The Role of Small, Secreted Proteins from Zymoseptoria tritici during Disease Progression in Wheat

Zymoseptoria tritici causes the devastating disease Septoria Tritici Blotch (STB) in wheat. This fungal pathogen is of high economic importance and can reduce yields by up to 50%. The Z. tritici genome includes ∼490 genes which encode small, secreted proteins (ZtSSPs), called effectors. These proteins can suppress host immunity and facilitate infection. In this study, the subcellular localisation of 100 ZtSSPs was analysed in silico using online localisation prediction tools. This analysis, along with subsequent biolistic studies, identified ZtSSPs with a potential non-apoplastic localisation. The Pseudomonas fluorescens Effector-to-Host Analyser System (EtHAn) was tested for ZtSSP delivery and subsequent screening for induction of cell death in wheat. The Z. tritici candidate effector ZtARLP1 was found sharing structural homology with abscisic acid (ABA) receptors from Arabidopsis thaliana and wheat. To investigate a potential role in hormone signalling, ZtARLP1 was ectopically expressed in Arabidopsis. ZtARLP1 +SP Arabidopsis lines were found significantly more sensitive to ABA, compared to ZtARLP1 -SP lines. In addition, exogenous application of ABA increased STB symptoms, while the ABA-marker genes TaRAB18-A, TaRAD29B-A, TaABI1, and TaNCED3, were found to be upregulated during the transition of Z. tritici from biotrophic/latent necrotrophic to necrotrophic growth. These results suggest that ABA is a key hormone in the wheat – Z. tritici interaction, and ZtARLP1 may manipulate ABA signaling to promote disease in wheat. Furthermore, the role of ZtSSP2 in Z. tritici pathogenicity was investigated by assessing three independently generated gene replacement fungal knock-out (KO) strains (ΔZtSSP2_Δ1H; ΔZtSSP2_Δ7A; ΔZtSSP2_Δ8A). In planta phenotyping data showed that these ZtSSP2 KO mutants had significantly reduced disease symptoms and produced fewer fruiting bodies in wheat, compared to wildtype Z. tritici. This suggests that ZtSSP2 plays a role in pathogenicity. In conclusion, ZtSSPs are key in host defence manipulation. The present study identified the potential intrinsic role of Z. tritici effectors in disease. Additionally, novel pathogenicity strategies employed by this fungus were discovered. Results from this research work can help develop efficient STB control strategies in the future.