REGULATION OF THE BROAD-SPECTRUM DISEASE RESISTANCE PROTEIN RPW8.2 BY PHOSPHORYLATION AND 14-3-3 IN ARABIDOPSIS

Abstract

Ascomycete powdery mildew (PM) fungi belonging to the order of Erysiphales cause diseases on more than 10,000 plant species including economically important staple food crops and numerous horticultural plants. The Arabidopsis protein RPW8.2 confers broad-spectrum resistance against all infectious powdery mildew pathogens. RPW8.2 is unique among characterized plant R proteins in that it activates broad-spectrum resistance to PM fungi, and the protein is specifically targeted to the extra-haustorium membrane (EHM). However, how RPW8.2 is regulated to exert haustorium-targeted defenses remains poorly characterized. To understand how RPW8.2 is regulated, I first performed a thorough site-specific mutagenesis of potential serine or threonine residues in RPW8.2 and identified two residues, threonine at 64 and serine at 138 to be critical for RPW8.2’s function. While the T64A mutation makes RPW8.2 auto-active, the S138A mutation abolishes RPW8.2’s ability to activate cell death and defense, with S138A being dominant over T64A. This suggests that RPW8.2 is negatively and positively regulated by (de)phosphorylation at T64 and S138, respectively. One candidate phosphatase and two kinases were genetically and biochemically tested for a potential role in (de)phosphorylation of RPW8.2. I also investigated how an RPW8.2-interacting protein 14-3-3λ regulates RPW8.2’s function. To this end, I developed a novel, divalent 14-3-3-sequestering protein named RYC to circumvent likely functional redundancy among different 14-3-3 isoforms. Our results demonstrate that RYC can effectively sequester multiple 14-3-3 isoforms from plants and human. When expressed in guard cells of Arabidopsis, RYC sequestered 14-3-3s away from H+-ATPases, thereby inducing stomatal closure, which in turn increased drought tolerance of transgenic Arabidopsis. When expressed in powdery-mildew-invaded cells, RYC abrogated RPW8.2-mediared resistance to powdery mildew, yet did not grossly affect its EHM-specific localization, suggesting that the C-terminus of RPW8.2 may exert a self-inhibition function which can be relieved when 14-3-3λ binds to the C-terminus during fungal infection. Taken together, our results lead to a better understanding of the molecular mechanisms regulating RPW8.2.