Examination of a necrosis and chlorosis inducing protein produced by Zymoseptoria tritici

Zymoseptoria tritici causes Septoria tritici blotch (STB), a devastating hemibiotrophic fungal disease of bread and durum wheat (Triticum aestivum L. and T. turgidum L. ssp. durum). The pathogen inflicts high costs in crop protection and substantial reduction of yield, making STB the most important disease of wheat in Europe and Russia and a top ranking wheat disease worldwide. Current measures of control are limited to resistance breeding and fungicide application, both of which have been rapidly overcome by Z. tritici in the past. Therefore, substantial research into the molecular mechanisms underlying STB development is being undertaken to support development of new sustainable methods for crop protection.In previous work, specific in vitro liquid culture growth conditions for Z. tritici were established, which induced production of a phytotoxic activity. Upon infiltration into host leaves, the sterile culture filtrates (secretome) caused necrosis and chlorosis reminiscent of STB disease symptoms. The work reported in this thesis aimed at investigating this necrosis and chlorosis activity (NCA). A preliminarily characterisation of the NCA was undertaken and the proteinaceous nature of the NCA agent confirmed. Fungal culture and in planta assay conditions were studied for sources of the observed variability in intensity of NCA. Subsequently, a method to reduce the complexity in the secretome sample was established using fast protein liquid chromatography (FPLC). This reduced complexity secretome (eluate) was analysed using mass spectrometry (MS) and N-terminal sequencing for the presence of an NCA effector candidate. Effectors are small, secreted and cysteine rich proteins involved in pathogenicity of a disease-inflicting organism. Such an NCA effector candidate, with protein ID 69140, was identified in the eluate and underwent comprehensive analysis.In silico analysis for the 69140 protein revealed properties consistent with the above stated effector characteristics. Consequently, molecular methods were applied to disrupt the 69140 gene in Z. tritici to investigate the effects on pathogenicity. Since 69140 knock-out mutants of Z. tritici retained at least partial NCA, no unequivocal conclusion for involvement of this protein in NCA could be drawn. Heterologous expression of the candidate effector was attempted to resolve this question, but was unsuccessful. Furthermore, the analysis of the effect of the pure protein in planta is needed to confirm or refute the hypothesis that 69140 is involved in the NCA.In order to generate comprehensive effector candidate lists for the remaining observed NCA, transcriptomic and proteomic analyses were carried out comparing cultures producing the NCA effector with those that do not. RNA sequencing was used to reveal genes higher expressed in cultures inducing the NCA than in non-NCA inducing cultures. Using the same two distinct conditions, this approach was complemented by MS analysis of the secretome, identifying proteins with higher abundance in secretome inducing NCA.Several top ranking effector candidates on the generated lists have no detectable homology in protein databases, suggesting unknown functionality. One top effector candidate, protein 104571, allows speculation on mechanisms consistent with the observed NCA and accrued data about the wheat host immune system. Proteins 69140 and 104571, collectively or as individual effectors, are strong candidates for involvement in NCA, but other effector candidates are equally important. Future research on these effector candidates is anticipated to improve understanding of the molecular mechanism underlying the NCA and may facilitate resistance screening.