2011 Annual Report 1a.Objectives (from AD-416) Objective 1: Analyze pathogen gene expression during disease development to identify the mechanisms of pathogenicity or virulence of fungal pathogens to maize and wheat. Sub-objective 1a. Assess the role of genes that regulate conidiation and phytotoxin synthesis during pathogenesis of maize and wheat by fungal pathogens, including Exserohilum turcicum, Cercospora zeae-maydis, and Mycosphaerella graminicola. Sub-objective 1b. Identify pathogen proteins secreted into apoplastic fluids and test whether they function as virulence factors in pathogens of maize and wheat, e.g., Exserohilum turcicum and Mycosphaerella graminicola. Sub-objective 1c. Assess the impact of mating-type gene evolution on speciation in fungal plant pathogens, including Mycosphaerella graminicola and Septoria passerinii. Objective 2: Analyze the function and chromosomal location of host genes predicted to be involved in disease resistance in wheat and maize. Sub-objective 2a. Investigate mechanisms of host-specific resistance of wheat to Mycosphaerella graminicola and of maize to Exserohilum turcicum. Sub-objective 2b. Elucidate mechanisms of non-host resistance focusing on resistance of barley to Mycosphaerella graminicola and resistance of wheat to Septoria passerinii. Sub-objective 2c. Discover closely linked markers on wheat chromosome 3BS for marker-assisted selection and eventual positional cloning of the Stb2 gene for resistance to Mycosphaerella graminicola. 1b.Approach (from AD-416) Fungal genes expressed during critical stages of pathogenesis will be identified by microarray analysis and by subtractive suppressive hybridization; involvement of selected genes, including those for phytotoxin synthesis and conidiation, will be assessed by transformation and gene disruption and silencing methods. Patterns of gene expression in resistant and susceptible genotypes of maize and wheat will be determined with microarrays composed primarily of cDNAs identified from a database of ESTs. The chromosomal location of genes for resistance to fungal pathogens will be determined by employing a variety of PCR-based, molecular methods. 3.Progress Report Objective 1. Based on our analyses of the genome sequence for Mycosphaerella graminicola, numerous genes were selected for functional analysis. Constructs to knock out gene function were made and introduced into the pathogen. Problems with the technology for functional analysis were identified and this reduced the number of genes that could be analyzed successfully. However, several were successful including genes for important cellular functions that have been shown to be involved in pathogenicity in some other systems. Our results showed extensive changes to secondary metabolism and, for one gene, on detection and regulation of gene expression by light, the first time this was shown for a species of Mycosphaerella. Novel effects on pigment production also were identified. This work led to two manuscripts that were published in peer-reviewed journals during 2011. Work on mating type evolution is almost complete and all of the required sequences have been obtained. The work exceeded what was planned for most species by the acquisition of complete genome sequences instead of just the mating type region. The analysis of those sequences is being initiated and hopefully will be completed by the end of the FY. So far it appears that mating type genes in Mycosphaerella evolve even faster than anticipated with large structural changes and rearrangements in addition to the expected point mutations and small insertions/deletions. In addition, it appears that in some species there are partial duplications of the mating type locus which can lead to incorrect characterizations and may give rise to unexpected mating type behavior in populations. This could affect the epidemiology of plant diseases so is being analyzed in a larger sample to confirm and test whether it has implications for disease management. Work on maize was terminated due to retirement in December 2010 of the SY and technician working on that part of the project. Objective 2. On the host wheat side of the interaction, we are continuing to isolate different genes for resistance to Septoria tritici blotch in a highly susceptible genetic background. Backcrossing of the genes is continuing. This has been slowed by unexpected recessiveness for one of the genes and dubious linkages with some of the molecular markers, which have required extensive retesting of materials to verify incorporation of the resistance into the next generation. Most lines only need one or two backcross generations to be finished, and then will be ready for analyses of gene function. Two additional resistance genes from wheatgrass are being moved into wheat. Those lines are being tested. So far, recombination was less than expected so some of the wheatgrass chromosomes are remaining intact and may require additional cycles of crossing to make these materials useful to wheat breeders. 4.Accomplishments 1. Functional analysis of the velvet gene of Mycosphaerella graminicola. Septoria tritici blotch, caused by the fungus Mycosphaerella graminicola, is one of the most important diseases of wheat worldwide, yet very little is known about how this pathogen causes disease or which genes are involved. To address this problem, ARS researchers at West Lafayette, IN, used the genome sequence of M. graminicola to clone genes and eliminate their expression. A knock-out mutant of a velvet gene homolog, known to be involved in pathogenicity and light signaling in other pathogens, showed changes in pigment production and light signaling but did not affect the pathogenicity of M. graminicola towards wheat. The effect on pigment production and the lack of function in pathogenicity are both unique and extend the known effects of this gene. This information will be used by plant pathologists trying to control septoria tritici blotch and by fungal geneticists to better understand the molecular basis for host-pathogen interactions. Review Publications Dhillon, B., Cavaletto, J.R., Wood, K.V., Goodwin, S.B. 2010. Accidental Amplification and Inactivation of a Methyltransferase Gene Eliminates Cytosine Methylation in Mycosphaerella Graminicola. Genetics. 186:67-77. Garcia, S.L., Van Der Lee, T.J., Ferreira, C.F., Hekkert, B., Carlier, J., Goodwin, S.B., Guzman, M., Souza, M.T., Kema, G.J. 2010. Variable Number of Tandem Repeat Markers in the Genome Sequence of Mycosphaerella Fijiensis, the Causal Agent of Black Leaf Streak Disease of Banana (Musa spp.). Genetics and Molecular Research. 9:2207-2212. Choi, Y., Goodwin, S.B. 2011. MVE1 Encoding the velvet gene product homolog in Mycosphaerella graminicola is associated with aerial mycelium formation, melanin biosynthesis, hyphal swelling, and light signaling. Applied and Environmental Microbiology. 77:942-953. Choi, Y., Goodwin, S.B. 2011. Gene encoding a C-type cyclin in Mycosphaerella graminicola is involved in aerial mycelium formation, filamentous growth, hyphal swelling, melanin biosynthesis, stress response, and pathogenicity. Molecular Plant-Microbe Interactions. 24:469-477. Goodwin, S.B., M'Barek, S., Dhillon, B., Wittenberg, A.J., Crane, C.F., Van Der Lee, T.J., Grimwood, J., Aerts, A., Antoniw, J., Bailey, A., Bluhm, B., Bowler, J., Bristow, J., Canto-Canche, B., Churchill, A., Conde-Ferraez, L., Cools, H., Coutinho, P.M., Csukai, M., Dehal, P., De Wit, P., Donzelli, B., Foster, A.J., Hammond-Kosack, K., Hane, J., Henrissat, B., Killian, A., Koopmann, E., Kourmpetis, Y., Kuzniar, A., Lindquist, E., Lombard, V., Maliepaard, C., Martins, N., Mahrabi, R., Oliver, R., Ponomarenko, A., Rudd, J., Salamov, A., Schmutz, J., Schouten, H.J., Shapiro, H., Stergiopoulos, I., Torriani, S.F., Tu, H., De Vries, R.P., Wiebenga, A., Zwiers, L., Grigoriev, I.V., Kema, G.J. 2011. Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity and stealth pathogenesis. PLoS Genetics. Available at: http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1002070. Dhillon, B., Goodwin, S.B. 2011. Identification and annotation of repetitive sequences in fungal genomes. Methods in Molecular Biology. In: Xu, J.R., Bluhm, B.H., editors. Methods in Molecular Biology: Fungal Genomics. New York, NY: Humana Press. p. 33-50. Rouxel, T., Grandaubert, J., Hane, J.K., Hoede, C., Van De Wouw, A. ., Couloux, A., Dominguez, V., Anthouard, V., Bally, P., Bourras, S., Cozijnsen, A.J., Ciuffetti, L.M., Degrave, A., Dilmaghani, A., Duret, L., Fudal, I., Goodwin, S.B., Gout, L., Glaser, N., Linglin, J., Kema, G.G., Lapalu, N., Lawrence, C.B., May, K., Meyer, M., Ollivier, B., Poulain, J., Simon, A., Stachowiak, A., Turgeon, G.B., Tyler, B.M., Vincent, D., Weissenbach, J., Amselem, J., Balesdent, M., Howlett, B., Oliver, R.P., Quesneville, H., Wincker, P. 2011. Effector diversification within compartments of the Leptosphaeria maculans genome affected by repeat induced point mutations. Nature Communications. 2:202.