Genomic Characterization of Necrotrophic Effector Sensitivity Genes in Wheat
Abstract
The necrotrophic fungal pathogens Parastagonospora nodorum and Pyrenophora tritici-repentis cause the diseases septoria nodorum blotch (SNB) and tan spot, respectively, reducing yield by decreasing the photosynthetic area of the plant. The pathogens produce necrotrophic effectors (NEs) that target host genes to induce cell death. The Tsc1-Ptr ToxC and Tsn1-Ptr ToxA interactions contribute to plant susceptibility to tan spot, while the Tsn1-SnToxA and Snn5-B1-SnTox5 interactions contribute to SNB susceptibility. The three main goals of this dissertation were to clone susceptibility genes Tsc1 and Snn5 and develop robust genetic markers for use in marker-assisted elimination of Tsn1. A genetic linkage map was developed delineating the Tsc1 region to 184 kb. Structural and gene content comparisons of the identified Tsc1 and Tsn1 regions in using the sequenced wheat genomes revealed gene content variation correlating with host phenotypes, reducing the Tsc1 candidate gene list to just two genes. Comparative sequence analysis in two generated mutant populations revealed the identity of Tsc1, which has protein kinase and leucine-rich repeat domains. The structural and gene content comparison of the sequenced genomes in the Tsn1 region identified two conserved haplotypes in accessions with presence/absence variation corresponding with ToxA sensitivity. Genetic markers flanking Tsn1 were designed in segments syntenic between Tsn1+ and Tsn1- accessions, allowing the codominant detection of Tsn1. The Tsn1 markers were validated on over 1,500 wheat accessions, demonstrating a near perfect ability to determine if an accession would be insensitive to ToxA. The application of these markers in wheat breeding programs can effectively reduce susceptibility to ToxA-producing pathogens. Snn5-B1 candidates were identified in the Chinese Spring genome and validated using random mutagenesis, targeted mutagenesis, and the Cadenza TILLING mutants. Snn5-B1 contains protein kinase and major sperm protein domains. Furthermore, a second SnTox5 sensitivity locus, designated Snn5-B2, was mapped to the short arm of chromosome 2B in durum wheat. The cloning of susceptibility genes Tsc1 and Snn5-B1 allows for the development of molecular markers based on causal polymorphisms and for gene-disruption though gene-editing methods for the selection or creation of nonfunctional alleles that cannot be targeted by NE to induce cell death and disease.