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. Analysis of the non-host and R-gene resistance projects was completed. The results identified many differentially expressed genes and showed that the non-host resistance response is completely different from that triggered by major resistance genes Stb2 and Stb3, which also are different from each other. This is important because it indicates that non-host resistance may be more durable compared to the specific resistance genes, which can be overcome rapidly when deployed in the field. A manuscript on these results is pending. Additional mutants of the pathogen with defects in genes involved in sensing and responding to light were generated and tested for alterations in growth and development. This is important because it may identify vulnerabilities in the life history of the pathogen that can be targeted for control measures. Two additional manuscripts on that work are now being prepared. From the initial RNA sequencing experiment, which was published during July of 2020, we have identified a list of ten additional genes that are most likely involved in light responses yet are not annotated in the genome sequence and may have novel properties. Those genes all have been targeted for knockout analyses, and we are in the process of generating the mutants needed to test their function. This work is needed to augment annotation of the genome sequence and identify targets for disease control as well as to better understand the biology of light responses in the pathogen. Objective 2. Currently seven genes have been screened by BSMV-VIGS to assay if they make critical contributions to resistance to Fusarium crown rot. So far, none of these genes have shown strong effect. Objective 3. Two manuscripts on comparative analyses of fungal genomes were completed through collaborative research and published. One analysis of fungi classified as 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 and living off of dead plants rather than causing disease. This work also showed conclusively that ability to infect plants arose independently multiple times from ancestors that were saprobes. Objective 4. Our previous analyses plus results from two other labs in Australia and the UK showed that host-induced gene silencing (HIGS) may be difficult or impossible against our targeted wheat pathogen. Therefore, we refocused the project to try to determine at which point the effect on the pathogen is being blocked. Our first goal was to test for an effect on fungal growth in culture using antisense RNAs made against several essential genes identified in previous work. These antisense RNAs should bind to sense RNAs produced by the pathogen, leading to their destruction and a lack of gene expression.The antisense RNAs were generated, and preliminary feeding experiments showed no effect. Objective 5. Progeny developed from a cross between two of our advanced lines that differ only for the specific resistance gene Stb2 were tested for resistance or susceptibility to the pathogen. RNA samples from 30 each of the resistant and susceptible lines were bulked, and the bulks plus parents were sent for sequencing. The sequencing is complete and the results are being analyzed. This experiment should identify genes that are very closely linked to the Stb2 resistance gene. To identify additional molecular markers linked to these genes and look for new sources of resistance, a genome-wide association study (GWAS) was performed through collaborative research on a large collection of Purdue University wheat cultivars with different origins. The population was tested with three replications of two fungal isolates that were selected because they gave clear but different responses in preliminary analyses. Many peaks associated with Septoria tritici blotch resistance were identified, and their approximate chromosomal locations were obtained based on the known locations of the linked molecular markers. Several of these likely corresponded to previously known Stb resistance genes, but several others appear 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 be targeted for functional analysis with VIGS once we are able to get back into the lab full time. 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. Part of the focus for corn pathology was switched to include tar spot, a new disease in the US since 2015, about which almost nothing is known, in addition to previously planned work on gray leaf spot, northern corn leaf blight and possibly stalk rot. Several new experiments were initiated, including one to look for resistance against tar spot among the parents of previously made mapping populations. Scoring of those corn accessions for disease identified a huge variability for tar spot resistance. Based on those results, two segregating populations were selected to be grown during the summer of 2020. They now have been planted and will be scored for tar spot resistance later this summer or fall, along with a repetition of the parental lines. An additional experiment was set up to test whether there were differences in the microbiomes between lines that were resistant versus susceptible to tar spot. Leaf material was collected and sent for sequencing of associated fungi and bacteria. The results revealed a huge difference between the microbiomes on the resistant and susceptible lines of one of the mapping populations. Material from a second population was collected and is ready for sequencing. A third project involves doing RNA sequencing on resistant versus susceptible lines based on tar spot resistance to try to identify genes involved during the resistance response. Those materials were collected and will soon be sequenced.
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