Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / 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.

La polarité cellulaire est un phénomène biologique essentiel du monde vivant. Chez la levure Candida albicans, sa capacité à croître sous une forme hyperpolarisée semble être un élément déterminant de sa pathogénicité. Nous avons entrepris d’identifier les éléments moléculaires d’une structure essentielle à cette croissance hyphale, le Spitzenkörper, afin de mieux comprendre le rôle de ce corps apical dans la croissance polarisée. Nous nous sommes également intéressés à la régulation des protéines Rho3 et Rho4 impliquées dans la croissance polarisée de C. albicans, à travers l’identification et l’étude de la protéine RhoGAP commune à ces deux protéines Rhos, la protéine Rgd1.Chez la levure Saccharomyces cerevisiæ, les protéines Rho3 et Rho4 sont également impliquées dans le contrôle de la croissance polarisée, et sont régulées par la protéine Rgd1. Le laboratoire, à l’origine de la découverte de ce régulateur commun, étudiait des aspects de croissance polarisée contrôlée par les protéines Rho3 et Rho4, à travers l’étude de la régulation de la protéine Rgd1. Nous avons notamment mis en évidence que Rgd1p est modifiée au niveau post-traductionnel par des phosphorylations. La kinase Ipl1 de la famille des kinases Aurora est un des acteurs de cette modification. Différents éléments indiquent que le complexe phosphatase Glc7-Bud14 serait également impliqué dans le contrôle de l’état de phosphorylation de Rgd1p, de façon antagoniste à la kinase Ipl1 / Cell polarity is an essential process for living organisms. In the yeast Candida albicans, its ability of hyperpolarized growth seems to be a decisive element for its pathogenicity. We undertook to identify molecular elements of an essential structure for hyphal growth, named Spitzenkörper, to better understand the role of this apical body in polarised growth. We also studied regulation of Rho3 and Rho4 proteins implicated in C. albicans polarised growth, through identification and study of a shared RhoGAP protein, named Rgd1.In the yeast Saccharomyces cerevisiæ, Rho3 and Rho4 proteins are also implicated in polarised growth control, and are regulated by Rgd1 protein. The laboratory, which identified this shared regulator, studied polarised growth aspects controlled by Rho3 and Rho4 proteins, through study of Rgd1p regulation. We showed that Rgd1p is post-translationally modified, by phosphorylations. The Ipl1 kinase, an Aurora family member, is implicated in this modification. Several elements indicate that the Glc7-Bud14 phosphatase complex would be also implicated in the control of Rgd1 phosphorylation state, antagonistically to Ipl1p