Objective 1: Identify candidate genes expressed by the host and fungal pathogens during resistant, susceptible and non-host interactions to elucidate mechanisms of host resistance of wheat, corn and barley. Sub-objective 1a. Identify genes expressed by hosts containing different resistance genes to identify the mechanisms involved in each resistance response. Sub-objective 1b. Identify genes expressed by the pathogens during different growth conditions that are involved in survival and pathogenicity. Objective 2: Determine whether selected genes function in fungal resistance responses by virus-induced gene silencing (VIGS) in wheat, and test if the best candidates confer improved resistance in transgenic plants. Sub-objective 2a. Utilize BSMV-VIGS to assess function of candidate genes in FCR and Septoria leaf blotch resistance. Sub-objective 2b. Attempt to engineer FCR, FHB or STB resistance in transgenic wheat utilizing genes confirmed in VIGS analysis. Objective 3: Analyze multiple fungal genomes to identify effectors and other biologically important genes involved in pathogenicity on wheat and corn. Objective 4: Determine the effectiveness of Host-Induced Gene Silencing (HIGS) to control Septoria and Fusarium diseases in wheat. Objective 5: Refine the map locations of genes for resistance to Septoria diseases in wheat and fungal diseases in corn to identify tightly linked molecular markers for marker-assisted selection in cereal improvement programs. Sub-objective 5a. Identify molecular markers tightly linked to the Stb2 and Stb3 resistance genes in wheat. Sub-objective 5b. Identify molecular markers linked to resistance genes in corn.
Diseases caused by fungal pathogens pose significant economic threats to grain crop production. Currently, little is known about the molecular and genetic mechanisms that govern host resistance and fungal virulence in wheat. Research objectives and approaches in this project focus on identifying genes expressed by the host and the fungal pathogens during infection. The primary subjects of research will be septoria tritici blotch (STB) and Fusarium head blight (FCHB) and crown rot (FCR) of wheat. We will utilize RNA sequencing to identify wheat genes expressed during different types of resistance responses and fungal genes involved in pathogenicity and other important biological processes. Some of the host materials will include recently developed isogenic lines for resistance genes against STB. These genes are on different wheat chromosomes and the isogenic lines will allow us to test the hypothesis that they use different mechanisms for resistance. We will analyze non-host resistance responses in interactions between barley and wheat inoculated with Mycosphaerella graminicola and Septoria passerinii, respectively. Gene function in the pathogens will be confirmed by generating knockout mutants and testing for phenotype and in the host by Virus-Induced Gene Silencing (VIGS). We also will use comparative genomics of resequenced isolates to identify essential genes in M. graminicola and will use these plus others identified from the RNA-seq experiments for both pathogens to identify genes that can be targeted for Host-Induced Gene Silencing (HIGS) to increase the level of resistance in wheat. Additional objectives are to develop a CRISPR/Cas9 system for M. graminicola and to do fine-scale genetic mapping for developing additional molecular markers linked to the resistance genes. Successful completion of the objectives will contribute to the basic understanding of diseases caused by plant-pathogenic fungi and will provide clues about potential targets for genetic modification of the crop to prevent or circumvent damage resulting from fungal pathogens.
Objective 1. The final RNA samples for the non-host and R-gene resistance projects were sequenced. Analyses for both projects are underway. Results have shown a nice increase in pathogen gene expression in the susceptible interactions, and a low proportion of pathogen RNA in all of the resistant interactions, as expected. Mapping of sequencing reads to the host and pathogen genomes has been completed and comparative analyses are mostly completed. With the large datasets involved each comparison takes many days to complete and with dozens of comparisons the analysis time is long. So far it looks like many differentially expressed genes will be identified and we hope to have the analysis of the non-host project completed by September of 2019. Development of a CRISPR-based transformation system for the pathogen has not been successful. Similar attempts have been made in three laboratories in Europe and one in Australia, also without success so it appears that molecular manipulations with these organisms are much more difficult than anticipated. Although the other laboratories have now given up we are now attempting a novel approach that should have a much higher frequency of homologous recombination, a possible barrier to successful implementation of the technique. Experiments are now underway to generate the ku70 mutant and several types of CRISPR methods will be tried again once the mutant becomes available. The mating-type experiments were not performed because all of the available Indiana isolates had the same mating type. Part of the protocol involves placing inoculated plants outside so the experiment can only be done with local isolates. Additional Indiana isolates are being obtained and screened to find some of the opposite mating type that can be used to complete those experiments. The first part of the light response in the pathogen was completed and a manuscript was submitted for publication. Work on generating knockout mutants of the selected genes is continuing. One potential knockout is being evaluated and several hundred potential transformants are being screened. Objective 2. Substantial work has gone into optimizing the Fusarium crown rot – VIGS assay, so that it is as reproducible as possible. A number of genes have been identified as candidates for encoding functions required for Fusarium Crown Rot resistance in initial VIGS assays. These VIGS assays are being repeated as well as confirmed by tests with a new silencing fragments derived from the same candidate genes. Objective 3. The planned comparative analyses were completed through collaborative research. One analysis of Dothideomycetes analyzed early evolutionary divergence and showed that the entire class likely derived from rock-inhabiting ancestors, indicating that pathogenicity to plants was a later, derived character. A second comparative analysis of genome sequences in the class used machine learning to predict lifestyle from gene content. This work identified six clusters of genes that are associated with a plant-pathogenic lifestyle; other genes were associated with being a saprobe. This work also showed conclusively that ability to infect plants arose independently multiple times from ancestors that were saprobes. Both of these analyses were written up and the manuscripts have been submitted to peer-reviewed journals. A third analysis was a genome-wide association study (GWAS) to identify genes associated with pathogenicity to wheat. The preliminary analysis identified several peaks associated with pathogenicity to different wheat cultivars, some of which were on dispensable chromosomes, the first time a possible function for a dispensable chromosome has been shown in this fungus. Those analyses need to be confirmed by additional testing and plans for a manuscript on this work are being developed. Objective 4. Our preliminary analyses plus results from two other labs in Australia and the United Kingdom have shown that host-induced gene silencing (HIGS) may be difficult or impossible against our targeted wheat pathogen. Therefore, we are refocusing the project to try to determine at which point the effect on the pathogen is being blocked. Our first goal now is to test for an effect on fungal growth in culture using antisense RNAs made against several essential genes identified in previous work. If we can get gene silencing to work on the fungus in pure culture then we will proceed with the planned testing in recombinant wheat plants. So far we have designed primers to make the RNAs and expect to finish the initial feeding experiments by the end of summer or beginning of fall 2019. Objective 5. A new population was made for precise identification of genes involved in the response initiated by the Stb2 resistance gene in wheat. A homozygous resistant and a homozygous susceptible sister line that were developed after six generations of backcrossing the resistance source to a recurrent susceptible parent, were crossed with each other and a segregating F2 population was analyzed. Segregation of resistance in this population showed an almost exact 3:1 ratio, as expected for a dominant gene. We are now making bulks of the resistant and susceptible lines from this population and will perform RNA sequencing to identify which genes are associated only with resistance. This should be an extremely clean experiment since we started with two sibling lines that were already almost identical except for the resistance gene. Sequencing of these materials should be completed by the end of 2019. To identify additional molecular markers linked to these genes and look for new sources of resistance, a GWAS was initiated on a large collection of Purdue University wheat cultivars with different origins through collaborative research. The population was tested with three replications of one isolate and one replication of a second. Initial analyses identified several peaks associated with Septoria tritici blotch resistance, at least one of which appears to be new. Analysis of the wheat genome near the markers associated with these peaks identified numerous likely candidate genes, many of which have potential functions in disease resistance. These genes will now be targeted for functional analysis with VIGS. Two additional populations, one on historical wheat cultivars and another on eastern wheat cultivars also will be tested pending availability of space in the growth chamber being used for the phenotyping. A manuscript on the results from the first round of testing is being planned.
Gunupuru, L.R., Perochon, A., Ali, S.S., Scofield, S.R., Doohan, F.M. 2018. Virus-induced gene silencing (VIGS) for functional characterization of disease resistance genes in barley seedlings. Methods in Molecular Biology. 1900:95-114. https://doi.org/10.1007/978-1-4939-8944-7_7.