Functional and structural analyses of plant ATG8-RabGAP interactions

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Due to the lack of circulatory system and mobile immune cells, plant immunity hinges on cell autonomous immunity. A key component of this immunity is the catabolic process of autophagy, once thought to be a non-specific degradation pathway. The discovery of pathogen-specific plant cargo receptors suggested a role for autophagy in immunity. Like many plant pathogens, Phytophthora infestans, the oomycete responsible for late blight disease in potato, relies on host modulation and suppression of immunity through proteinaceous effectors to infect its host. A recent study has shown that the P. infestans secreted effector PexRD54 outcompetes the host cargo receptor Joka2 for binding of the core autophagy protein ATG8CL through their common ATG8 interacting motif (AIM). Both the effector and cargo receptor trigger autophagosome formation in an AIM dependent manner. With such an essential pathway being activated by binding alone, it was suggested that there could exist a negative regulator. Mass spectrometry analysis of ATG8CL revealed a strong interaction with a RabGTPase activating protein, Rab3GAP. Bioinformatic analysis has shown that Rab3GAP carries a putative AIM, pointing towards a regulatory role in the ATG8CL autophagy pathway. Previous work showed that not only does Rab3GAP interact with ATG8CL in vivo but also reduces ATG8CL autophagosomes in an AIM dependent manner. Furthermore, preliminary data shows that Rab3GAP increases susceptibility to P. infestans.The primary objective of this study is to analyse Rab3GAP's role in the ATG8CL autophagy at the cellular, molecular and structural level. Furthermore, I aim to elucidate Rab3GAP's role in immunity against P. Infestans and to a wider extent, the role of the ATG8CL autophagy. I intend to use Nicotiana Benthamiana as a model system for infections, expression of proteins and cell biology. I also intend to make extensive use of the Agrobacterium transformation system for transient expression assays. This will pave the way for the creation of CRISPR/Cas9 transgenic lines. To achieve this, I will focus on three aims for this project:1. Elucidating Rab3GAP's role in the ATG8CL pathway.In order to investigate Rab3GAP's role in autophagy, I intend to use a multi-faceted approach of cellular and molecular biology. First, I aim to confirm Rab3GAP's interaction with ATG8CL's AIM through in vitro interaction assays. Furthermore, I will employ Co-IP followed by mass spectrometry analysis to determine the interacting partners of Rab3GAP that are associated to the ATG8CL pathway. I will investigate the role of candiate Rab3GAP interactors in autophagy via monitoring autophagic activity upon silencing or overexpression of the Rab3GAP interactors.2. Dissecting Rab3GAP's role in immunity against P. Infestans.We aim to elucidate the mechanisms behind the increased suscepbility to P. Infestans caused by Rab3GAP. The first step would be to determine if the effect on immunity is due to the ATG8CL pathway. Secondly, we intend to look into the other domains of Rab3GAP. The GAP active site is of particular interest and indentifying the associated Rab(s) would give us insight into Rab3GAP's role in the network of membrane traffic pathways.3. Structural and functional analyses of Rab3GAP and its partners in autophagyThe final facet of our approach is to structurally characterise Rab3GAP's interactions and gain insights into its suppresive role in autophagosome formation. Understanding Rab3GAP's binding dynamics with other members of the ATG8CL autophagic pathway could help us understand the network of interactions in the pathways. We intend to identify the Rab(s) associated with Rab3GAP by modifying the catalytic arginine in the GAP active site and GTP-locking any potential Rabs to stabilise the interaction. Linking structural observations with protein function would allow us to describe essential motifs in Rab3GAP and maybe even in other members of the Rab superfamily.