Characterizing the molecular mechanism of the AvrRpt2-RIN4- RPS2 defense-activation module
Pathogens deploy virulence effectors to perturb host processes. Plants utilize intracellular resistance (R) proteins to recognize pathogen effectors either by direct interaction or indirectly via effector-mediated perturbations of host components. RPM1-INTERACTING PROTEIN4 (RIN4) is a plant immune regulator that mediates the indirect activation of multiple, independently evolved R-proteins by multiple, unrelated effector proteins. One of these, RPS2 (RESISTANT TO P. SYRINGAE2), is activated upon cleavage of Arabidopsis (At)RIN4 by the Pseudomonas syringae effector AvrRpt2. The currently favored model for RPS2 activation states that RPS2 is activated post AvrRpt2 mediated elimination of RIN4. However, this model does not reconcile with the hypothesized link between the virulence activity of AvrRpt2 and the activation of RPS2. In this study we have demonstrated that in the presence of wild type RIN4, nonmembrane tethered derivatives of RIN4 activate RPS2. Additionally we demonstrate that the AvrRpt2 induced cleavage fragments of RIN4 take up new roles in RPS2 regulation. The membrane-tethered fragment retains the ability to suppress RPS2, while the non-membrane tethered fragment is able to overcome this suppression. These results link the AvrRpt2-induced cleavage fragments of RIN4 to the activation of RPS2. To further gain insight into the AvrRpt2-RIN4-RPS2 defense-activation module, we compared the function of AtRIN4 with RIN4 homologs present in a diverse range of plant species. We selected seven homologs containing conserved features of AtRIN4, including two NOI (Nitrate induced) domains, each containing a predicted cleavage site for AvrRpt2, and a C-terminal palmitoylation site predicted to mediate membrane tethering of the proteins. Palmitoylation-mediated tethering of AtRIN4 to the plasma membrane is required for suppression of RPS2. We have demonstrated that while all seven homologs localized at the plasma membrane, only four suppressed RPS2 when transiently expressed in Nicotiana benthamiana. All seven homologs were cleaved by AvrRpt2 and, for those homologs that were able to suppress RPS2, cleavage relieved suppression of RPS2. Lastly, we show that the membrane localization of RPS2 is unaffected by its suppression or activation status.
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