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. Non-host plant-pathogen inoculations were completed. RNA prepared from those samples are being sequenced and those results will be used to complete the analysis. Analysis of the initial sequencing results, as expected, showed that pathogen sequences were very few in the earliest time points and increased in the susceptible interaction. Many host genes responded and the data are still being analyzing to determine the biochemical mechanisms involved in the non-host resistance response. Similar analysis of the R-gene responses is also underway. Initial results show that many genes are responding and, interestingly, significantly different from the non-host resistance response. Objective 2. VIGS analysis is underway testing whether expression of candidate genes is essential for resistance to Fusarium crown rot (FCR), but as yet genes making strong contributions to resistance have not been identified. The FCR assay must be performed in very young seedlings and there are some concerns about getting sufficiently reliable gene silencing at such an early stage. Therefore, additional effort is being expended to optimize the FCR-VIGS assay and a second VIGS virus system is being tested to see if it is superior for this application. Objective 3. Analysis of fungal RNAseq experiments identified many genes that appear to be affected by light, the first time that this has been proven for this fungus. Genes involved in light responses in other fungi plus several others that showed a response to light but have no useful annotations were chosen for functional analyses. The relevant genes were cloned using the previously generated genome sequences as a guide and constructs were made to knock out the genes in the pathogen. Transformations have been performed, but so far have not been successful. Work now is focusing on the transformation protocol to identify problems. Work was also started on CRISPR/Cas9 transformations but has been halted until Agrobacterium-mediated transformations are successful. To further understand the effect of light on fungal sporulation, samples were collected from field plants where the flag leaves had either grown normally or had twisted so that the bottom side was facing up. Analysis of spore bodies on the top versus bottoms of the leaves showed a small but significant difference for spores to be produced on the side facing the light. This trait requires further analysis but might be useful for phenotyping the knockout lines once they are generated. Our previously developed pipeline for annotating mitochondrial genomes of fungi was applied to numerous new species leading to a much better understanding of the evolution of this molecule among fungi in the class Dothideomycetes. To provide a background and a comparison with plant pathogens, the mitochondrial genomes of ten species of fungi were analyzed, most of which are saprobes. The results revealed a huge diversity of mitochondrial genome structural variation and limited variability of gene content. So far there were no great differences between these and the plant pathogens that are available, other than for presence of introns. Mitochondrial genomes from the plant-pathogenic fungi usually have many more introns compared to the saprobes, almost always due to insertions of self-splicing, homing endonuclease genes, one of which has been shown to affect the ability to develop fungicide resistance. One of the endonuclease genes was found to have a possible homolog in a host genome, which could indicate horizontal transfer between host and pathogen genomes. Analysis of more genomes is needed to test this hypothesis and to determine whether these changes affect fungal evolutionary rate or pathogenicity. Objective 4. Genes from six categories were chosen for future Host-induced gene silencing (HIGS) analysis. Sequences of all genes were identified within the fungal genome sequence and are being used to assemble the HIGS constructs. These genes also are being used to make small RNAs that will be used in direct feeding experiments to test for growth repression. This has never been done for this fungus so experimentation is still being done to optimize the protocols. Objective 5. Analysis of the septoria resistance mapping populations continued and all of the readily available molecular markers have been exhausted. To find additional markers, the focus will switch slightly to perform RNAseq analyses of resistant and susceptible bulks. Lines to be included in the bulks have been chosen and the RNAs will be extracted pending an additional test to be sure that the materials respond as expected. To augment this project, lines from the Purdue wheat breeder were utilized. Lines that were planted in the field for a Genome-Wide Association Study (GWAS) of other traits were scored for resistance for several diseases including two species of rust pathogens plus Septoria tritici blotch. Analysis of the data identified molecular markers associated with all of the traits. Those results will be confirmed by additional testing in the greenhouse this fall and scoring the GWAS population in the field again next spring.