Identification and characterization of novel factors involved in dimorphism and pathogenicity of fungal wheat pathogen Zymoseptoria tritici
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Abstract
In the current study Zymoseptoria tritici (teleomorph: Mycosphaerella graminicola) was used as a model organism, representing one of the most economically important fungal pathogens worldwide. This fungus causes Septoria blotch disease on wheat plants, responsible for massive crop losses every year. One of the characteristic features of this fungus is its ability to switch between two distinct growth forms, which can be induced by different environmental conditions. The fungus differentiates a yeast-like growth form, which is observed on nutrient-rich culture media. On the other hand, the fungus propagates in filamentous growth form, which is induced by nutrient deprivation or elevated temperature. The latter form is associated with pathogenicity, allowing the fungus to infect and colonize the host plant. A forward genetics approach was applied, aiming to investigate the molecular basis of the morphological transition of the wheat pathogenic fungus Z. tritici. A previously developed Agrobacterium tumefaciens mediated transformation protocol was used to generate a mutant library by insertional mutagenesis including more than 10000 random insertional transformants. A plate-based screening system was established, to identify mutant strains harbouring defects in dimorphic transition. Hence, with this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing a high amount of mycelium under different incubation conditions. Sequence analysis of flanking regions for T-DNA insertions revealed potential candidate genes responsible for phenotypes observed. Relying on selection criteria defined, four transformants were characterized in detail. Three of them are non-pathogenic and one mutant exhibits a drastically impaired virulence within infection assays on whole wheat plants. Using genome walking, the T-DNA integration sites could be recovered, and the disrupted genomic loci of corresponding mutants were validated by reverse genetics approach. We present four functionally characterized genes involved in the dimorphic switch of the fungal plant pathogen Z. tritici, yet not described in the literature. In addition, transcriptome analysis of selected mutant strains by RNA-Seq was performed to get first insight into regulons associated with yeast-to-hyphal transition. The outcome of the RNA-Seq analysis provides a substantial list of target genes proposed to be implicated in regulation of dimorphic switch. Among the genes obtained, were those, whose role in the pathomorphogenic processes was previously established in other fungal pathogens. Moreover, making use of bioinformatic analysis, we also identified sets of differentially expressed genes previously not described and likely having unique role for dimorphic switch in Z. tritici. Based on these data, one further gene was characterized. This gene probably encodes an essential transcription factor, responsible for the coordination of meiotic division and spore formation. Based on results obtained from in silico analysis, its role for cellular processes in Z. tritici will be discussed.