2008 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 We recently obtained and analyzed over 27,000 ESTs from Cercospora zeae-maydis grown under a variety of light conditions and growth media. Among the ESTs were sequences corresponding to a variety of photoreceptors, some of which belong to families that have not been described in fungi. Thus, we are using reverse genetics to determine the role of light and fungal light receptors in pathogenesis by disrupting each of the five putative photoreceptors and characterizing the pathogenicity phenotype of each mutant strain. For example, in our initial experiments, disruption of a gene designated CRP1, an ortholog of the blue-light receptor WC1 in Neurospora crassa, resulted in a mutant that is unable to infect and cause disease due to an inability to locate stomata and correctly position appressoria for penetration of the leaf. We are analyzing the response of maize to infection by fungal foliar pathogens, C. zeae-maydis and Exserohilum turcicum, by microarray analysis to reveal genes expressed during various stages of infection and disease development. Our ultimate goal is to understand the molecular communication between maize and fungal pathogens and to identify host genes that play significant roles in dictating the outcome of the plant-fungal interaction leading to the expression of resistance. Analysis of molecular markers linked to the Stb2 gene for resistance to Mycosphaerella graminicola was slowed due to unforeseen problems with the population being analyzed. This population appears to contain an inversion that suppresses recombination rates. While developing another population, we discovered an error in a previous mapping for the Stb3 gene. Recent work identified a new location for this gene on wheat chromosome 7A rather than 6D as reported previously. This location was validated in collaborative work with another USDA location plus researchers at North Dakota State University and the University of California, Davis. A poster on this work will be presented at a meeting during August of 2008 and a manuscript for submission to a peer-reviewed journal is in preparation. Sequencing of mating type genes from the poplar pathogen Septoria musiva (Mycosphaerella populorum) identified a gene of almost 8,000 base pairs in length, which is very large compared to related fungi in which this region is less than 3,000 bases long. This great length was unexpected and slowed down progress on the project. However, the final result is very interesting and shows that the MAT-1 locus of this species appears to have acquired a second gene that is not linked to mating type in related species such as the wheat pathogen M. graminicola. This result bolsters the hypothesis that mating type genes evolve rapidly in this genus and may help to drive speciation. Sequencing of the second mating type from M. populorum continues and hopefully will be completed early during FY09. Identification of mating-type frequencies among almost 400 isolates of the pathogen in several populations was completed and will help to fill out a manuscript that we hope to submit for publication by the end of 2008. 4.Accomplishments 1. Characterization of a blue-light photoreceptor from Cercospora zeae-maydis. The gray leaf spot fungus Cercospora zeae-maydis requires blue light to infect maize, but the molecular mechanism involved is not known. We searched our EST database for putative photoreceptors involved in light-dependent infection, and identified and characterized PHL1, an ortholog of the cryptochrome/photolyase family of blue-light photoreceptors whose function in fungi is unknown. DNA photolyases harvest light energy to repair genomic lesions induced by UV irradiation, whereas cryptochromes have evolved in plants and animals as blue-light photoreceptors that function exclusively in signal transduction. Disruption of the PHL1 gene in C. zeae-maydis revealed that it is a global regulator of DNA repair (photoreactivation) in response to UV irradiation, including the expression of at least two genes involved in general DNA repair, but the PHL1 disruption mutants were fully virulent on maize, indicating that PHL1 is dispensable for pathogenesis. Although similar genes are predicted to be present in the sequenced genomes of other fungi, this is the first characterization of a member of this gene family in a filamentous fungus. This information contributes to our knowledge of virulence determinants in fungal pathogens and can be used to unravel the molecular events that influence the outcome of plant-pathogen encounters for eventual disease control strategies. This accomplishment is aligned with NP 303 Component 2 - Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A – Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. 2. Corrected mapping of the Stb3 gene for resistance to Mycosphaerella graminicola in wheat. Breeding for resistance to Septoria tritici blotch (STB) caused by Mycosphaerella graminicola is the only economically viable means of disease management in many parts of the world. The Stb3 gene for resistance could be very useful to increase resistance, but previous genetic mapping identified only a single marker linked to this gene and others are needed for efficient marker-assisted selection. To confirm the published map location and find additional linked markers, more than 250 markers were tested on the parents and progeny of a mapping population for Stb3. Results of this analysis revealed that the previously published map location for Stb3 was incorrect. The correct chromosomal location was identified with ten linked molecular markers, and this location was verified in two independent populations. This information will be very useful to plant breeders for moving the resistance gene into new cultivars and to plant pathologists to better understand the organization of resistance genes in wheat. This accomplishment is aligned with NP 303 Component 3 - Plant Disease Resistance, Problem Statement 3A - Mechanisms of Plant Disease Resistance. 3. Mitochondrial genome sequence of the wheat pathogen Mycosphaerella graminicola. Mitochondria are essential for cell functioning and can be targets of fungicides and other compounds in plant protection. However, relatively little is known about gene content of mitochondrial genomes or about the levels of genetic variation among mitochondria within species, particularly for plant pathogenic fungi. To rectify this situation, complete mitochondrial genomes were sequenced from two strains of the septoria tritici blotch pathogen of wheat, Mycosphaerella graminicola, and compared to assess the level and degree of polymorphism. This analysis showed that the complete mitochondrial genome of M. graminicola is a circular molecule of 43,961 base pairs containing genes for 14 proteins related to energy production, one gene for making RNA, two ribosomal RNA genes and 27 transfer RNAs. Approximately one third of the genome was inverted relative to what was expected from other fungi, and nucleotide variation was low between strains originating from Europe and North America. This information will be useful to mycologists and population geneticists to understand the mating behavior and migration patterns of this fungus, to plant pathologists trying to identify potential new fungicide targets, and to evolutionary biologists to understand the past evolutionary history of this organism. This accomplishment is aligned with NP 303 Component 2 - Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A – Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. 4. Genome sequence of a banana pathogen. Mycosphaerella fijiensis causes black Sigatoka (or leaf streak) of banana. To aid comparative analyses with a previously sequenced wheat pathogen, the genome sequence of M. fijiensis was generated through the Community Sequencing Program of the U.S. Department of Energy’s Joint Genome Institute (JGI). The genome sequence contains 73.4 million bases of DNA organized into 395 pieces and coding for approximately 10,327 genes. All available Expressed Sequence Tags were placed on the finished sequence to assess completeness and to identify the correct zondares between introns and exons. This is the first genome sequence of a banana pathogen and is of great importance to plant pathologists and evolutionary biologists for comparative genomics involving related fungal pathogens in this group, such as the closely related wheat pathogen M. graminicola, which was sequenced through the same program previously. This accomplishment is aligned with NP 303 Component 2 - Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and Their Relationships with Hosts and Vectors, Problem Statement 2A – Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of Other Technology Transfer 2 Review Publications Adhikari, T.B., Ali, S., Burlakoti, R., Singh, P., Mergoum, M., Goodwin, S.B. 2007. Genetic structure of Phaeosphaeria nodorum populations in the north-central and mid-western United States. Journal of Phytopathology. 98:101-107. Torriani, S.F., Goodwin, S.B., Kema, G.H., Pangilinan, J.L., Mcdonald, B.A. 2008. Intraspecific comparison and annotation of two complete mitochondrial genome sequences from the plant pathogenic fungus Mycosphaerella graminicola. Fungal Genetics and Biology. 45:628-637. Adhikari, T.B., Balaji, B., Breeden, J., Goodwin, S.B. 2007. Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression. Physiological and Molecular Plant Pathology. 71:55-68. Goodwin, S.B., Van Der Lee, T., Cavaletto, J.R., Hekkert, B., Crane, C.F., Kema, G.H. 2006. Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the Septoria tritici blotch pathogen Mycosphaerella graminicola. Fungal Genetics and Biology. 44:398-414.