@article{crouch_beirn_boehm_carbone_clarke_kerns_malapi-wight_mitchell_venu_tredway_2021, title={Genome Resources for Seven Fungal Isolates That Cause Dollar Spot Disease in Turfgrass, Including Clarireedia jacksonii and C. monteithiana}, volume={105}, ISSN={["1943-7692"]}, DOI={10.1094/PDIS-06-20-1296-A}, abstractNote={Fungi in the genus Clarireedia are widespread and destructive pathogens of grasses worldwide, and are best known as the causal agents of dollar spot disease in turfgrass. Here, we report genome assemblies of seven Clarireedia isolates, including ex-types of the two most widespread species, Clarireedia jacksonii and C. monteithiana. These datasets provide a valuable resource for ongoing studies of the dollar spot pathogens that include population diversity, host–pathogen interactions, marker development, and disease control.}, number={3}, journal={PLANT DISEASE}, author={Crouch, Jo Anne and Beirn, Lisa A. and Boehm, Michael J. and Carbone, Ignazio and Clarke, Bruce B. and Kerns, James P. and Malapi-Wight, Martha and Mitchell, Thomas K. and Venu, R. C. and Tredway, Lane P.}, year={2021}, month={Mar}, pages={691–694} }
 @article{nunes_gowda_sailsbery_xue_chen_brown_oh_mitchell_dean_2011, title={Diverse and tissue-enriched small RNAs in the plant pathogenic fungus, Magnaporthe oryzae}, volume={12}, DOI={10.1186/1471-2164-12-288}, abstractNote={Abstract Background Emerging knowledge of the impact of small RNAs as important cellular regulators has prompted an explosion of small transcriptome sequencing projects. Although significant progress has been made towards small RNA discovery and biogenesis in higher eukaryotes and other model organisms, knowledge in simple eukaryotes such as filamentous fungi remains limited. Results Here, we used 454 pyrosequencing to present a detailed analysis of the small RNA transcriptome (~ 15 - 40 nucleotides in length) from mycelia and appressoria tissues of the rice blast fungal pathogen, Magnaporthe oryzae . Small RNAs mapped to numerous nuclear and mitochondrial genomic features including repetitive elements, tRNA loci, rRNAs, protein coding genes, snRNAs and intergenic regions. For most elements, small RNAs mapped primarily to the sense strand with the exception of repetitive elements to which small RNAs mapped in the sense and antisense orientation in near equal proportions. Inspection of the small RNAs revealed a preference for U and suppression of C at position 1, particularly for antisense mapping small RNAs. In the mycelia library, small RNAs of the size 18 - 23 nt were enriched for intergenic regions and repetitive elements. Small RNAs mapping to LTR retrotransposons were classified as LTR retrotransposon-siRNAs (LTR-siRNAs). Conversely, the appressoria library had a greater proportion of 28 - 35 nt small RNAs mapping to tRNA loci, and were classified as tRNA-derived RNA fragments (tRFs). LTR-siRNAs and tRFs were independently validated by 3' RACE PCR and northern blots, respectively. Conclusions Our findings suggest M. oryzae small RNAs differentially accumulate in vegetative and specialized-infection tissues and may play an active role in genome integrity and regulating growth and development.}, journal={BMC Genomics}, author={Nunes, C. C. and Gowda, M. and Sailsbery, J. and Xue, M. F. and Chen, F. and Brown, D. E. and Oh, Y. and Mitchell, T. K. and Dean, Ralph}, year={2011} }
 @misc{meng_brown_ebbole_torto-alalibo_oh_deng_mitchell_dean_2009, title={Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae}, volume={9}, ISSN={["1471-2180"]}, DOI={10.1186/1471-2180-9-s1-s8}, abstractNote={Abstract Background Magnaporthe oryzae , the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html . However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. Methods A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. Results In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi . Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html . The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. Conclusion Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae .}, journal={BMC MICROBIOLOGY}, author={Meng, Shaowu and Brown, Douglas E. and Ebbole, Daniel J. and Torto-Alalibo, Trudy and Oh, Yeon Yee and Deng, Jixin and Mitchell, Thomas K. and Dean, Ralph A.}, year={2009}, month={Feb} }
 @article{craven_velez_cho_lawrence_mitchell_2008, title={Anastomosis is required for virulence of the fungal necrotroph Alternaria brassicicola}, volume={7}, ISSN={["1535-9786"]}, DOI={10.1128/EC.00423-07}, abstractNote={ABSTRACT A fungal mycelium is typically composed of radially extending hyphal filaments interconnected by bridges created through anastomoses. These bridges facilitate the dissemination of nutrients, water, and signaling molecules throughout the colony. In this study, we used targeted gene deletion and nitrate utilization mutants of the cruciferous pathogen Alternaria brassicicola and two closely related species to investigate hyphal fusion (anastomosis) and its role in the ability of fungi to cause disease. All eight of the A. brassicicola isolates tested, as well as A. mimicula and A. japonica , were capable of self-fusion, with two isolates of A. brassicicola being capable of non-self-fusion. Disruption of the anastomosis gene homolog ( Aso1 ) in A. brassicicola resulted in both the loss of self-anastomosis and pathogenicity on cabbage. This finding, combined with our discovery that a previously described nonpathogenic A. brassicicola mutant defective for a mitogen-activated protein kinase gene ( amk1 ) also lacked the capacity for self-anastomosis, suggests that self-anastomosis is associated with pathogenicity in A. brassicicola .}, number={4}, journal={EUKARYOTIC CELL}, author={Craven, Kelly D. and Velez, Heriberto and Cho, Yangrae and Lawrence, Christopher B. and Mitchell, Thomas K.}, year={2008}, month={Apr}, pages={675–683} }
 @misc{oh_donofrio_pan_coughlan_brown_meng_mitchell_dean_2008, title={Transcriptome analysis reveals new insight into appressorium formation and function in the rice blast fungus Magnaporthe oryzae}, volume={9}, ISSN={["1474-760X"]}, DOI={10.1186/gb-2008-9-5-r85}, abstractNote={Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by this and many other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. The molecular processes regulating appressorium formation are incompletely understood. We analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (approximately 21%) genes showed differential expression, with the majority being up-regulated. During appressorium formation, 357 genes were differentially expressed in response to both stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into Gene Ontology categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection. We present the first comprehensive genome-wide transcript profile study and functional analysis of infection structure formation by a fungal plant pathogen. Our data provide novel insight into the underlying molecular mechanisms that will directly benefit efforts to identify fungal pathogenicity factors and aid the development of new disease management strategies.}, number={5}, journal={GENOME BIOLOGY}, author={Oh, Yeonyee and Donofrio, Nicole and Pan, Huaqin and Coughlan, Sean and Brown, Douglas E. and Meng, Shaowu and Mitchell, Thomas and Dean, Ralph A.}, year={2008} }
 @article{meng_patel_heist_betts_tucker_galadima_donofrio_brown_mitchell_li_et al._2007, title={A systematic analysis of T-DNA insertion events in Magnaporthe oryzae}, volume={44}, ISSN={["1096-0937"]}, DOI={10.1016/j.fgb.2007.04.002}, abstractNote={We describe here the analysis of random T-DNA insertions that were generated as part of a large-scale insertional mutagenesis project for Magnaporthe oryzae. Chromosomal regions flanking T-DNA insertions were rescued by inverse PCR, sequenced and used to search the M. oryzae genome assembly. Among the 175 insertions for which at least one flank was rescued, 137 had integrated in single-copy regions of the genome, 17 were in repeated sequences, one had no match to the genome, and the remainder were unassigned due to illegitimate T-DNA integration events. These included in order of abundance: head-to-tail tandem insertions, right border excision failures, left border excision failures and insertion of one T-DNA into another. The left borders of the T-DNA were frequently truncated and inserted in sequences with micro-homology to the left terminus. By contrast the right borders were less prone to degradation and appeared to have been integrated in a homology-independent manner. Gross genome rearrangements rarely occurred when the T-DNAs integrated in single-copy regions, although most insertions did cause small deletions at the target site. Significant insertion bias was detected, with promoters receiving two times more T-DNA hits than expected, and open reading frames receiving three times fewer. In addition, we found that the distribution of T-DNA inserts among the M. oryzae chromosomes was not random. The implications of these findings with regard to saturation mutagenesis of the M. oryzae genome are discussed.}, number={10}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Meng, Yan and Patel, Gayatri and Heist, Melanie and Betts, Melania F. and Tucker, Sara L. and Galadima, Natalia and Donofrio, Nicole M. and Brown, Doug and Mitchell, Thomas K. and Li, Lei and et al.}, year={2007}, month={Oct}, pages={1050–1064} }
 @article{betts_tucker_galadima_meng_patel_li_donofrio_floyd_nolin_brown_et al._2007, title={Development of a high throughput transformation system for insertional mutagenesis in Magnaporthe oryzae}, volume={44}, ISSN={["1087-1845"]}, DOI={10.1016/j.fgb.2007.05.001}, abstractNote={Towards the goal of disrupting all genes in the genome of Magnaporthe oryzae and identifying their function, a collection of >55,000 random insertion lines of M. oryzae strain 70-15 were generated. All strains were screened to identify genes involved in growth rate, conidiation, pigmentation, auxotrophy, and pathogenicity. Here, we provide a description of the high throughput transformation and analysis pipeline used to create our library. Transformed lines were generated either by CaCl2/PEG treatment of protoplasts with DNA or by Agrobacterium tumefaciens-mediated transformation (ATMT). We describe the optimization of both approaches and compare their efficiency. ATMT was found to be a more reproducible method, resulting in predominantly single copy insertions, and its efficiency was high with up to 0.3% of conidia being transformed. The phenotypic data is accessible via a public database called MGOS and all strains are publicly available. This represents the most comprehensive insertional mutagenesis analysis of a fungal pathogen.}, number={10}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Betts, Melania F. and Tucker, Sara L. and Galadima, Natalia and Meng, Yan and Patel, Gayatri and Li, Lei and Donofrio, Nicole and Floyd, Anna and Nolin, Shelly and Brown, Doug and et al.}, year={2007}, month={Oct}, pages={1035–1049} }
 @article{torto-alalibo_tripathy_smith_arredondo_zhou_li_chibucos_qutob_gijzen_mao_et al._2007, title={Expressed sequence tags from Phytophthora sojae reveal genes specific to development and infection}, volume={20}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI-20-7-0781}, abstractNote={Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.}, number={7}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Torto-Alalibo, Trudy A. and Tripathy, Sucheta and Smith, Brian M. and Arredondo, Felipe D. and Zhou, Lecong and Li, Hua and Chibucos, Marcus C. and Qutob, Dinah and Gijzen, Mark and Mao, Chunhong and et al.}, year={2007}, month={Jul}, pages={781–793} }
 @article{cho_cramer_kim_davis_mitchell_figuli_pryor_lemasters_lawrence_2007, title={The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola}, volume={44}, ISSN={["1087-1845"]}, DOI={10.1016/j.fgb.2006.11.015}, abstractNote={Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway.}, number={6}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Cho, Yangrae and Cramer, Robert A., Jr. and Kim, Kwang-Hyung and Davis, Josh and Mitchell, Thomas K. and Figuli, Patricia and Pryor, Barry M. and Lemasters, Emily and Lawrence, Christopher B.}, year={2007}, month={Jun}, pages={543–553} }
 @article{jeong_mitchell_dean_2007, title={The Magnaporthe grisea snodprot1 homolog, MSPI, is required for virulence}, volume={273}, ISSN={["1574-6968"]}, DOI={10.1111/j.1574-6968.2007.00796.x}, abstractNote={Secreted proteins play central roles in plant-microbe interactions acting as signals, toxins, and effectors. One important group of small secreted proteins is the snodprot1 family, members of which have demonstrated phytotoxic properties. A split-marker transformation system was applied for gene deletion of the snodprot1 homolog, MSP1, in the rice blast fungus Magnaporthe grisea. msp1 mutants were phenotypically indistinguishable from wild type and elaborated apparently normal appressoria. However, the deletion mutants were greatly reduced in virulence primarily due to impaired growth in planta. Western blot analysis showed that the protein was secreted and not associated with the fungal cell wall. When purified MSP1 protein was applied to wounded leaf tissue, no apparent phytotoxic effects were noted. This is the first report to the authors' knowledge that directly implicates a snodprot1 protein as a virulence factor.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Jeong, Jun Seop and Mitchell, Thomas K. and Dean, Ralph A.}, year={2007}, month={Aug}, pages={157–165} }
 @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 The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora . The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora -resistant plants by using a suite of genes.}, 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} }
 @article{cramer_la rota_cho_thon_craven_knudson_mitchell_lawrence_2006, title={Bioinformatic analysis of expressed sequence tags derived from a compatible Alternaria brassicicola-Brassica oleracea interaction}, volume={7}, ISSN={["1364-3703"]}, DOI={10.1111/J.1364-3703.2006.00324.X}, abstractNote={SUMMARY Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on members of the Brassicaceae plant family. In order to identify candidate fungal pathogenicity genes and characterize a compatible host response, a suppression subtractive hybridization (SSH) cDNA library enriched for A. brassicicola and Brassica oleracea genes expressed during the interaction was created, along with a fungal cDNA library representing genes expressed during nitrogen starvation (NS). A total of 3749 and 2352 expressed sequence tags (ESTs) were assembled into 2834 and 1264 unisequence sets for the SSH and NS libraries, respectively. We compared two methods to identify the origins (plant vs. fungal) of ESTs in the SSH library using different classification procedures, with and without the availability of a database representing the A. brassicicola whole genome sequence and Brassicaceae-specific genes. BLASTX analyses of the 2834 unisequence set using the GenBank non-redundant database identified 114 fungal genes. Further BLASTN analyses of the genes with unidentifiable origin using a database consisting of the 1264 fungal unisequence set from the nitrogen-starved library identified 94 additional fungal genes. By contrast, BLASTN analyses of the same SSH unisequence set using a partially assembled A. brassicicola whole genome draft sequence identified a total of 310 unisequenes of fungal origin. Our results indicated that even a small number of organism-specific EST sequences can be very helpful to identify pathogen genes in a library derived from infected tissue, partially overcoming the limitation of the public databases for little studied organisms. However, using the whole genome draft sequence of A. brassicicola we were able to identify approximately 30% more fungal genes in the SSH library than without utilizing this resource. The putative role of specific fungal and plant genes identified in this study in a compatible interaction is discussed.}, number={2}, journal={MOLECULAR PLANT PATHOLOGY}, author={Cramer, RA and La Rota, CM and Cho, Y and Thon, M and Craven, KD and Knudson, DL and Mitchell, TK and Lawrence, CB}, year={2006}, month={Mar}, pages={113–124} }
 @article{donofrio_oh_lundy_pan_brown_jeong_coughlan_mitchell_dean_2006, title={Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea}, volume={43}, ISSN={["1087-1845"]}, DOI={10.1016/j.fgb.2006.03.005}, abstractNote={Efficient regulation of nitrogen metabolism likely plays a role in the ability of fungi to exploit ecological niches. To learn about regulation of nitrogen metabolism in the rice blast pathogen Magnaporthe grisea, we undertook a genome-wide analysis of gene expression under nitrogen-limiting conditions. Five hundred and twenty genes showed increased transcript levels at 12 and 48 h after shifting the fungus to media lacking nitrate as a nitrogen source. Thirty-nine of these genes have putative functions in amino acid metabolism and uptake, and include the global nitrogen regulator in M. grisea, NUT1. Evaluation of seven nitrogen starvation-induced genes revealed that all were expressed during rice infection. Targeted gene replacement on one such gene, the vacuolar serine protease, SPM1, resulted in decreased sporulation and appressorial development as well as a greatly attenuated ability to cause disease. Data are discussed in the context of nitrogen metabolism under starvation conditions, as well as conditions potentially encountered during invasive growth in planta.}, number={9}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Donofrio, N. M. and Oh, Y. and Lundy, R. and Pan, H. and Brown, D. E. and Jeong, J. S. and Coughlan, S. and Mitchell, T. K. and Dean, R. A.}, year={2006}, month={Sep}, pages={605–617} }
 @article{walker_mitchell_marek_2006, title={Influence of temperature and time of year on colonization of bermudagrass roots by Ophiosphaerella herpotricha}, volume={90}, ISSN={["1943-7692"]}, DOI={10.1094/PD-90-1326}, abstractNote={The influence of temperature on the infection of bermudagrass seedlings by Ophiosphaerella herpotricha and colonization of plants in the field was investigated. Bermudagrass seedlings (cv. Jackpot) inoculated with O. herpotricha exhibited dark lesions after 8 days. Root lesion length was greatest at 17°C and was similar for all temperatures examined below 21°C. Seedlings grown at 25 or 30°C had small lesions that remained similar in size when evaluated at 8 and 10 days post inoculation. Colonization of bermudagrass roots from field plots were examined in July, October, and November of 2003 and 2004. In 2003, no differences between sampling dates were observed for plants sampled from the edge of the spring patch in 5.4-cm increments to a total distance of 21.6 cm. In 2004, July and October samples were similar; however, an increase in root colonization was found between the October and November samplings. These studies suggest that infection and colonization of bermudagrass roots by O. herpotricha occurs over a wide range of cool soil temperatures, occurs in the spring, and can be variable in the autumn.}, number={10}, journal={PLANT DISEASE}, author={Walker, N. R. and Mitchell, T. K. and Marek, S. M.}, year={2006}, month={Oct}, pages={1326–1330} }
 @article{soderlund_haller_pampanwar_ebbole_farman_orbach_wang_wing_xu_brown_et al._2006, title={MGOS: A resource for studying Magnaporthe grisea and Oryza sativa interactions}, volume={19}, ISSN={["1943-7706"]}, DOI={10.1094/MPMI-19-1055}, abstractNote={The MGOS (Magnaporthe grisea Oryza sativa) web-based database contains data from Oryza sativa and Magnaporthe grisea interaction experiments in which M. grisea is the fungal pathogen that causes the rice blast disease. In order to study the interactions, a consortium of fungal and rice geneticists was formed to construct a comprehensive set of experiments that would elucidate information about the gene expression of both rice and M. grisea during the infection cycle. These experiments included constructing and sequencing cDNA and robust long-serial analysis gene expression libraries from both host and pathogen during different stages of infection in both resistant and susceptible interactions, generating >50,000 M. grisea mutants and applying them to susceptible rice strains to test for pathogenicity, and constructing a dual O. sativa-M. grisea microarray. MGOS was developed as a central web-based repository for all the experimental data along with the rice and M. grisea genomic sequence. Community-based annotation is available for the M. grisea genes to aid in the study of the interactions.}, number={10}, journal={MOLECULAR PLANT-MICROBE INTERACTIONS}, author={Soderlund, Carol and Haller, Karl and Pampanwar, Vishal and Ebbole, Daniel and Farman, Mark and Orbach, Marc J. and Wang, Guo-Liang and Wing, Rod and Xu, Jin-Rong and Brown, Doug and et al.}, year={2006}, month={Oct}, pages={1055–1061} }
 @article{thon_pan_diener_papalas_taro_mitchell_dean_2006, title={The  role of transposable element clusters in genome evolution and loss of synteny in the rice blast fungus Magnaporthe oryzae}, volume={7}, number={2}, journal={Genome Biology}, author={Thon, M. R. and Pan, H. Q. and Diener, S. and Papalas, J. and Taro, A. and Mitchell, T. K. and Dean, R. A.}, year={2006} }
 @article{craven_peterson_windham_mitchell_martin_2005, title={Molecular identification of the turf grass rapid blight pathogen}, volume={97}, ISSN={["1557-2536"]}, DOI={10.3852/mycologia.97.1.160}, abstractNote={Rapid blight is a newly described disease on turf grasses, primarily found on golf courses using suboptimal water for irrigation purposes. On the basis of shared morphological characteristics, it has been proposed that the rapid blight pathogen belongs to a genus of stramenopiles, Labyrinthula, which had been known to cause disease of marine plants only. We have collected 10 isolates from four species of turf grass in five states and sequenced portions of the SSU (18S) rDNA gene from each to provide a definitive taxonomic placement for rapid blight pathogens. We also included sequences from Labyrinthuloides yorkensis, Schizochytrium aggregatum, Aplanochytrium sp., Thraustochytrium striatum, Achlya bisexualis and several nonturf-grass isolates of Labyrinthula. We found that rapid blight isolates indeed are placed firmly within the genus Labyrinthula and that they lack detectable genetic diversity in the 18S rDNA region. We propose that the rapid blight pathogens share a recent common ancestor and might have originated from a single, infected population.}, number={1}, journal={MYCOLOGIA}, author={Craven, KD and Peterson, PD and Windham, DE and Mitchell, TK and Martin, SB}, year={2005}, pages={160–166} }
 @article{donofrio_rajagopalon_brown_diener_windham_nolin_floyd_mitchell_galadima_tucker_et al._2005, title={PACLIMS: A component LIM system for high-throughput functional genomic analysis}, volume={6}, journal={BMC Bioinformatics}, author={Donofrio, N. and Rajagopalon, R. and Brown, D. and Diener, S. and Windham, D. and Nolin, S. and Floyd, A. and Mitchell, T. and Galadima, N. and Tucker, S. and et al.}, year={2005} }
 @article{dean_talbot_ebbole_farman_mitchell_orbach_thon_kulkarni_xu_pan_et al._2005, title={The genome sequence of the rice blast fungus Magnaporthe grisea}, volume={434}, ISSN={["1476-4687"]}, DOI={10.1038/nature03449}, abstractNote={Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.}, number={7036}, journal={NATURE}, author={Dean, RA and Talbot, NJ and Ebbole, DJ and Farman, ML and Mitchell, TK and Orbach, MJ and Thon, M and Kulkarni, R and Xu, JR and Pan, HQ and et al.}, year={2005}, month={Apr}, pages={980–986} }
 @article{diener_dunn-coleman_foreman_houfek_teunissen_solingen_dankmeyer_mitchell_ward_dean_2004, title={Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei}, volume={230}, ISSN={["1574-6968"]}, DOI={10.1016/S0378-1097(03)00916-9}, abstractNote={Trichoderma reesei is a filamentous fungus widely used as an efficient protein producer and known to secrete large quantities of biomass degrading enzymes. Much work has been done aimed at improving the secretion efficiency of this fungus. It is generally accepted that the major bottlenecks in secretion are protein folding and ornamentation steps in this pathway. In an attempt to identify genes involved in these steps, the 5' ends of 21888 cDNA clones were sequenced from which a unique set of over 5000 were also 3' sequenced. Using annotation tools Gene Ontology terms were assigned to 2732 of the sequences. Homologs to the majority of Aspergillus niger's Srg genes as well as a number of homologs to genes involved in protein folding and ornamentation pathways were identified.}, number={2}, journal={FEMS MICROBIOLOGY LETTERS}, author={Diener, SE and Dunn-Coleman, N and Foreman, P and Houfek, TD and Teunissen, PJM and Solingen, P and Dankmeyer, L and Mitchell, TK and Ward, M and Dean, RA}, year={2004}, month={Jan}, pages={275–282} }
 @article{diener_dunn-coleman_foreman_houfek_teunissen_solingen_dankmeyer_mitchell_ward_dean_2004, title={Characterization of the protein processing and secretion pathways in a comprehensive set of expressed sequence tags from Trichoderma reesei (vol 230, pg 275, 2004)}, volume={235}, DOI={10.1111/j.1574-6968.2004.tb09588.x}, number={1}, journal={FEMS Microbiology Letters}, author={Diener, S. E. and Dunn-Coleman, N. and Foreman, P. and Houfek, T. D. and Teunissen, P. J. M. and Solingen, P. Van and Dankmeyer, L. and Mitchell, T. K. and Ward, M. and Dean, Ralph}, year={2004}, pages={209} }
 @article{diener_chellappan_mitchell_dunn-coleman_ward_dean_2004, title={Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization}, volume={41}, ISSN={["1096-0937"]}, DOI={10.1016/j.fgb.2004.08.007}, abstractNote={Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei’s secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.}, number={12}, journal={FUNGAL GENETICS AND BIOLOGY}, author={Diener, SE and Chellappan, MK and Mitchell, TK and Dunn-Coleman, N and Ward, M and Dean, RA}, year={2004}, month={Dec}, pages={1077–1087} }
 @article{takano_choi_mitchell_okuno_dean_2003, title={Large scale parallel analysis of gene expression during infection-related morphogenesis of Magnaporthe grisea}, volume={4}, ISSN={["1464-6722"]}, DOI={10.1046/J.1364-3703.2003.00182.X}, abstractNote={SUMMARY The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice. To initiate the infection of host tissues, conidia elaborate germ tubes that differentiate specialized infection structures called appressoria. Microarrays composed of 3500 cDNAs of M. grisea were prepared for the identification of genes that are specifically up‐ or down‐regulated during appressorium formation. Gene expression in ungerminated conidia, during appressorium formation, and during mycelial growth was investigated with a novel highly sensitive dendrimer based detection system. Transcripts of 85 different genes were found to be more abundant in ungerminated conidia and/or in conidia with developing appressoria than in vegetative mycelia. Nineteen of these showed higher expression in both ungerminated conidia and developing appressoria than in mycelia, suggesting that their expression remains elevated during the early stage of fungal infection. The expression of 18 genes was higher in ungerminated conidia than in developing appressoria, indicating their possible role in the germination process or maintaining dormancy. Transcripts of 47 genes were found to be more abundant in developing appressoria than in ungerminated conidia, suggesting that their expression is induced during appressorium formation. Several of these genes, including a chitin binding protein and infection structure specific protein MIF23, were previously shown to be preferentially expressed during appressorium formation. However, the expression of many of these genes has not been reported prior to this analysis. In contrast, transcripts of 38 different genes were found to be more abundant in mycelia than in developing appressoria. A Northern blot analysis of selected genes was consistent with the microarray results. Results from this study provide a powerful resource for furthering our understanding of gene expression during infection‐related morphogenesis and for the functional analysis of M. grisea genes involved in fungal infection.}, number={5}, journal={MOLECULAR PLANT PATHOLOGY}, author={Takano, Y and Choi, WB and Mitchell, TK and Okuno, T and Dean, RA}, year={2003}, month={Sep}, pages={337–346} }
 @misc{mitchell_thon_jeong_brown_deng_dean_2003, title={The rice blast pathosystem as a case study for the development of new tools and raw materials for genome analysis of fungal plant pathogens}, volume={159}, ISSN={["0028-646X"]}, DOI={10.1046/j.1469-8137.2003.00787.x}, abstractNote={Summary Fungi have an astounding and diverse impact on this planet. While they are agents of human diseases and the cause of allergic reactions, factories for the conversion of carbon in environmental and industrially adapted systems, and potential biological weapons, their importance as plant pathogens is unparalleled. In plants alone, fungi cause tens of thousands of different diseases and are responsible for massive losses of food, fiber and forestry at an estimated annual cost of hundreds of billions of dollars. These losses are not only realized in the incomes of individual farmers and state economies, but contribute significantly to world hunger problems and issues relating to safeguarding a global food supply. Our collective understanding of how fungi, particularly plant pathogens, grow, reproduce, identify a host and cause disease is still at a formative stage. There is an equal lack of detailed knowledge about how a plant recognizes that it is being attacked and then mounts an adequate defense response. The advent of genomic technologies has given researchers an unprecedented opportunity to address these mysteries in a powerful and more holistic manner. Where the genetic revolution of only a few years ago allowed for the characterization of single genes, today's genomic technologies are facilitating the evaluation of the entire complement of genes in an organism and the discovery of the suites of genes that act during any one time or particular condition. This review will describe the recent development of tools for whole or partial genome analysis and multigenome comparisons. Th discussion focuses on the rice blast pathosystem as a case study.}, number={1}, journal={NEW PHYTOLOGIST}, author={Mitchell, TK and Thon, MR and Jeong, JS and Brown, D and Deng, JX and Dean, RA}, year={2003}, month={Jul}, pages={53–61} }
 @article{foreman_brown_dankmeyer_dean_diener_dunn-coleman_goedegebuur_houfek_england_kelley_et al._2003, title={Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei}, volume={278}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M304750200}, abstractNote={The filamentous fungus Trichoderma reesei produces and secretes profuse quantities of enzymes that act synergistically to degrade cellulase and related biomass components. We partially sequenced over 5100 random T. reesei cDNA clones. Among the sequences whose predicted gene products had significant similarity to known proteins, 12 were identified that encode previously unknown enzymes that likely function in biomass degradation. Microarrays were used to query the expression levels of each of the sequences under different conditions known to induce cellulolytic enzyme synthesis. Most of the genes encoding known and putative biomass-degrading enzymes were transcriptionally co-regulated. Moreover, despite the fact that several of these enzymes are not thought to degrade cellulase directly, they were coordinately overexpressed in a cellulase overproducing strain. A variety of additional sequences whose function could not be ascribed using the limited sequence available displayed analogous behavior and may also play a role in biomass degradation or in the synthesis of biomass-degrading enzymes. Sequences exhibiting additional regulatory patterns were observed that might reflect roles in regulation of cellulase biosynthesis. However, genes whose products are involved in protein processing and secretion were not highly regulated during cellulase induction. The filamentous fungus Trichoderma reesei produces and secretes profuse quantities of enzymes that act synergistically to degrade cellulase and related biomass components. We partially sequenced over 5100 random T. reesei cDNA clones. Among the sequences whose predicted gene products had significant similarity to known proteins, 12 were identified that encode previously unknown enzymes that likely function in biomass degradation. Microarrays were used to query the expression levels of each of the sequences under different conditions known to induce cellulolytic enzyme synthesis. Most of the genes encoding known and putative biomass-degrading enzymes were transcriptionally co-regulated. Moreover, despite the fact that several of these enzymes are not thought to degrade cellulase directly, they were coordinately overexpressed in a cellulase overproducing strain. A variety of additional sequences whose function could not be ascribed using the limited sequence available displayed analogous behavior and may also play a role in biomass degradation or in the synthesis of biomass-degrading enzymes. Sequences exhibiting additional regulatory patterns were observed that might reflect roles in regulation of cellulase biosynthesis. However, genes whose products are involved in protein processing and secretion were not highly regulated during cellulase induction. Saprophytic microorganisms produce and secrete a variety of hydrolytic enzymes, including proteases, amylases, cellulases, and hemicellulases. These enzymes degrade organic biological substrates, providing nutrients for growth and contributing to carbon recycling in nature. Recently, a great deal of attention has focused on cellulases and hemicellulases produced by these organisms because of their potential to be produced industrially and used in degradation of biomass for a number applications, most notably biofuel production (1Biely P. Tenkanen M. Harman G.E. Kubicek C.P. Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications. Vol. 2. Taylor & Francis Ltd., London1998: 25-47Google Scholar, 2Dove A. Nat. Biotechnol. 2000; 18: 490Crossref PubMed Scopus (2) Google Scholar, 3Gerngross T.U. Nat. Biotechnol. 1999; 17: 541-544Crossref PubMed Scopus (126) Google Scholar, 4Himmel M.E. Adney W.S. Baker J.O. Elander R. McMillan J.D. Nieves R.A. Sheehan J. Thomas S.R. Vinzant T.B. Zhang M. Woodward J. Saha B. Fuels and Chemicals from Biomass. American Chemical Society, Washington D. C.1997: 2-45Google Scholar, 5Mielenz J.R. Curr. Opin. Microbiol. 2001; 4: 324-329Crossref PubMed Scopus (366) Google Scholar, 6Sheehan J. Himmel M. Biotechnol. Prog. 1999; 15: 817-827Crossref PubMed Scopus (179) Google Scholar, 7Wyman C.E. Appl. Biochem. Biotechnol. 2001; 91: 5-21Crossref PubMed Scopus (85) Google Scholar). The principal component of biomass is cellulose. It consists of polymers of β-1,4-linked glucose residues that are organized into higher order fibrillar structures (8Bayer E.A. Chanzy H. Lamed R. Shoham Y. Curr. Opin. Struct. Biol. 1998; 8: 548-557Crossref PubMed Scopus (465) Google Scholar). In addition to cellulose, biomass contains large quantities of hemicelluloses, heteropolysaccharides composed of two or more monosaccharides such as d-xylose, l-arabinose, d-mannose, d-glucose, d-galactose, and 4-O-methyl-d-glucuronic acid (1Biely P. Tenkanen M. Harman G.E. Kubicek C.P. Trichoderma and Gliocladium: Enzymes, Biological Control and Commercial Applications. Vol. 2. Taylor & Francis Ltd., London1998: 25-47Google Scholar). Among the most prolific producers of biomass-degrading enzymes is the filamentous fungus Trichoderma reesei. The cellulase activity produced by T. reesei is composed of a complement of endoglucanases (EGI/Cel7B, EGII/Cel5A, EGIII/Cel12A, EGIV/Cel61A, and EGV/Cel45A) and exoglucanases (the cellobiohydrolases, CBHI/Cel7A, and CBHII/Cel6A) that act synergistically to break down cellulose to cellobiose (glycosyl β-1,4-glucose) (9Okada H. Tada K. Sekiya T. Yokoyama K. Takahashi A. Tohda H. Kumagai H. Morikawa Y. Appl. Environ. Microbiol. 1998; 64: 555-563Crossref PubMed Google Scholar, 10Saloheimo M. Lehtovaara P. Penttila M. Teeri T.T. Stahlberg J. Johansson G. Pettersson G. Claeyssens M. Tomme P. Knowles J.K. Gene (Amst.). 1988; 63: 11-22Crossref PubMed Scopus (266) Google Scholar, 11Teeri T.T. Lehtovaara P. Kauppinen S. Salovuori I. Knowles J. Gene (Amst.). 1987; 51: 43-52Crossref PubMed Scopus (272) Google Scholar, 12Ward M. Wu S. Dauberman J. Weiss G. Larenas E. Bower B. Rey M. Clarkson K. Bott R. Aubert J.-P. Beguin P. Millet J. Biochemistry and Genetics of Cellulose Degradation. Academic Press, New York1993: 53-70Google Scholar, 13Saloheimo M. Nakari-Setala T. Tenkanen M. Penttila M. Eur. J. Biochem. 1997; 249: 584-591Crossref PubMed Scopus (160) Google Scholar, 14Saloheimo A. Henrissat B. Hoffren A.M. Teleman O. Penttila M. Mol. Microbiol. 1994; 13: 219-228Crossref PubMed Scopus (150) Google Scholar). Two β-glucosidases (BGLI/Cel3A and BGLII/Cel1A) have been identified that are implicated in hydrolyzing cellobiose to glucose (15Barnett C.C. Berka R.M. Fowler T. Bio/Technology. 1991; 9: 562-567Crossref PubMed Scopus (128) Google Scholar, 16Takashima S. Nakamura A. Hidaka M. Masaki H. Uozumi T. J. Biochem. (Tokyo). 1999; 125: 728-736Crossref PubMed Scopus (114) Google Scholar). An additional protein, swollenin (encoded by the gene swo1), has been described that disrupts crystalline cellulose structures, presumably making polysaccharides more accessible to hydrolysis (17Saloheimo M. Paloheimo M. Hakola S. Pere J. Swanson B. Nyyssonen E. Bhatia A. Ward M. Penttila M. Eur. J. Biochem. 2002; 269: 4202-4211Crossref PubMed Scopus (336) Google Scholar). The four most abundant components of T. reesei cellulase CBHI/Cel7A, CBHII/Cel6A, EGI/Cel7B, and EGII/Cel5A together constitute greater than 50% of the protein produced by the cell under inducing conditions and can be secreted in excess of 40 g/liter (18Durand H. Baron M. Calmels T. Tiraby G. FEMS Symp. 1998; 43: 135-152Google Scholar). The regulation of cellulolytic enzyme expression in T. reesei is complex and only partially understood. Transcription of the major components of cellulase (CBHI/Cel7A, CBHII/Cel6A, EGI/Cel7B, EGII/Cel5A, EGIII/Cel12A, EGIV/Cel61A, and EGV/Cel45A) is induced not only by cellulose but also by a variety of disaccharides including lactose, cellobiose, and sophorose (glycosyl β-1,2-glucose) (13Saloheimo M. Nakari-Setala T. Tenkanen M. Penttila M. Eur. J. Biochem. 1997; 249: 584-591Crossref PubMed Scopus (160) Google Scholar, 19Nogawa M. Goto M. Okada H. Morikawa Y. Curr. Genet. 2001; 38: 329-334Crossref PubMed Scopus (82) Google Scholar, 20Ilmen M. Saloheimo A. Onnela M.L. Penttila M.E. Appl. Environ. Microbiol. 1997; 63: 1298-1306Crossref PubMed Google Scholar). Induction by these molecules is antagonized by the presence of the preferred carbon sources, glucose and fructose. Sophorose is by far the most potent inducer of cellulase expression (21Mandels M. Parrish F.W. Reese E.T. J. Bacteriol. 1962; 83: 400-408Crossref PubMed Google Scholar). However, it is unclear whether this high potency is an innate characteristic of the molecule or whether other disaccharides are less effective because they are more readily cleaved by cellular glucosidases. The balance between intact disaccharides and inhibitory monosaccharide cleavage products might then influence the transcriptional state of these genes. In accordance with this notion both lactose and cellobiose fail to fully induce cellulase gene expression when present at high concentrations. In addition to enzymes with cellulolytic activity, a number of enzymes have been identified in T. reesei that degrade hemicellulose (17Saloheimo M. Paloheimo M. Hakola S. Pere J. Swanson B. Nyyssonen E. Bhatia A. Ward M. Penttila M. Eur. J. Biochem. 2002; 269: 4202-4211Crossref PubMed Scopus (336) Google Scholar, 22Margolles-Clark E. Tenkanen M. Nakari-Setala T. Penttila M. Appl. Environ. Microbiol. 1996; 62: 3840-3846Crossref PubMed Google Scholar, 23Margolles-Clark E. Tenkanen M. Luonteri E. Penttila M. Eur. J. Biochem. 1996; 240: 104-111Crossref PubMed Scopus (71) Google Scholar, 24Margolles-Clark E. Saloheimo M. Siika-aho M. Penttila M. Gene (Amst.). 1996; 172: 171-172Crossref PubMed Scopus (28) Google Scholar, 25Margolles-Clark E. Tenkanen M. Soderlund H. Penttila M. Eur. J. Biochem. 1996; 237: 553-560Crossref PubMed Scopus (96) Google Scholar, 26Mach R.L. Strauss J. Zeilinger S. Schindler M. Kubicek C.P. Mol. Microbiol. 1996; 21: 1273-1281Crossref PubMed Scopus (150) Google Scholar, 27Zeilinger S. Mach R.L. Schindler M. Herzog P. Kubicek C.P. J. Biol. Chem. 1996; 271: 25624-25629Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 28Xu J. Takakuwa N. Nogawa M. Okada H. Morikawa Y. Appl. Microbiol. Biotechnol. 1998; 49: 718-724Crossref Scopus (75) Google Scholar, 29Den Haan R. Van Zyl W.H. Appl. Microbiol. Biotechnol. 2001; 57: 521-527Crossref PubMed Scopus (25) Google Scholar, 30Rose S.H. van Zyl W.H. Appl. Microbiol. Biotechnol. 2002; 58: 461-468Crossref PubMed Scopus (43) Google Scholar, 31Torronen A. Mach R.L. Messner R. Gonzalez R. Kalkkinen N. Harkki A. Kubicek C.P. Bio/Technology. 1992; 10: 1461-1465Crossref PubMed Scopus (192) Google Scholar). These enzymes include four xylanases (Xyn1, Xyn2, Xyn3, and Xyn4) and mannanase (Man1), which cleave the xylan and mannan main chains of hemicellulose. Acetyl xylan esterase (Axe1), α-glucuronidase (Glr1), and arabinofuranosidase (Abf1) digest side chains containing acetyl, methylglucuronic acid, and arabinose moieties, respectively. Additionally, enzymes that digest oligosaccharides derived from hemicellulose have been identified. These are β-xylosidase (Bxl1) and three α-galactosidases (Agl1, Agl2, and Agl3). Among the hemicellulases that have been studied, all except arabinofuranosidase are expressed at a substantially higher level when T. reesei is grown in medium containing cellulose than when it is grown in medium containing non-inducing carbon sources such as sorbitol (28Xu J. Takakuwa N. Nogawa M. Okada H. Morikawa Y. Appl. Microbiol. Biotechnol. 1998; 49: 718-724Crossref Scopus (75) Google Scholar, 32Margolles-Clark E. Ilmen M. Penttila M. J. Biotechnol. 1977; 57: 167-179Crossref Scopus (140) Google Scholar). Additionally, expression of many of the genes encoding these enzymes, with the notable exception of man1, is induced by xylans. Sugars such as sophorose, arabitol, xylobiose, cellobiose, and galactose also induce expression to varying extents, particularly of enzymes that degrade substrates related to these sugars (32Margolles-Clark E. Ilmen M. Penttila M. J. Biotechnol. 1977; 57: 167-179Crossref Scopus (140) Google Scholar). The molecular mechanisms by which T. reesei senses the composition of the extracellular milieu and modulates the expression of these enzymes are unknown. Moreover, although expression of some of these genes is modified by certain substrates, it is unclear to what extent each gene has a unique regulatory apparatus and to what extent expression of these genes are coupled among themselves and with the cellulases via sharing of regulatory pathways. The mechanisms by which the cellulase and hemicellulase genes are regulated are likely to influence the ecological niches that T. reesei occupies and are of interest in the commercial production of these enzymes. The very large quantity of biomass-degrading enzymes synthesized by T. reesei requires a significant investment of cellular resources. Evidence suggests that a primary means by which the cell manages these demands is to regulate transcription of the genes encoding these enzymes according to the availability of different carbon sources. However, the degree to which gene products involved in other cellular processes, such as secretion, must be regulated to accommodate the substantial burden of cellulase biosynthesis has not been systematically investigated. In this study, aspects of these questions are addressed by determining the transcriptional effects of cellulase inducers on a genomic scale. Over 5100 cDNAs from T. reesei were partially or fully sequenced. Twelve cDNAs encoding new enzymes with putative roles in biomass degradation were discovered. Microarrays were used to examine the regulation of these and previously identified genes encoding biomass-degrading enzymes in the context of the extensive repertoire of newly identified genes. The results presented shed light on the coregulation of cellulases and hemicellulases and the mechanisms by which the cell copes with synthesizing very large quantities of these secreted enzymes. Media, Strains, and Culture Growth Conditions—T. reesei strains used in this study were obtained from the American Type Culture Collection. Liquid minimal medium was as described previously (20Ilmen M. Saloheimo A. Onnela M.L. Penttila M.E. Appl. Environ. Microbiol. 1997; 63: 1298-1306Crossref PubMed Google Scholar), except that 100 mm piperazine-N, N-bis(3-propanesulfonic acid) (Calbiochem) was included to maintain the pH at 5.5. Vogel's medium was described by Davis and DeSerres (33Davis R.H. DeSerres F.J. Methods Enzymol. 1970; 17: 79-143Crossref Scopus (930) Google Scholar). YEG medium contains 0.5% yeast extract (Difco), 2% glucose. For Northern blot analysis, ∼1 × 108 spores were inoculated into 50 ml of minimal medium supplemented with 5% glucose and grown at 30 °C for 24 h. Mycelia were collected by centrifugation, washed in carbon-free medium, and resuspended to an optical density of ∼0.3 in 50 ml of minimal medium supplemented with 5% glucose, 2% avicel (FMC), 2% glycerol, or 2% glycerol containing 1 mm sophorose (Sigma). Cultures were grown in flasks with vigorous agitation for 20 h. For microarray analysis, inoculation was performed as above. Cultures were grown in triplicate overnight. Mycelia were collected by centrifugation and washed with minimal medium containing 2% glycerol. They were then resuspended to an OD of 0.15–0.2 and grown for 9 h (OD 0.5). The cultures were then divided in half, and 0.01 volume of 100 mm sophorose was added to 1 flask of each pair immediately and 10 h later. The cultures were grown an additional 2 h to an OD ∼2–4. cDNA Library and Sequencing—T. reesei strain QM6a mycelia were grown in baffled flasks at 30 °C for 24 h in YEG medium with vigorous aeration. 5 ml of this culture was added to 50 ml of the following media and grown under the following conditions: Vogel's liquid medium, 2% avicel, 3 and 6 days; Vogel's liquid medium, 2% Solka floc (International Fiber Corp., North Tonawanda, NY), 3 and 6 days; Vogel's liquid medium, 2% wheat bran (Skidmore Sales and Distributing Co., Inc., West Chester, OH), 3 and 6 days; Vogel's liquid medium, 2% beet pulp (D&D Ingredients Distributors, Inc., Delphos, OH), 6 days; Vogel's liquid medium, 2% glucose, 24 h; Vogel's liquid medium, 2% lactose, 24 h; Vogel's liquid medium, 2% xylose, 24 h; Vogel's liquid medium, 2% fructose, 24 h; Vogel's liquid medium, 2% maltose, 24 h; Vogel's liquid medium, no carbon source added, 24 h; Vogel's liquid medium, no nitrogen source added, 24 h; Vogel's liquid medium, 2% phosphoric acid swollen cellulose, 3 days; YEG medium, 42 °C, 1.5 h; YEG medium, 20 mm dithiothreitol, 1.5 h; YEG, room temperature, closed container with no agitation (anoxia), 1.5 h; solid state, 15 g of wheat bran, 1 g of proflo, 1 g of solkafloc, 30 ml of water, 6 and 7 days; solid state, 15 g of beet pulp, 1 g of proflo, 1 g of solkafloc, 30 ml of water, 9 days. RNA was prepared from the mycelia by grinding under liquid nitrogen with a mortar and pestle and extracting using Trizol reagent (Invitrogen) according to manufacturer's instructions. cDNA libraries were constructed by Invitrogen in the vector pREP3Y, which is a derivative of pREP3X (33Davis R.H. DeSerres F.J. Methods Enzymol. 1970; 17: 79-143Crossref Scopus (930) Google Scholar) containing additional restriction sites at the multiple cloning site. ESTs 1The abbreviations used are: EST, expressed sequence tag; GH, glycoside hydrolase; ORF, open reading frame. were generated by sequencing cDNA clones from the 5′ end. Template DNA was extracted in a 96-well format using a modified alkaline lysis protocol. Sequencing reactions were performed following standard Big Dye (Applied Biosystems) protocols for a 0.25× reaction. Cycle sequencing was performed over 35 cycles (96 °C for 10 s; 50 °C for 5 s; and 60 °C for 4 min) in an Applied Biosystems GenAmp 9700 thermocycler. DyeEx 96-well plates (Qiagen) were used for dyeterminator removal. Samples were sequenced using an ABI 3700 capillary sequencer (Applied Biosystems). Fermentation—Duplicate fermentations were run for each of the strains displayed in Fig. 3. 0.8 liters of minimal media containing 5% glucose was inoculated with 1.5 ml of frozen spore suspension. After 48 h, each culture was transferred to 6.2 liters of the same media in a 14-liter Biolafitte fermenter. The fermenter was run at 25 °C, 750 rpm, and 8 standard liters per min of air flow. One hour after the initial glucose was exhausted, a 25% (w/w) lactose feed was started and was fed in a carbon-limiting fashion to prevent lactose accumulation. The concentrations of glucose and of lactose were monitored using a glucose oxidase assay kit or a glucose hexokinase assay kit with β-galactosidase added to cleave lactose, respectively (Instrumentation Laboratory Co., Lexington, MA). Samples were obtained at regular intervals to monitor the progress of the fermentation. Samples obtained before (20–35 g/liter glucose) and just after glucose exhaustion, and 24 and 48 h after the lactose feeding commenced were used for microarray analysis. Isolation of RNAs, Labeling, and Hybridization—Mycelia were harvested by filtration through miracloth (Northern blots and fermentation samples) or by vacuum filtration through Whatman No. 1 paper and were quick frozen in liquid nitrogen. For Northern blotting, RNA was prepared as described above for construction of cDNA libraries. Polyadenylated RNA was selected 2 times using Oligotex (Qiagen). Blotting was performed using a NorthernMax-Gly Kit (Ambion). 32P-Labeled probes were generated using a DECAprime Kit (Ambion). Hybridization was performed using ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion). In all of the microarray experiments performed, the relative expression levels were determined between two RNA samples derived from growth under two different conditions. RNA was prepared using a FastRNA (Red) Kit (Qbiogene). Two aliquots of total RNA were taken from each sample. One aliquot was labeled with cyanine 3-CTP, and the other aliquot was labeled with cyanine 5-CTP (PerkinElmer Life Sciences) using a fluorescent linear amplification kit (Agilent Technologies). 250 ng of cyanine 5-labeled RNA derived from mycelia grown under one condition of interest were combined with 250 ng of cyanine 3-labeled RNA derived from mycelia grown under a second condition of interest, and the pooled RNAs were hybridized to the microarrays using an in situ hybridization kit (Agilent Technologies). The relative fluorescent intensities of cyanine 5- and cyanine 3-labeled species bound to each probe of the microarray were determined using an Agilent Technologies microarray scanner and software. The log of the ratio (log ratio) of the two fluorescent species bound to each of the probes reflects the relative expression levels of the cognate genes in the two samples (34Hughes T.R. Mao M. Jones A.R. Burchard J. Marton M.J. Shannon K.W. Lefkowitz S.M. Ziman M. Schelter J.M. Meyer M.R. Kobayashi S. Davis C. Dai H. He Y.D. Stephaniants S.B. Cavet G. Walker W.L. West A. Coffey E. Shoemaker D.D. Stoughton R. Blanchard A.P. Friend S.H. Linsley P.S. Nat. Biotechnol. 2001; 19: 342-347Crossref PubMed Scopus (1039) Google Scholar, 35DeRisi J. Penland L. Brown P.O. Bittner M.L. Meltzer P.S. Ray M. Chen Y. Su Y.A. Trent J.M. Nat. Genet. 1996; 14: 457-460Crossref PubMed Scopus (1738) Google Scholar). To avoid possible bias resulting from differences in incorporation of the dyes, duplicate microarrays were hybridized in which reciprocal dye combinations were used. Thus, for example, in sophorose induction experiments, cyanine 5-labeled RNA derived from sophorose-induced cultures was combined with cyanine 3-labeled RNA from uninduced cultures; and in replicate microarrays, cyanine 3-labeled RNA derived from sophorose-induced cultures was combined with cyanine 5-labeled RNA from uninduced cultures. A total of 6 microarrays was performed for each condition in the sophorose induction experiments (duplicate reciprocally labeled arrays for triplicate cultures). In the lactose induction experiments 4 microarrays were performed for each time point (duplicate reciprocally labeled arrays for duplicate fermenters). For all figures the mean values across replicates are presented. Microarray Design—60-mer oligonucleotides corresponding to the assembled ESTs were designed and synthesized in situ by Agilent Technologies. Bioinformatics and Data Analysis—Sequence chromatograms were assigned base quality values by Phred (version 0.990722, www.phrap.org). Sequences containing >100 bases with Phred quality values ≥20 or an average base quality ≥12 were retained. High quality sequences were assembled using the PhredPhrap (version 0.990329) script provided by Consed (version 11.0) (36Gordon D. Abajian C. Green P. Genome Res. 1998; 8: 195-202Crossref PubMed Scopus (2817) Google Scholar). Contigs were virtually translated in 6 reading frames and were annotated using the BioSCOUT system from LION Biosciences as in Andrade et al. (37Andrade M.A. Brown N.P. Leroy C. Hoersch S. de Daruvar A. Reich C. Franchini A. Tamames J. Valencia A. Ouzounis C. Sander C. Bioinformatics. 1999; 15: 391-412Crossref PubMed Scopus (162) Google Scholar). Briefly, biasdb (37Andrade M.A. Brown N.P. Leroy C. Hoersch S. de Daruvar A. Reich C. Franchini A. Tamames J. Valencia A. Ouzounis C. Sander C. Bioinformatics. 1999; 15: 391-412Crossref PubMed Scopus (162) Google Scholar) and Seq (38Wooton J.C. Federhen S. Methods Enzymol. 1996; 266: 554-571Crossref PubMed Google Scholar) were used to identify compositionally biased regions and regions of low compositional complexity, respectively. For each sequence, homology search results using FASTA (39Pearson W.R. Lipman D.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2444-2448Crossref PubMed Scopus (9328) Google Scholar) and BLAST (40Altschul S.F. Gish W. Miller W. Myers E. Lipman D. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68351) Google Scholar) were pooled, and a lexical analysis procedure was applied to the descriptive field of the set of homologs (37Andrade M.A. Brown N.P. Leroy C. Hoersch S. de Daruvar A. Reich C. Franchini A. Tamames J. Valencia A. Ouzounis C. Sander C. Bioinformatics. 1999; 15: 391-412Crossref PubMed Scopus (162) Google Scholar). Annotations were classified as direct, clear, tentative, marginal, or unknown according to the following criteria: 10<–70, 10–10 to 10–70, 10–4 to 10–10, 0.1 to 10–4 and >0.1 for BLAST; and >500, >130, >90, and >0 for FASTA, respectively. Selected sequences were further analyzed by aligning them with similar genes using ClustalW (clustalw.genome.ad.jp/). All tools were used with default parameters. Secretion signal sequence prediction was performed according to Ref. 41Nielsen H. Engelbrecht J. Brunak S. von Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4911) Google Scholar. Glycoside hydrolase families and carbohydrate-binding module families are were assigned as in Ref. 42Coutinho P.M. Henrissat B. Ohmiya K. Hayashi K. Sakka K. Kobayashi Y. Karita S. Kimura T. Genetics, Biochemistry, and Ecology of Cellulose Degradation. Uni Publishers Co., Tokyo1999: 15-23Google Scholar. To identify genes involved in protein processing and secretion, the sequences of gene products of interest from other organisms were compared by BLAST to the translated ESTs. Microarray data were quantified using Feature Extraction software (Agilent Technologies). The data were visualized and analyzed using Genespring version 4.2 (Silicon Genetics). Clustering was performed using a standard correlation and Genespring default settings. Identification of New Genes Encoding Hydrolytic Enzymes— The inducible expression of the very abundant cellulolytic and hemicellulolytic enzymes requires the coordination of a variety of cellular processes. To identify T. reesei genes that participate in these processes and to identify new enzymes that might play a role in biomass utilization, we sequenced the 5′ ends of 18,000 random cDNAs from mycelia grown on a wide variety of carbon sources and conditions. The sequences of individual sequence reads were compared, and overlapping segments were assembled to form 2101 contigs consisting of two or more 5′ reads. 3030 individual reads did not have significant sequence overlap with any other reads in the data set. The predicted coding regions of the EST set were compared with all publicly available sequence data bases. Twelve new sequences were identified encoding proteins with significant similarity to known enzymes whose substrates are commonly found in biomass. Full-length sequences corresponding to these gene products were determined. The genes encoding these sequences along with relevant previously identified enzymes from T. reesei are listed in Table I.Table ICharacterized and predicted biomass degrading activities and their genes in T. reeseiGeneFamilyaGH, glycosylhydrolase family; CE, carbohydrate esterase family.FunctionbFunction and features of proteins identified in this study are predicted from encoded amino acid sequence.FeaturesbFunction and features of proteins identified in this study are predicted from encoded amino acid sequence.,cSS, N-terminal signal sequence; CBM, carbohydrate binding module; GPI, glycosylphosphatidyloinositol anchor.Ref.GenBank™ accession no.cbh1/cel7aGH7CellobiohydrolaseSS, CBM58Shoemaker S. Schweickart V. Ladner M. Gelfand D. Kwok S. Myambo K.A.I.M. Bio/Technology. 1983; 1: 691-696Crossref Scopus (309) Google Scholarcbh2/cel6aGH6CellobiohydrolaseSS, CBM11Teeri T.T. Lehtovaara P. Kauppinen S. Salovuori I. Knowles J. Gene (Amst.). 1987; 51: 43-52Crossref PubMed Scopus (272) Google ScholarM16190egl1/cel7bGH7Endo-1,4-glucanaseSS, CBM59Penttila M. Lehtovaara P. Nevalainen H. Bhikhabhai R. Knowles J. Gene (Amst.). 1986; 45: 253-263Crossref PubMed Scopus (252) Google ScholarM15665egl2/cel5aGH5Endo-1,4-glucanaseSS, CBM10Saloheimo M. Lehtovaara P. Penttila M. Teeri T.T. Stahlberg J. Johansson G. Pettersson G. Claeyssens M. Tomme P. Knowles J.K. Gene (Amst.). 1988; 63: 11-22Crossref PubMed Scopus (266) Google ScholarM19373egl3/cel12aGH12Endo-1,4-glucanaseSS9Okada H. Tada K. Sekiya T. Yokoyama K. Takahashi A. Tohda H. Kumagai H. Morikawa Y. Appl. Environ. Microbiol. 1998; 64: 555-563Crossref PubMed Google ScholarAB003694egl4/cel61aGH61Endo-1,4-glucanaseSS, CBM13Saloheimo M. Nakari-Setala T. Tenkanen M. Penttila M. Eur. J. Biochem. 1997; 249: 584-591Crossref PubMed Scopus (160) Google ScholarY11113egl5/cel45aGH45Endo-1,4-glucanaseSS, CBM14Saloheimo A. Henrissat B. Hoffren A.M. Teleman O. Penttila M. Mol. Microbiol. 1994; 13: 219-228Crossref PubMed Scopus (150) Google ScholarZ33381cel74aGH74Endo-1,4-glucanaseSS, CBMThis studyAY281371cel61bGH61Endo-1,4-glucanaseSSThis studyAY281372cel5bGH5Endo-1,4-glucanaseSS, GPIThis studyAY281373bgl1/cel3aGH3β-GlucosidaseSS15Barnett C.C. Berka R.M. Fowler T. Bio/Technology. 1991; 9: 562-567Crossref PubMed Scopus (128) Google Scholar, 60Mach R.L. Mikrobielle Biochemie. Ph.D. thesis, Institute of Biochemistry and Technology, Vienna, Austria1993Google ScholarU09580bgl2/cel1aGH1β-Glucosidase16Takashima S. Nakamura A. Hidaka M. Masaki H. Uozumi T. J. Biochem. (Tokyo). 1999; 125: 728-736Crossref PubMed Scopus (114) Google ScholarAB003110cel3bGH3β-GlucosidaseSSThis studyAY281374cel3cGH3β-GlucosidaseThis studyAY281375cel1bGH1β-GlucosidaseThis studyAY281377cel3dGH3β-GlucosidaseThis studyAY281378cel3eGH3β-GlucosidaseSSThis studyAY281379xyn1GH11XylanaseSS31Torronen A. Mach R.L. Messner R. Gonzalez R. Kalkkinen N. Harkki A. Kubicek C.P. Bio/Technology. 1992; 10: 1461-1465Crossref PubMed Scopus (192) Google Scholar, 61Saarelainen R. Paloheimo M. Fagerstrom R. Suominen P.L. Nevalainen K.M. Mol. Gen. Genet. 1993; 241: 497-503Crossref PubMed Scopus (61) Google ScholarX69574xyn2GH11XylanaseSS61Saarelai}, number={34}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Foreman, PK and Brown, D and Dankmeyer, L and Dean, R and Diener, S and Dunn-Coleman, NS and Goedegebuur, F and Houfek, TD and England, GJ and Kelley, AS and et al.}, year={2003}, month={Aug}, pages={31988–31997} }
 @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 Cercosporin is a non-host-specific polyketide toxin produced by many species of plant pathogens belonging to the genus Cercospora . This red-pigmented, light-activated toxin is an important pathogenicity determinant for Cercospora species. In this study, we screened 244 bacterial isolates representing 12 different genera for the ability to degrade cercosporin. Cercosporin degradation was determined by screening for the presence of cleared zones surrounding colonies on cercosporin-containing culture medium and was confirmed by assaying the kinetics of degradation in liquid medium. Bacteria belonging to four different genera exhibited the cercosporin-degrading phenotype. The isolates with the greatest cercosporin-degrading activity belonged to Xanthomonas campestris pv. zinniae and X. campestris pv. pruni. Isolates of these pathovars removed over 90% of the cercosporin from culture medium within 48 h. Bacterial degradation of red cercosporin was accompanied by a shift in the color of the growth medium to brown and then green. The disappearance of cercosporin was accompanied by the appearance of a transient green product, designated xanosporic acid. Xanosporic acid and its more stable lactone derivative, xanosporolactone, are nontoxic to cercosporin-sensitive fungi and to plant tissue and are labile in the presence of light. Detailed spectroscopic analysis (to be reported in a separate publication) of xanosporolactone revealed that cercosporin loses one methoxyl group and gains one oxygen atom in the bacterial conversion. The resulting chromophore (4,9-dihydroxy-3-oxaperlylen-10H-10-one) has never been reported before but is biosynthetically plausible via oxygen insertion by a cytochrome P-450 enzyme.}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Mitchell, TK and Chilton, WS and Daub, ME}, year={2002}, month={Sep}, pages={4173–4181} }
 @article{martin_blackmon_rajagopalan_houfek_sceeles_denn_mitchell_brown_wing_dean_2002, title={MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea}, volume={30}, ISSN={["0305-1048"]}, DOI={10.1093/nar/30.1.121}, abstractNote={We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIIIdigestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.}, number={1}, journal={NUCLEIC ACIDS RESEARCH}, author={Martin, SL and Blackmon, BP and Rajagopalan, R and Houfek, TD and Sceeles, RG and Denn, SO and Mitchell, TK and Brown, DE and Wing, RA and Dean, RA}, year={2002}, month={Jan}, pages={121–124} }