1a. Objectives (from AD-416):
Objective 1: Discover and analyze the function of genes involved in pathogenicity of foliar fungal pathogens of wheat and other grain crops. Sub-objective 1a. Provide gene-expression annotations for the approximately 44% of the genes in the M. graminicola genome that have no predicted function, particularly those that share conserved domains of unknown function in other fungi. Sub-objective 1b. Analyze genes expressed under many conditions including during early, transitional and late stages of pathogenicity to identify those that may be important for disease development and other biological processes. Sub-objective 1c. Test the function of candidate pathogenicity genes or others involved in important biological processes by knocking out their expression and analyzing changes in phenotype. Objective 2: Characterize the genetic and biochemical bases for resistance of wheat to the foliar fungal pathogens responsible for Septoria and Stagonospora leaf blotches. Sub-objective 2a. Analyze genes expressed at several time points after inoculation with the pathogens to identify those associated with the major-gene resistance response of wheat. Sub-objective 2b. Analyze genes expressed at several time points after inoculation with the pathogens to identify those associated with non-host resistance responses of wheat and barley. Sub-objective 2c. Test the function of candidate resistance-associated genes by virus-induced gene silencing (VIGS) or RNA interference (RNAi). Objective 3: Develop genetic markers to be used by associated breeding programs in the development of disease-resistant germplasm of wheat and other grain crops. Sub-objective 3a. Develop large recombinant-inbred populations segregating for the Stb2 and Stb3 genes for resistance to Septoria tritici blotch in wheat and identify additional closely linked molecular markers. Sub-objective 3b. Develop mapping populations to identify quantitative resistance against the Septoria pathogens of wheat. Sub-objective 3c. Identify and validate molecular markers linked to quantitative resistance against the Septoria pathogens of wheat. Sub-objective 3d. Test Chinese wheat cultivars for resistance against the Septoria pathogens of wheat.
1b. Approach (from AD-416):
The preferred approach will be sequencing of messenger RNAs produced during several stages of pathogenesis by Mycosphaerella graminicola and Stagonospora nodorum on wheat, and of non-host resistance responses on barley. Numerous libraries will be analyzed from as many treatments as possible to obtain the greatest number of expressed genes, which was shown to be very effective in a previous analysis of EST sequences. Some treatments will consist of the pathogen exclusively in pure culture to identify genes involved in fungal development and responses to light, while others will include the pathogen in its host to identify genes involved in fungal pathogenicity and in host resistance. Most experiments will be done with isolate IPO323 of M. gramincola, the strain that was sequenced. Availability of a completely sequenced genome for M. graminicola provides a unique opportunity to analyze global gene expression to identify candidate genes for pathogenicity, sporulation, mating, light reception/regulation and other important biological processes. For some host-pathogen interactions isolates T48 from Indiana and isolate Pasco from Australia may be used. Isolate T48 is avirulent on all resistance sources tested so far but gives good infection on susceptible controls. It was used to map genes Stb1, Stb4 and Stb8. The Pasco isolate was used to map resistance genes Stb2 and Stb3 and can be used as a backup in case of problems with one of the other isolates.
3. Progress Report:
Objective 1. RNA sequencing experiments were begun for the fungus grown under blue, red and white light and of mutants that have only filamentous or only yeast-like growth. Sequencing of the samples from the light treatments has been done and the analysis is underway. After beginning the analysis, we realized that there was a flaw in the experimental design because it did not include a dark treatment. A dark treatment is now being initiated and will be analyzed with the other experiments. One of the mutants could not be revived from cold storage so that part of the project will be modified. Experiments with the other mutant have been completed and sent off for RNA sequencing. Initial results with an experimental-evolution population of the fungus showed that changes were induced by fungicide treatments. The entire experiment is now being repeated for more robust statistical analysis. A process for annotating the mitochondrial genomes of fungi was developed. The process involves using two tools for initial predictions of genes and their introns, manually validating the predicted genes against those in other fungi, identifying the intron types and exporting the results to a program that draws circular genomes and another program that prepares all of the annotations for submission to the organelle genome database at GenBank. Ultimately we hope to automate the entire process but for now much of it is still done manually. We have used this process to annotate numerous fungal genomes in the class Dothideomycetes, including both the smallest and the largest mitochondrial genomes for filamentous fungi. Several publications on this work are in preparation. Objective 2. Work on making isogenic lines for the Stb2 and Stb3 genes for resistance to M. graminicola is continuing. We now have both resistance genes in a common susceptible background of wheat and the lines are almost at the stage where they can be used experimentally. Experiments for RNA sequencing of resistance (R) gene and non-host responses are underway. The R-gene experiments did not show good infection on the susceptible controls so will be repeated once the greenhouses cool off in the fall. Infection of the susceptible checks during the non-host experiments was good and those samples are being prepared for RNA sequencing and will be analyzed this fall. Objective 3. The progeny set segregating for the Stb2 gene was advanced to another generation and is now ready to begin analyses this fall. We will test approximately 100 of the lines for resistance phenotype and use those results to make bulks for screening molecular markers. The first goal is to test the markers that were linked to Stb2 in a different progeny set. If the markers are not linked, then we will use the bulks to screen additional markers covering the entire wheat genome to determine the location of our gene, which was recessive in previous work in contrast to the dominant nature of the gene that segregates in the other progeny set. Once we have established whether the genes are the same we will use the rest of the lines for fine-scale mapping of all markers that are
1. Identification of mitochondrial genes in the fungal class Dothideomycetes. Genome sequencing projects generate sequences for both nuclear and mitochondrial genomes, including for many species of Dothideomycetes, the largest and most diverse class of fungi, yet there are no defined methods for identification of mitochondrial genes so they usually are ignored. To address this problem, ARS researchers in West Lafayette, IN, developed a process for identifying fungal mitochondrial genes. The process involves using available computer tools plus manual intervention and results in complete lists of the genes present within mitochondrial genomes. Output is in a form suitable for drawing a circular map and for preparing the gene catalogs for submission to the GenBank database. The process has been applied to numerous mitochondrial genomes of fungi in the Dothideomycetes including new gene lists for numerous species. The results identified two Dothideomycetes (the warehouse-staining fungus Baudoinia compniacensis and the wine cellar fungus Zasmidium cellare) with the smallest mitochondrial genomes in their fungal group and two other plant-pathogenic species (the wheat pathogen Pyrenophora tritici-repentis and the Brassica pathogen Leptosphaeria maculans) with the largest mitochondrial genomes reported for fungi, among many others. Use of this process will make these mitochondrial gene lists accessible to the worldwide scientific community and will facilitate comparative analyses of their evolutionary relationships. This information will be of interest to bioinformaticists trying to catalog fungal mitochondrial genomes, to evolutionary biologists studying fungal evolution, to fungal geneticists analyzing mitochondrial biology and to plant pathologists interested in controlling diseases caused by fungi.
Ohm, R., Feau, N., Henrissat, B., Schoch, C.L., Horwitz, B.A., Barry, K.W., Condon, B.J., Copeland, A.C., Dhillon, B., Glaser, F., Goodwin, S.B. et al. 2012. Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi. PLoS Pathogens. 8(12): e1003037. DOI:10.1371/journal.ppat.1003037.