Dissecting the mechanisms of Joka2 mediated defence-related autophagy

Abstract

Phytophthora infestans forms infection structures, haustoria, that enable translocation of virulence proteins, effectors. Invaded cells respond with a spatially confined defence response known as focal immunity, a poorly understood process that is implicated in the concentration of plant immune responses around haustoria. P. infestans counteracts this by deploying PexRD54, an effector that antagonizes Joka2 mediated immunity across the extra-haustorial membrane (EHM). However, how Joka2 contributes to immunity and molecular mechanisms enhancing resistance remain unknown. In this study, I dissect the role of Joka2 in defence-related autophagy. I first investigated recruitment of defence-related host autophagy machinery, including the plant autophagy cargo receptor Joka2 and ATG9 towards the EHM (Chapter 3), I showed that trafficking of Joka2 perihaustorial region is necessary for Joka2-mediated immunity. Then, I identified candidate Joka2 interactors which were obtained from a mass spectrometry analyses of Joka2 pull-downs. Remarkably, this revealed potential Joka2 interactors, including defence-related enzymes and signalling components that are implicated in both basal plant immunity and effector triggered immunity. By using a co-immunoprecipitation approach, I validated that PAL and MAP kinases 3, 6, and 9 interact with Joka2 in vivo, but not PDX. I showed that PAL-Joka2 interaction is UBA domain dependent and MAPK-Joka2 association is through the uncharacterized domain(s) of Joka2 (Chapter 4). Consistent with this, Joka2 co-expression together with MAPK cascade activation stabilised MAPKs protein levels which shows MAPKs are not autophagy substrates recruited by Joka2. Furthermore, abundant Joka2 enhanced hypersensitive response triggered by these MAPK’s pathway (Chapter 5). I also found that Joka2 loses its ability to contribute to immunity upon removal of the domain responsible for MAPK binding, indicating that Joka2-MAPK interaction is essential for activation of defence-related autophagy. These results implicate selective autophagy in plant focal immunity and suggest more complex functions for autophagy than the widely known degradative roles.