Identifying pathogenicity factors in the wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici is a necrotrophic fungal pathogen causing the disease Septoria tritici blotch (STB) on leaves of bread wheat and durum wheat. It is one of the most devastating wheat pathogens in Europe and causes significant yield losses globally. Current management of STB is limited to resistance breeding and fungicide application. However, no commercial wheat line is resistant to all Z. tritici isolates and the pathogen is reported to be increasingly resistant to fungicides. Thus, many efforts have been made to elucidate the STB disease mechanisms. Quantitative and qualitative differences in virulence among different isolates are commonly observed within Z. tritici populations. Recent studies have identified Avr3D1 which quantitatively affects the STB disease severity, as well as Mg3LysM and AvrStb6 that qualitatively impact the STB disease outcome. Yet, the genetic components of Z. tritici that underpin the differences in its quantitative/qualitative virulence remain largely elusive. This thesis aimed to identify the Z. tritici genes contributing to both quantitative and qualitative virulence. The first experimental chapter presents the analyses of an intra-specific comparative transcriptomic study consisting of three Australian Z. tritici isolates. All the three strains caused necrotic lesions on the WW2449 (wheat cultivar) leaves, but their ability to sporulate on the WW2449 leaves differed dramatically. Differential expression analysis was performed using the data obtained from this RNA-seq experiment, and a list of differentially expressed genes potentially involved in the quantitative virulence difference of these isolates was generated. Within this list, a multi-copy gene down-regulated in the Z. tritici strain showing the most abundant number of pycnidia during infection on WW2449 was identified. This gene was named REP9-1 and was chosen for further study. In silico analysis revealed that REP9-1 resides in a novel Class II transposable element in the Zymoseptoria genus. Silencing and overexpression of this gene in two distinct genetic backgrounds demonstrated that the expression of REP9-1 negatively influences the asexual reproduction of Z. tritici during infection. The impact of REP9-1 is independent of the host genotype, adding to the complexity of this pathogen. In addition to the identification of Z. tritici genes with quantitative impacts on virulence, I also attempted to identify genes influencing the virulence of Z. tritici qualitatively via UV-mutagenesis. The wildtype isolate WAI332 used in this experiment is non-pathogenic on several wheat cultivars, including Arina and Israel_493. After the generation and screen of 1,000 UV mutants, I obtained 18 mutants that have gained virulence on the resistant wheat varieties (eight on Arina, eight on Isral_493, and two on both). The genomes of the mutants have been sequenced; variants within each mutant have been identified. Although no genes were commonly disrupted in all the mutants from the same virulent group(s) and no potential avirulence gene candidates were identified, the variants data will contribute to the future discovery of genes underpinning the qualitative virulence of WAI332. Collectively, I aimed to identify genes impacting the quantitative/qualitative virulence of Z. tritici. This work has identified a gene REP9-1 residing in a novel transposon that has a quantitative effect on the pycnidiation of Z. tritici. These results improved our understanding of the quantitative factors that influence this disease, in this case a transposable element gene whose expression limits asexual reproduction in the host. In addition, several gain-of-virulence mutants were generated on two wheat cultivars. Further, work is required to establish whether this phenotype is due to a quantitative or qualitative virulence factor. Together this work contributes several genes and isolates for future study on the genetic components affecting the virulence of Z. tritici