Investigating the delivery and secretion of effectors in the rice blast fungus Magnaporthe oryzae

Abstract

Magnaporthe oryzae is a hemi-biotrophic fungus and the causal agent of rice blast disease, which is a serious threat to global rice production. During its biotrophic phase, the fungus feeds and develops within living plant cells. To facilitate this state, the fungus secretes effectors that suppress plant immunity and modify host cell structure, metabolism and function. Effectors in M. oryzae can be classified as either cytoplasmic effectors or apoplastic effectors, depending on where they are localised during host colonisation. Cytoplasmic effectors accumulate in a membrane-rich plant structure called the biotrophic interfacial complex (BIC), while apoplastic effectors are found between the fungal cell wall and the extra-invasive hyphal membrane which surrounds invasive hyphae. A previous report has provided evidence that different secretion pathways operate to drive effector secretion from fungal hyphae, including a nonconventional Golgi-independent pathway for secretion of cytoplasmic effectors. Little is known, however regarding how these secreted effectors are delivered to the correct domains and, in particular, how cytoplasmic effectors are translocated to plant cells. In this thesis, I report that the promoter and signal peptide sequences of effector-encoding genes are involved in delivering an effector into the correct domain. I generated a library of chimeric effectors that were systematically tested for localisation and translocation during M. oryzae growth inside the rice cell. This showed that when the promoter and signal peptide of a cytoplasmic effector gene was used to control expression of an apoplastic effector, then it was re-directed to the BIC and translocated into plant cells. Conversely, cytoplasmic effectors could be re-directed to the apoplast when expressed under control of the promoter and signal peptide region of an apoplastic effector gene.I also observed M. oryzae invasive hyphae in live cell imaging experiments with stable transgenic rice plants in which early endosomal compartments and the plant plasma membrane were fluorescent tagged. These transgenic rice lines allow the use of plasmolysis assays to observe effector translocation inside host cells and to visualise endosomal trafficking at the BIC structure. When considered together, the thesis provides evidence that effector secretion is controlled by sequences at the 5’ end of effector genes that are sufficient to direct effector delivery into the appropriate pathway during plant infection. These signals are independent of the nature of the protein being secreted.