Molecular and Mechanistic Study of Phytophthora RxLR Effector PSR2 in Arabidopsis

Phytophthora belong to a group of fungus-like and zoospore-forming microorganisms, which are important plant pathogens that cause diseases on a broad range of crop and tree species worldwide. However, the control of Phytophthora diseases remains challenging due to the lack of understanding of their pathogenesis. Phytophthora are successful plant pathogens since they encode hundreds of effectors to suppress plant immune responses. Among them, the PSR2 family effectors are evolutionarily conserved among several Phytophthora species. Both PSR2 (encoded by Phytophthora sojae) and PiPSR2 (encoded by Phytophthora infestans) function as RNA silencing suppressors and are able to promote Phytophthora infection in plants. To understand the molecular mechanisms by which PSR2 suppresses RNA silencing and increase disease susceptibility in plants, I identified serine/threonine protein phosphatase 2A (PP2A) as a PSR2-associating protein in plants. PP2A is a heterotrimeric enzyme consisting of scaffold A, regulatory B, and catalytic C subunits, where each subunit is encoded by gene families with multiple members. PSR2 has stronger associations with A subunits, weaker associations with C subunits, and no association with B subunits. Arabidopsis transgenic plant expressing PSR2 showed reduced production of phasiRNAs, which might be one of the underlying mechanisms suppressing plant immunity. To determine the functional involvement of PP2A subunits in PSR2-mediated RNA silencing suppression, I examined small RNA accumulation in transgenic Arabidopsis with PSR2-expressed in pp2a mutation backgrounds. Interestingly, the reduction of the phasiRNAs caused by PSR2 was rescued by the scaffold subunit rcn1 and pdf1 mutations, while these rcn1 and pdf1 mutants alone did not alter small RNA biogenesis. In addition, PSR2 deletion mutants that had reduced interaction with PP2A partially lost the phasiRNA suppression activity, suggesting the functional involvement of PP2A in PSR2-mediated small RNA suppression.Lastly, mass spectrometry analyses revealed plenty of PP2A B subunits in RCN1-, but not PSR2-, associated protein complexes. Thus, PSR2 may serve as a regulatory B subunit to modulate the function of PP2A core enzyme (consisting of A and C subunits). This hypothesis was further supported by that PSR2 structurally mimicked PP2A B family subunits and it also shared similar binding sites to the scaffold with B family subunits. Furthermore, PSR2 was able to compete out a B subunit from the PP2A by an initial replacement pull-down assay. Together, my thesis research provides novel mechanistic insights into the pathogenesis of Phytophthora PSR2 effector by hijacking PP2A core enzyme in plants to suppress plant RNA silencing