In planta characterization of Magnaporthe oryzae biotrophy-associated secreted (BAS) proteins and key secretion components

Abstract

Rice blast caused by the ascomycetous fungus Magnaporthe oryzae remains a threat to global sustainable agriculture and food security. This pathogen infects staple cereal crops such as rice, wheat, barley and millets, as well as turf grasses, in a distinct way among fungal plant pathogens, which we described in the first chapter. In addition to economical importance, rice blast is a model pathosystem for difficult-to-study biotrophic fungi and fungal-plant interactions. We are studying proteins that fungi secrete inside living cells to block plant defenses and control host cell processes; these proteins are called effectors. To date mechanisms for secretion and delivery of effectors inside host cells during disease establishment remain unknown. This step is critical to ensure the successful infection. So far, the only commonality found among all unique small-secreted blast effector proteins is their accumulation in a novel in planta structure called the biotrophic-interfacial complex (BIC). Identifying effectors and understanding how they function inside rice cells are important for attaining durable disease control. In the second chapter, we presented one approach to address this challenge. We characterized four candidate effector genes that were highly expressed specifically during the rice cell invasion. Using transgenic fungi that secrete fluorescently-labeled versions of each protein allowed me to follow them during invasion in vivo by live cell imaging. These candidates show distinct secretion patterns suggesting a spatially-segregated secretion mechanism for effectors. Results revealed a BIC-located strong candidate cytoplasmic blast effector, two putative cell-to-cell movement proteins and a putative extrainvasive hyphal membrane (EIHM)-matrix protein, which has become a valuable tool for assessing successful infection sites. In the third chapter, we test if normal secretion components of filamentous fungi are involved in accumulation of effectors into BICs. We report localization studies with M. oryzae orthologs of conserved secretion machinery components to investigate secretion mechanisms for effectors showing preferential BIC accumulation and for non-BIC proteins such as BAS4. Especially bright fluorescence adjacent to BICs from Mlc1p (Myosin Light Chain, a Spitzenkörper marker), from Snc1p (a secretory vesicle marker), and from Yup1p (a putative t-SNARE endosomal protein) suggest secretion actively occurs in the BIC-associated cells. Localization of Spa2p (a polarisome marker), as a distinct spot at the tips of the bulbous invasive hyphae (IH) in planta, suggests the existence of two secretion complexes after the fungus switches growth from the polarized filamentous primary hyphae to bulbous IH. In the final chapter on future perspectives, we present some strategies towards the molecular understanding of the M. oryzae secretion mechanism during biotrophic invasion, which will lead to novel strategies for disease control.