2010 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. Analysis of repetitive elements in the genomes of Mycosphaerella graminicola and M. fijiensis continued, and a comparative genomics analysis was initiated. A project to sequence the genomes of additional species in the class Dothideomycetes, picked up by the Joint Genome Institute of DOE, has proceeded, and samples have been submitted for sequencing. Those sequences will greatly expand our understanding of fungal biology. We are using the genomic sequence of M. graminicola to select genes for functional analysis. A post-doc has developed an efficient protocol for introducing foreign genes into M. graminicola and has used it to knock out or over express certain genes. That work has identified several genes involved in fungal pathogenicity. 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. This work was slowed by recessive inheritance of one gene and loss of effectiveness in some genetic backgrounds. Two potentially new genes for resistance are being moved from the genetic relative wheatgrass into wheat. That project should result in a new source of durable resistance. Objective 1. In efforts to identify genes that impact pathogenesis in Cercospora zeae-maydis (gray leaf spot of corn), we have concentrated on genes predicted to be involved in regulating light responses because light, specifically blue light, influences fungal virulence by affecting multiple stages of pathogenesis – spore production, synthesis of cercosporin, and appressorium formation. Mutants with a disrupted gene encoding a putative blue-light receptor are unable to infect corn plants. Thus, this gene is a virulence gene. We have resumed our efforts to complement the mutation with the wild-type gene to determine whether it is responsible for the multiple phenotypes. We isolated from infected leaves numerous strains of C. zeae-maydis that do not produce cercosporin. Inoculations of corn in the greenhouse indicated that these strains are capable of causing disease symptoms with levels of virulence indistinguishable from the cercosporin-producing strains, suggesting that cercosporin is not a pathogenicity factor, an observation that impacts disease management strategies based on screening corn germplasm for reaction to cercosporin or genetically engineering cercosporin resistance. We have used a set of simple sequence repeat markers to analyze the population structure of C. zeae-maydis. Although no sexual stage is known, we detected the presence of two mating types in nearly equal proportions throughout the U.S. Attempts to induce sexual reproduction by culturing compatible mating types in a variety of media and physical environments have not been successful. Thus, the mechanism involved in generating unexpected levels of genetic variation remains unexplained. The occurrence of a sexual stage and associated genetic recombination impacts approaches for screening corn germplasm for resistance to gray leaf spot. 4.Accomplishments 1. Functional analysis of genes involved in pathogenicity 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 either eliminate or increase their expression. A knock-out mutant of a c-type cyclin gene and over expression of a gene involved in producing reactive oxygen species showed distinct phenotypes indicating that both genes are involved in the pathogenicity of M. graminicola towards wheat. Two other genes that are involved in nutrient acquisition and were thought to be involved in pathogenicity did not show the expected phenotype, indicating that nutrition of M. graminicola during the early stages of infection occurs by a different mechanism than believed previously. This information will be of interest to plant pathologists trying to control septoria tritici blotch and to fungal geneticists trying to understand the molecular basis for host-pathogen interactions. 2. Circadian rhythm regulates hyphal melanization in the fungal pathogen Cercospora kikuchii. Light is one of the most important environmental factors influencing plant disease development. Circadian rhythms are typically triggered by light and have been thoroughly studied in plants and animals but not in plant pathogenic fungi. We identified a circadian rhythm governing colony morphogenesis in Cercospora kikuchii, an important foliar and seed-borne pathogen of soybean. After growth in light or light:dark cycles, colonies transferred to complete darkness produced zonate bands of thick, melanized hyphae interspersed with bands of hyaline hyphae, which 24-hour rhythm persisted for at least seven days after transfer to constant darkness. Blue light but not red light was sufficient to entrain the circadian rhythm in C. kikuchii. This represents the first documented circadian rhythm among Dothideomycete fungi and provides a new opportunity to dissect the molecular basis of circadian rhythms in filamentous fungi and facilitate investigations that focus on the role of light-regulated biological rhythms in plant pathogenesis. Such information will be useful to scientists in devising innovative strategies for disease control by targeting expression of key plant defense genes and fungal virulence genes, some of which are involved in melanin biosynthesis. Review Publications Dunkle, L.D., Crane, C.F., Goodwin, S.B. 2009. Development of Simple Sequence Repeat Markers from Expressed Sequence Tags of the Maize Gray Leaf Spot Pathogen, Cercospora Zea-Maydis. Molecular Ecology Resources. 9:1375-1379. Anderson, J.M., Bucholtz, D.L., Sardesai, N., Santini, J.B., Gyulai, G., Williams, C.E., Goodwin, S.B. 2009. Potential New Genes for Resistance to Mycosphaerella Graminicola Identified in Triticum Aestivum x Lophopyrum Elongatum Disomic Substitution Lines. Euphytica. 172:251-262. Goodwin, S.B., Dunkle, L.D. 2010. Cercosporin production in Cercospora and Related Anamorphs. In: Lartey, R.T., Weiland, J.J., Panella, L., Crous, P.W., and Windels, C.E. editors. Cercospora Leaf Spot of Sugar Beet and Related Species. St. Paul, MN:APS Press. p. 97-108.