Identification and functional characterization of putative (a)virulence factors in the fungal wheat pathogen Zymoseptoria tritici

Zymoseptoria tritici (Desm.) Quaedvlieg &amp; Crous (previously known as Mycosphaerella graminicola) is the causal agent of septoria tritici blotch (STB), which is a devastating foliar wheat disease worldwide. It is responsible for significant yield losses occurring annually in all major wheat-growing areas and threatens global food security. Z. tritici is a hemi-biotrophic fungal pathogen that, after stomatal penetration, establishes a stealthy biotrophic and symptomless relation with its host plant that is followed by a sudden switch to a necrotrophic growth phase coinciding with chlorosis that eventually develops in large necrotic blotches containing many pycnidia producing asexual splash-borne conidia. Under natural conditions - once competent mating partners are present and conditions are conducive- pseudothecia are formed producing airborne ascospores. Disease management of STB is primarily achieved through fungicide applications and growing commercial cultivars carrying Stb resistance genes. However, the efficacy of both strategies is limited as strains resistant to fungicides frequently develop and progressively dominate natural populations, which hampers disease management; also&nbsp;the deployed Stb genes are often overcome by existing or newly developed isolates of the fungus. Hence, there is a need for discovery research to better understand the molecular basis of the host-pathogen interaction that enables breeders to identify and deploy new Stb genes, which will eventually contribute to more sustainable disease control.&nbsp;&nbsp; <strong>Chapter 1 </strong>introduces the subject of the thesis and describes various aspects of the lifestyle&nbsp;of Z. tritici with emphasis on dissecting the various stages and physiological processes during pathogenesis on&nbsp;wheat. In addition, it includes a short summary and discussion of the current understanding of the role of (a)virulence factors in the Z. tritici&ndash;wheat pathosystem.&nbsp; <strong>Chapter 2 </strong>describes new gateway technology-driven molecular tools comprising 22 entry constructs facilitating rapid construction of binary vectors for functional analyses of fungal genes. The entry vectors for single, double or triple gene deletion mutants were developed using hygromycin, geneticin and nourseothricin resistance genes as selection markers. Furthermore, these entry vectors contain the genes encoding green fluorescent (GFP) or red fluorescent (RFP) protein in combination with the three selection markers, which enables simultaneous tagging of gene deletion mutants for microscopic analyses. The functionality of these entry vectors was validated in Z. tritici and described in <strong>Chapters 3, 4 and 5</strong>.&nbsp; <strong>Chapter 3 </strong>describes the functional characterization of ZtWor1, the orthologue of Wor1 in the fungal human pathogen Candida albicans. ZtWor1 is up-regulated during&nbsp; initiation of colonization and fructification, and regulates expression of candidate effector genes, including one that was discovered after comparative proteome analysis of Z. tritici wild-type and &Delta;ZtWor1 strains. Cell fusion and anastomosis occurred frequently in &Delta;ZtWor1 strains, which is reminiscent of mutants of MgGpb1, the &beta;-subunit of the heterotrimeric G protein. Comparative expression profiling of &Delta;ZtWor1, &Delta;MgGpb1 and &Delta;MgTpk2 (the catalytic subunit of protein kinase A) strains, suggests that ZtWor1 is downstream of the cyclic adenosine monophosphate (cAMP) pathway that is crucial for pathogenicity of many fungal plant pathogens.&nbsp; <strong>Chapter 4 </strong>describes combined bioinformatics and expression profiling studies during pathogenesis in order to discover candidate effectors of Z. tritici important for virulence. In addition, a genetic approach was followed to map quantitative trait loci (QTLs) in Z. tritici carrying putative effectors. Functional analysis of two top effector candidates, small-secreted proteins SSP15 and SSP18, which were selected based on their expression profile in planta, showed that they are dispensable for virulence of Z. tritici. These analyses suggest that generally adopted criteria for effector discovery, such as protein size, number of cysteine residues and up-regulated expression during pathogenesis, should be taken with caution and cannot be applied to every pathosystem, as they likely represent only a subset of effector genes.&nbsp;&nbsp; <strong>Chapter 5 </strong>describes the functional characterization of ZtCpx1 and ZtCpx2 encoding a secreted and a cytoplasmic catalase-peroxidase (CP) in Z. tritici, respectively. Gene replacement of ZtCpx1 resulted in mutant strains that were sensitive to exogenously added H2O2 and in planta phenotyping showed they are significantly less&nbsp;virulent compared to wild-type. All mutant phenotypes could be restored to wild-type by&nbsp;complementation&nbsp;with the wild-type allele of ZtCpx1 driven by its native promoter. Additionally, functional analysis of ZtCpx2 confirmed that this gene encodes&nbsp;a secreted CP and is, however, dispensable for virulence of&nbsp;Z. tritici on wheat. However, we showed that both genes act&nbsp;synergistically, as the generated double knock-out strain showed a significantly stronger reduction in virulence than the individual single knock-out&nbsp;strains. Hence, both genes are required by Z. tritici for successful infection and colonization of&nbsp;wheat.&nbsp; In <strong>Chapter 6 </strong>I discuss and summarize the genetic approaches used in this study, reflect on the major findings and bottlenecks encountered, and propose new&nbsp; strategies to identify effectors of Z. tritici in the future.&nbsp; &nbsp