On the mechanism of translocation of Magnaporthe oryzae effectors into rice cells

Rice blast caused by Magnaporthe oryzae is the most destructive disease of rice worldwide. During the infection process, M. oryzae secretes various effectors, which are hypothesized to be involved in effective host infection. Notably, cytoplasmic effectors of M. oryzae are associated with a specialized interfacial structure, the biotrophic interfacial complex (BIC). To date, little is known about the mechanisms of effector uptake into plant cells during fungal infection. Here we show translocation of the cytoplasmic effectors Bas1, Pwl1 and Pwl2 in vesicles from BICs to rice cytoplasm during biotrophic development. Using fluorescent protein tagging, we found that cytoplasmic effectors Bas1, Pwl1 and Pwl2 are sorted into different vesicles in BICs formed on primary hyphae (PH), revealing new levels of functional complexity for this biotrophic structure. In contrast, most of the vesicles from BICs on mature bulbous hyphae showed colocalization of the cytoplasmic effectors. Whereas BICs on primary hyphae deliver effectors in micro-vesicles, BICs on mature bulbous hypha deliver effectors in macro-vesicles, at times reaching diameter sizes over 3 µm. Through FM4-64 labeling and Lit6b:eGFP transgenic rice colocalization assays, we demonstrated that the vesicles containing effectors originate from plant plasma membrane. Furthermore, we demonstrate that endocytosis inhibitors, Cantharidin, Triclosan and Wortmannin, induces abnormally shapes and swollen BICs as well as the accumulation of cytoplasmic effectors in the BICs. Moreover, Cantharidin, Triclosan and Wortamannin treatment induced the accumulation of the cytoplasmic effectors under penetration pores, suggesting that effector uptake begins even before host penetration. Based on these results we conclude that cytoplasmic effector translocation is mediated by vesicle formation and may be characteristic of appressoria as well as biotrophic invasive hyphae. Our results also suggest a potential role of M. oryzae effectors for manipulation of the host cell endocytosis process.