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Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast is a major fungal disease on rice, caused by the hemibiotrophic filamentous ascomycete fungus, Magnaporthe oryzae. This disease accounts for 157 million tons of grain loss annually. The fungus produces a specialized cell called appressorium to penetrate the host surface barrier and enter inside. It produces intracellular Invasive Hyphae (IH) that grow form cell to cell to colonize the host. The mechanisms of appressorium formation and host penetration have been studied in detail but the host colonization strategies remain largely unknown. We applied live-cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells. Early loading experiments with the endocytotic tracker, FM4-64, showed dynamic plant membranes around IH. These hyphae showed remarkable plasticity and recruited plant cell components. IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies have revealed insights into a novel hemibiotrophic strategy employed by the blast fungus. Few genes have been previously characterized that impact the biotrophic IH. To understand the molecular basis of the biotrophic infection strategy we employed Laser Microdissection (LM) technology to isolate and purify the IH at this early growth stage. We compared the gene expression of these samples with axenically-grown mycelium using M. oryzae whole genome microarrays. We identified several hundreds of infection specific genes. We have shown that LM technology can be used to isolate homogenous cells from the infected rice tissues to study the underlying molecular mechanisms of signaling during disease formation. These studies will be very critical to understand the host-pathogen interactions to eventually develop durable management strategies.

La communication intercellulaire est essentielle pour le développement et la survie d'organismes multicellulaires. Dans le règne végétale, une des voies privilégiée pour la communication intercellulaire est la voie symplastique qui implique des canaux aux dimensions nanométriques connectant les cellules entre elles, leur permettant d'échanger directement photo-assimilats, miARN, protéines, oligoéléments etc. Observés pour la première fois en 1880 par le botaniste autrichien Eduard Tangl (Tangl 1880; Kohler & Carr 2006), ils ont longtemps été considérés comme de simples trous perméables permettant la diffusion de matériel cellulaire (Lee & Lu 2011; Oparka & Roberts 2001). Etant donné leurs taille nanoscopique, ce n'est que dans les années 1960, avec la démocratisation de la Microscopie Électronique en Transmission (MET) qui permet d'atteindre , que les premiers modèles ultrastructuraux sont établis (Lopez-Saez 1965; Robards 1970). Ils font état d'un canal d'environ 30 à 40 nm de diamètre avec un élément central cylindrique traversant le pore, appelé le desmotubule, connecté au Réticulum Endoplasmique des deux cellules (Figure 1 of our review Tilsner et al. 2016). Dans les années 1980 notre compréhension des plasmodesmes a quelque peu évolué et nous savons maintenant que ces structures ne sont pas de simples trous mais des structures membranaires très spécialisées et régulées (Lucas & Lee 2004; Faulkner & Maule 2011; Furuta et al. 2012). Le modèle ultrastructural actuel découle de la congrégation d'études ultrastructurale, physiologiques et pharmacologiques plus ou moins anciennes dépeignant une structure morphologiquement très souple et changeant de conformation au cours du développement. Les plasmodesmes peuvent réguler leur ouverture/fermeture par la constriction de leurs extrémités grâce à l'accumulation entre la membrane plasmique et la paroi végétale d'un polymère de sucre, la callose qui va pousser la membrane plasmique contre le desmotubule et en obstruer les entrées. Cette modulation permettrait majoritairement de réguler les flux intercellulaires qui impliquent les plasmodesmes. Cependant nos connaissances sur les remaniements membranaires prenant place durant le développement des plasmodesmes et sur la régulation de leur perméabilité sont encore imparfaites.La microscopie électronique en transmission, malgré l'ancienneté de la technique, est l'une des plus résolutive, largement utilisée en biologie. Avec l'amélioration des techniques de préservation d'échantillons, notamment les cryo- méthodes, elle permet d'atteindre à l'heure actuelle des résolutions inférieures à 5 nm en condition contrastée et inclus en résine et peut descendre en dessous du nanomètre pour la cryo-microscopie. Ce potentiel permet aisément l'étude des sous-compartiments cellulaires de l'ordre du µm tel que mitochondries, chloroplastes, noyaux etc. (Frey et al. 2002) mais permet également l'étude ultrastructurale précise de structures de l'ordre de la dizaine de nm (Beck et al. 2007; Al-Amoudi et al. 2007).En revanche, dans son utilisation classique, la microscopie électronique ne permet pas d'accéder à la troisième dimension de l'espace, rendant difficile l'interprétation de structure à l'architecture quelque peu compliquée. En effet, les images produites ne sont que des projections en deux dimensions d'objets en trois dimensions. Cela a mené au développement de la tomographie électronique en transmission (Crowther et al. 1970), méthode basée sur un concept mathématiques formulé par Johann Radon au XIXe siècle. Ce n'est que dans les années 2000 que la tomographie électronique a pris un essor significatif grâce au couplage avec des méthodes d'automatisation informatiques. / Plasmodesmata were first observed by Austrian botanist Eduard Tangl in 1880. He devoted himself to studying the anatomy and cytology of plants and his greatest discovery, of course, was the observation and first characterization of plasmodesmata (Tangl 1880, 1884 and 1885). Despite not having access to their ultrastructure, he observed thin striations (see front page engraving) between cotyledon cells of Strychnos nuxvomica and in the endosperm of seeds and described them as being conductive ducts. Already at the time, he was evoking the idea that these strands "unite them [the cells] to an entity of higher order", in other words formulating the first definition of a symplastic domain. lt is only in 1901 that Strasburger finally names these canals "plasmodesmata". His discovery led to a radical change in our conception of the plant entity and brought in new concepts such as the symplasm (Munch 1930) and transmembrane fluxes between cells, which are now being tackled with great interest by numerous research teams around the globe.Because of their size, plasmodesmata ultrastructure was not accessible until the advent of electron microscopy and they were long thought to be simple holes connecting plant cells one-another with no specific regulation. lt is only with the advent of electron microscopy and chemical fixation that botanists started to gain interest in this structure again. And even with these methods allowing the observation of structures down to several nanometers in size, there are still debates on the nature of the canal, its constituents and physiology (Lopez-Saez J. 1965, Robards A. 1970, Ding et al. 1992, Tilney et al. 1991, Overall and Gunning 1982, Schulz et al. 1995).Nowadays, with the advent of modern cryopreservation and three-dimensional electron tomography methods, great improvements are to be done in the understanding of the ultrastructure and physiology of these mysterious canals. More particularly by understanding the link between the membranous rearrangements taking place in these pores and the molecular transit regulation.My work has led us to view plasmodesmata as specialised Membrane Contact Sites (MCS). Hence, by analogy with MCS found in mammals, yeast and plants, this work embraces an original angle on the speculation of the composition and role of the desmotubule-plasma-membrane tethering complex. The work produced during my thesis allowed me to contribute to the publication of one review and two articles, which will constitute the introduction and two main sub-sections of the results chapter, respectively. The introductory review has been published in 2016 in Annual Review of Plant Biology. The first one is still under reviewing at Nature Plant and the other has been published in The Plant Cell journal in April 2015.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25340号 / 農博第2606号 / 新制||農||1107(附属図書館) / 学位論文||R6||N5512 / DFAM / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.