using laser scanning confocal microscopy. This assay showed a uniform green fluorescence, indicative of reduced cercosporin, in the cytoplasm of hyphal cells treated with cercosporin. We hypothesize that the main protection mechanism against cercosporin phototoxicity in the fungus consists of transformation of cercosporin to a reduced state and localization of this reduced form in the aqueous compartment of the cell, thus decreasing intracellular 1O2 production to levels that can be tolerated by the fungus. In addition, we have, for the first time, directly detected 1O2 phosphorescence from fungal culture, either stained with the photosensitizer rose bengal or actively synthesizing cercosporin, demonstrating 1O2 production in vivo and from cercosporin in culture.}, number={2}, journal={PHOTOCHEMISTRY AND PHOTOBIOLOGY}, author={Daub, ME and Li, M and Bilski, P and Chignell, CF}, year={2000}, month={Feb}, pages={135–140} }
 @misc{daub_ehrenshaft_jenns_2000, title={Isolated genes and proteins encoding resistance to photosensitizers}, volume={6,063,987}, number={2000 May 16}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Daub, M. E. and Ehrenshaft, M. and Jenns, A. E.}, year={2000} }
 @article{herrero_culbreath_csinos_pappu_rufty_daub_2000, title={Nucleocapsid gene-mediated transgenic resistance provides protection against Tomato spotted wilt virus epidemics in the field}, volume={90}, DOI={10.1094/phyto.2000.90.2.139}, abstractNote={ Transformation of plants with the nucleocapsid (N) gene of Tomato spotted wilt tospovirus (TSWV) provides resistance to disease development; however, information is lacking on the response of plants to natural inoculum in the field. Three tobacco cultivars were transformed with the N gene of a dahlia isolate of TSWV (TSWV-D), and plants were evaluated over several generations in the greenhouse. The resistant phenotype was more frequently observed in ‘Burley 21’ than in ‘KY-14’ or ‘K-326’, but highly resistant ‘Burley 21’ transgenic lines were resistant to only 44% of the heterologous TSWV isolates tested. Advanced generation (R3 and R4) transgenic resistant lines of ‘Burley 21’ and a ‘K-326’ F1 hybrid containing the N genes of two TSWV isolates were evaluated in the field near Tifton, GA, where TSWV is endemic. Disease development was monitored by symptom expression and enzyme-linked immunosorbent assay (ELISA) analysis. Whereas incidence of TSWV infection in ‘Burley 21’ susceptible controls was 20% in 1996 and 62% in 1997, the mean incidence in transgenic lines was reduced to 4 and 31%, respectively. Three transgenic ‘Burley 21’ lines were identified that had significantly lower incidence of disease than susceptible controls over the two years of the study. In addition, the rate of disease increase at the onset of the 1997 epidemic was reduced for all the ‘Burley 21’ transgenic lines compared with the susceptible controls. The ‘K-326’ F1 hybrid was as susceptible as the ‘K-326’ nontransformed control. ELISA analysis demonstrated that symptomless plants from the most resistant ‘Burley 21’ transgenic lines accumulated detectable nucleocapsid protein, whereas symptomless plants from more susceptible lines did not. We conclude that transgenic resistance to TSWV is effective in reducing incidence of the disease in the field, and that accumulation of transgene protein may be important in broad-spectrum resistance. }, number={2}, journal={Phytopathology}, author={Herrero, S. and Culbreath, A. K. and Csinos, A. S. and Pappu, H. R. and Rufty, R. C. and Daub, M. E.}, year={2000}, pages={139–147} }
 @misc{daub_ehrenshaft_2000, title={The photoactivated Cercospora toxin cercosporin: Contributions to plant disease and fundamental biology}, volume={38}, ISSN={["1545-2107"]}, DOI={10.1146/annurev.phyto.38.1.461}, abstractNote={ Plant pathogenic fungi in eight genera produce light-activated perylenequinone toxins that are toxic to plants via the generation of activated oxygen species, particularly singlet oxygen. Studies on the cercosporin toxin produced by Cercospora species have documented an important role for this toxin in pathogenesis of host plants. Cercosporin-generated active oxygen species destroy the membranes of host plants, providing nutrients to support the growth of these intercellular pathogens. Resistance of Cercospora species to the toxic effects of their own toxin has allowed these organisms to be used as a model for understanding the cellular basis of resistance to singlet oxygen and to general oxidative stress. In particular, the recent discovery that pyridoxine (vitamin B6) quenches singlet oxygen has led to the understanding of a novel role for this vitamin in cells as well as the discovery of a novel pathway of biosynthesis. }, journal={ANNUAL REVIEW OF PHYTOPATHOLOGY}, author={Daub, ME and Ehrenshaft, M}, year={2000}, pages={461-+} }
 @article{bilski_li_ehrenshaft_daub_chignell_2000, title={Vitamin B-6 (pyridoxine) and its derivatives are efficient singlet oxygen quenchers and potential fungal antioxidants}, volume={71}, ISSN={["1751-1097"]}, DOI={10.1562/0031-8655(2000)071<0129:SIPVBP>2.0.CO;2}, abstractNote={Abstract Vitamin B6 (pyridoxine, 1) and its derivatives: pyridoxal (2), pyridoxal 5-phosphate (3) and pyridoxamine (4) are important natural compounds involved in numerous biological functions. Pyridoxine appears to play a role in the resistance of the filamentous fungus Cercospora nicotianae to its own abundantly produced strong photosensitizer of singlet molecular oxygen (1O2), cercosporin. We measured the rate constants (kq) for the quenching of 1O2 phosphorescence by 1–4 in D2O. The respective total (physical and chemical quenching) kq values are: 5.5 × 107 M−1 s−1 for 1; 7.5 × 107 M−1 s−1 for 2, 6.2 ×107 M−1 s−1 for 3 and 7.5 × 107 M−1 s−1 for 4, all measured at pD 6.2. The quenching efficacy increased up to five times in alkaline solutions and decreased ∼10 times in ethanol. Significant contribution to total quenching by chemical reaction(s) is suggested by the degradation of all the vitamin derivatives by 1O2, which was observed as declining absorption of the pyridoxine moiety upon aerobic irradiation of RB used to photosensitize 1O2. This photodegradation was completely stopped by azide, a known physical quencher of 1O2. The pyridoxine moiety can also function as a redox quencher for excited cercosporin by forming the cercosporin radical anion, as observed by electron paramagnetic resonance. All B6 vitamers fluoresce upon UV excitation. Compounds 1 and 4 emit fluorescence at 400 nm, compound 2 at 450 nm and compound 3 at 550 nm. The fluorescence intensity of 3 increased ∼10 times in organic solvents such as ethanol and 1,2-propanediol compared to aqueous solutions, suggesting that fluorescence may be used to image the distribution of 1–4 in Cercospora to understand better the interactions of pyridoxine and 1O2 in the living fungus.}, number={2}, journal={PHOTOCHEMISTRY AND PHOTOBIOLOGY}, author={Bilski, P and Li, MY and Ehrenshaft, M and Daub, ME and Chignell, CF}, year={2000}, month={Feb}, pages={129–134} }
 @article{ehrenshaft_bilski_li_chignell_daub_1999, title={A highly conserved sequence is a novel gene involved in de novo vitamin B6 biosynthesis}, volume={96}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.96.16.9374}, abstractNote={TheCercospora nicotianae SOR1(singlet oxygen resistance) gene was identified previously as a gene involved in resistance of this fungus to singlet-oxygen-generating phototoxins. Although homologues toSOR1occur in organisms in four kingdoms and encode one of the most highly conserved proteins yet identified, the precise function of this protein has, until now, remained unknown. We show thatSOR1is essential in pyridoxine (vitamin B6) synthesis inC. nicotianaeandAspergillus flavus,although it shows no homology to previously identified pyridoxine synthesis genes identified inEscherichia coli. Sequence database analysis demonstrated that organisms encode eitherSOR1orE. colipyridoxine biosynthesis genes, but not both, suggesting that there are two divergent pathways forde novopyridoxine biosynthesis in nature. Pathway divergence appears to have occurred during the evolution of the eubacteria. We also present data showing that pyridoxine quenches singlet oxygen at a rate comparable to that of vitamins C and E, two of the most highly efficient biological antioxidants, suggesting a previously unknown role for pyridoxine in active oxygen resistance.}, number={16}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Ehrenshaft, M and Bilski, P and Li, MY and Chignell, CF and Daub, ME}, year={1999}, month={Aug}, pages={9374–9378} }
 @article{chung_jenns_ehrenshaft_daub_1999, title={A novel gene required for cercosporin toxin resistance in the fungus Cercospora nicotianae}, volume={262}, DOI={10.1007/pl00008642}, abstractNote={Cercosporin, a photosensitizing perylenequinone toxin produced by the plant pathogenic Cercospora fungi, generates the highly toxic singlet oxygen (1O2) upon exposure to light. Cercosporin shows broad toxicity against a wide range of organisms, including bacteria, fungi, plants, and animals; however, Cercospora fungi are resistant to its effects. A novel gene, crg1 (cercosporin-resistance gene) was isolated from a wild-type strain of C. nicotianae by genetic complementation of a C. nicotianae mutant (CS10) which is cercosporin sensitive and down-regulated in cercosporin production. Sequence analysis indicated that crg1 encodes a putative protein of 550 amino acids with four putative transmembrane helical regions, however CRG1 shows no strong similarity to any other protein in sequence databases. Northern analysis identified two transcripts (4.5 and 2.6 kb) that are unaffected by the presence of light or cercosporin. Southern analysis demonstrated that crg1 is present in a single copy in the C. nicotianae genome and can be detected only in Cercospora species. Targeted disruption of crg1 resulted in mutants that, like CS10, are sensitive to cercosporin. However, unlike CS10, crg1 disruption mutants are not down-regulated in toxin production. Both CS10 and the crg1 disruption mutants are unaffected in their response to other 1O2-generating photosensitizers, suggesting that CRG1 functions specifically against cercosporin, rather than against 1O2.}, number={2}, journal={Molecular and General Genetics}, author={Chung, K. R. and Jenns, A. E. and Ehrenshaft, M. and Daub, M. E.}, year={1999}, pages={382–389} }
 @article{ehrenshaft_chung_jenns_daub_1999, title={Functional characterization of SOR1, a gene required for resistance to photosensitizing toxins in the fungus Cercospora nicotianae}, volume={34}, ISSN={["0172-8083"]}, DOI={10.1007/s002940050423}, abstractNote={The Cercospora nicotianae SOR1 gene is required for resistance to singlet oxygen-generating photosensitizers. SOR1 was characterized in the wild-type and in five photosensitizer-sensitive mutant strains which are complemented to photosensitizer resistance by transformation with SOR1. Sequence analysis determined that three of the mutants contain SOR1 copies with mutations encoding substitutions in the protein-coding sequence; however, two other mutants had wild-type SOR1 protein and promoter sequences. All five mutants accumulate SOR1 mRNA at levels comparable to that of the wild-type strain. In the wild-type strain, SOR1 accumulation is enhanced two-fold by light, but is unaffected by the presence of cercosporin, the photosensitizer synthesized by C. nicotianae. Southern analysis indicates that SOR1 is present in other fungi that synthesize structurally related perylenequinone photosensitizers.}, number={6}, journal={CURRENT GENETICS}, author={Ehrenshaft, M and Chung, KR and Jenns, AE and Daub, ME}, year={1999}, month={Jan}, pages={478–485} }
 @article{sherman_moyer_daub_1998, title={A regeneration and Agrobacterium-mediated transformation system for genetically diverse Chrysanthemum cultivars}, volume={123}, ISSN={["0003-1062"]}, DOI={10.21273/jashs.123.2.189}, abstractNote={An efficient, high-frequency regeneration and Agrobacterium-mediated transformation system was developed allowing the genetic engineering of three chrysanthemum (Dendranthema grandiflora Tzvelev) cultivars: the formerly recalcitrant and economically important cut-flower mum `Polaris' and two potted mums, `Hekla' and `Iridon'. The regeneration protocol used leaf explants on a sequence of media with four hormone regimes. Explants were first cultured on an embryogenesis-type medium containing a high concentration of 2,4-D, which promoted callus formation. Shoot primordia were induced by culture on medium lacking 2,4-D, followed by shoot elongation on a high-cytokinin plus gibberellic acid medium. Finally, elongated shoots were rooted on a low-auxin rooting medium. Transformed plants of the three cultivars were obtained following co-culture of leaf explants with A. tumefaciens strain EHA 105 harboring the plasmid pBI121 containing genes for neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS). Stable transformation of the three cultivars was verified via GUS assays and Southern analysis.}, number={2}, journal={JOURNAL OF THE AMERICAN SOCIETY FOR HORTICULTURAL SCIENCE}, author={Sherman, JM and Moyer, JW and Daub, ME}, year={1998}, month={Mar}, pages={189–194} }
 @article{daub_ehrenshaft_jenns_chung_1998, title={Active oxygen in fungal pathogenesis of plants: the role of cercosporin in Cercospora diseases}, volume={32}, number={1998}, journal={Recent Advances in Phytochemistry}, author={Daub, M. E. and Ehrenshaft, M. and Jenns, A. E. and Chung, K. R.}, year={1998}, pages={31–56} }
 @article{ehrenshaft_jenns_chung_daub_1998, title={SOR1, a gene required for photosensitizer and singlet oxygen resistance in Cercospora fungi, is highly conserved in divergent organisms}, volume={1}, ISSN={["1097-2765"]}, DOI={10.1016/S1097-2765(00)80060-X}, abstractNote={Filamentous Cercospora fungi are resistant to photosensitizing compounds that generate singlet oxygen. C. nicotianae photosensitizer-sensitive mutants were restored to full resistance by transformation with SOR1 (Singlet Oxygen Resistance 1), a gene recovered from a wild-type genomic library. SOR1 null mutants generated via targeted gene replacement confirmed the requirement for SOR1 in photosensitizer resistance. SOR1 RNA is present throughout the growth cycle. Although resistance to singlet oxygen is rare in biological systems, SOR1, a gene with demonstrated activity against singlet-oxygen-generating photosensitizers, is highly conserved in organisms from widely diverse taxa. The characterization of SOR1 provides an additional phenotype to this large group of evolutionarily conserved genes.}, number={4}, journal={MOLECULAR CELL}, author={Ehrenshaft, M and Jenns, AE and Chung, KR and Daub, ME}, year={1998}, month={Mar}, pages={603–609} }
 @article{sherman_moyer_daub_1998, title={Tomato spotted wilt virus resistance in chrysanthemum expressing the viral nucleocapsid gene}, volume={82}, ISSN={["0191-2917"]}, DOI={10.1094/PDIS.1998.82.4.407}, abstractNote={ Three tomato spotted wilt virus (TSWV) nucleocapsid (N) gene constructs were employed for Agrobacterium-mediated transformation of chrysanthemum (Dendranthema grandiflora) cv. Polaris. These constructs contained either a full-length N gene (pTSWVN+), a full-length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the full-length N gene (pTSWVN-), all derived from a dahlia isolate of TSWV (TSWV-D). Initial resistance screens were conducted on cuttings made from 152 pTSWVN+, 37 pTSWVNt, and 47 pTSWVN- transformed plants employing a highly virulent, heterologous strain of TSWV (TSWV-GB) isolated from chrysanthemum and vectored by thrips. This screening served to eliminate the majority of TSWV-susceptible transgenic lines. More rigorous resistance tests with three rounds of mechanical inoculation with TSWV-GB identified one pTSWVNt and two pTSWVN- transformed lines that exhibited a total lack of systemic symptoms and no virus accumulation. Six other lines, including some pTSWVN+, exhibited a lack of one or more of the destructive necrotic TSWV symptoms (stem canker and apical bud death) and a delay in symptom expression. Both sense and antisense constructs, therefore, were found to be effective at yielding TSWV resistance in chrysanthemum. Molecular analysis revealed that the highly TSWV-resistant pTSWVNt line had no detectable levels of N protein. All three resistant lines had low levels of N gene transcript and at least three transgene insertion sites within their genomes, although susceptible lines often had a similar number of insertion sites. The generation of Polaris lines resistant to TSWV transmitted either mechanically or by thrips represents the first time a major ornamental crop has been genetically engineered for disease resistance. }, number={4}, journal={PLANT DISEASE}, author={Sherman, JM and Moyer, JW and Daub, ME}, year={1998}, month={Apr}, pages={407–414} }
 @inbook{daub_jones_moyer_1997, title={Biotechnological approaches for virus resistance in floral crops}, booktitle={Biotechnology of ornamental plants}, publisher={Wallingford: CAB International}, author={Daub, M. E. and Jones, R. K. and Moyer, J. W.}, editor={R. L. Geneve, J. E. Preece and Merkle, S. A.Editors}, year={1997}, pages={335–351} }
 @inbook{daub_ehrenshaft_1997, title={The photoactivated toxin cercosporin: Toxicity, resistance, regulation and role in disease}, ISBN={1886106754}, booktitle={Toxins in plant disease development and evolving biotechnology}, publisher={Enfield, N.H., U.S.A. : Science Publishers, Inc}, author={Daub, M. E. and Ehrenshaft, M.}, editor={R. K. Upadhyay and Mukerji, K. G.Editors}, year={1997}, pages={37} }
 @article{daughtrey_jones_moyer_daub_baker_1997, title={Tospoviruses strike the greenhouse industry - INSV has become a major pathogen on flower crops}, volume={81}, ISSN={["0191-2917"]}, DOI={10.1094/PDIS.1997.81.11.1220}, abstractNote={HomePlant DiseaseVol. 81, No. 11Tospoviruses Strike the Greenhouse Industry: INSV Has Become a Major Pathogen on Flower Crops PreviousNext OPENOpen Access licenseTospoviruses Strike the Greenhouse Industry: INSV Has Become a Major Pathogen on Flower CropsMargery L. Daughtrey, Ronald K. Jones, James W. Moyer, Margaret E. Daub, and James R. BakerMargery L. DaughtreySearch for more papers by this author, Ronald K. JonesSearch for more papers by this author, James W. MoyerSearch for more papers by this author, Margaret E. DaubSearch for more papers by this author, and James R. BakerSearch for more papers by this authorAffiliationsAuthors and Affiliations Margery L. Daughtrey , L. I. Horticultural Research Laboratory, Cornell University, Riverhead, NY Ronald K. Jones James W. Moyer Margaret E. Daub , Department of Plant Pathology, North Carolina State University, Raleigh James R. Baker , Department of Entomology, North Carolina State University, Raleigh Published Online:22 Feb 2007https://doi.org/10.1094/PDIS.1997.81.11.1220AboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat DetailsFiguresLiterature CitedRelated Vol. 81, No. 11 November 1997SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 25 Jan 2008Published: 22 Feb 2007 Pages: 1220-1230 Information© 1997 The American Phytopathological SocietyPDF downloadCited byMolecular characterization and incidence of new tospovirus: Soybean Vein Necrosis Virus (SVNV) in Egypt1 January 2024 | Brazilian Journal of Biology, Vol. 84Soğuk Sıcaklıkta Fosfin Fümigasyonunun Karanfil üzerindeki Frankliniella occidentalis Perg. 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 @article{daub_jenns_ehrenshaft_1995, title={Fungal resistance to photosensitizers that generate singlet oxygen}, ISBN={0841233349}, journal={Light activated pest control}, publisher={Washington, DC : American Chemical Society}, author={Daub, M. E. and Jenns, A. E. and Ehrenshaft, M.}, editor={J. R. Heitz and Downum, K. R.Editors}, year={1995}, pages={201} }
 @article{daub_ehrenshaft_1993, title={The photoactivated toxin cercosporin as a tool in fungal photobiology}, volume={89}, DOI={10.1034/j.1399-3054.1993.890133.x}, number={1}, journal={Physiologia Plantarum}, author={Daub, M. E. and Ehrenshaft, M.}, year={1993}, pages={227} }
 @article{daub_jenns_1989, title={FIELD AND GREENHOUSE ANALYSIS OF VARIATION FOR DISEASE RESISTANCE IN TOBACCO SOMACLONES}, volume={79}, ISSN={["1943-7684"]}, DOI={10.1094/Phyto-79-600}, abstractNote={Daub, M. E., and Jenns, A. E. 1989. Field and greenhouse analysis of variation for disease resistance in tobacco somaclones. Phytopathology 79:600-605. A total of 854 somaclones of two flue-cured tobacco cultivars were parent cultivar in black shank resistance, but many had greater resistance generated from protoplast cultures, and their progeny analyzed in to bacterial wilt than did NC2326. Most cultivar Coker 319 somaclones greenhouse and field tests for yield, leaf chemistry, and resistance to black were more susceptible than the parent cultivar to black shank but had shank, bacterial wilt, tobacco mosaic virus, and root knot (Meloidogyne bacterial wilt resistance similar to that of Coker 319. Variation in black incognita). Under the culture conditions established in this study, shank and bacterial wilt resistance was slight, and few somaclones had approximately 55% of the somaclones were not self-fertile. Progeny of the responses equivalent to those of the susceptible and resistant control somaclones had normal phenotype and did not differ significantly from the cultivars. However, conditions used in this study reduced the amount of parent cultivars in yield and leaf chemistry. Significant variation was found potential variation. No somaclones were identified with resistance to in resistance to black shank and bacterial wilt, two diseases for which the tobacco mosaic virus or M. incognita. We conclude that genetic variation parent cultivars have low levels of resistance. The variation that was occurred in the somaclones, that the magnitude of variation was slight, and observed in response to these diseases depended on the disease as well as the that the variation depended on both the genotype of the parent cultivar and genotype of the parent. Somaclones of cultivar NC2326 were similar to the the trait. Additional keywords: Nicotiana tabacum, Phytophthora parasitica var. nicotianae, Pseudomonas solanacearum, tissue culture.}, number={5}, journal={PHYTOPATHOLOGY}, author={DAUB, ME and JENNS, AE}, year={1989}, month={May}, pages={600–605} }