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bioRxiv · 10.1101/2022.02.08.479400

PDR6-mediated camalexin efflux and disease resistance are regulated through direct phosphorylation by the kinases OXI1 and AGC2-2

Miao, G.·Han, J.·Liu, C.-x.·Liu, J.·Wang, C.-r.·Wang, S.-c.
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Abstract

Plant immune signaling largely relies on post-translational modifications to establish a rapid and appropriate defense response to different pathogen types and infection pressure. Specific pleiotropic drug resistance (PDR) transporters can transport secondary metabolites to contribute to pathogen invasion resistance. However, the establishment of the post-translational regulation of PDR transporters that efflux secondary metabolites is unclear. In this study, by detecting the camalexin contents on the leaf surfaces of mutants and overexpression lines, two AGC kinases, namely, OXI1 and its closest homologue AGC2-2, were found to be related to extracellular camalexin secretion. The overexpression of OXI1 or AGC2-2 resulted in an increase in camalexin contents on the leaf surface and a decrease in camalexin contents in the leaf interior. These effects increased the resistance of the transgenic lines to surface-inoculated Pseudomonas syringae and Botrytis cinerea. Through in vitro kinase assay and in vivo phosphorylation level detection, we confirmed that the two kinases were related to the phosphorylation modification of PDR6. Pull-down assays, bimolecular fluorescence complementation, and rapamycin-dependent delocalization assays indicated the existence of direct protein-protein interaction between the two kinases and PDR6. By using LC-MS/MS, we also identified the PDR6 phosphorylation sites that were modified by the two kinases in vitro. Through the expression of the dephosphorylated variants of PDR6 in the mutant background, the action site S31 of OXI1 and the action sites S33 and S827 of AGC2-2 were found to have positive effects on the efflux activity of PDR6. In addition, T832, the action site of OXI1, may contribute to the stability of PDR6 on the plasma membrane.

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Miao, G., Han, J., Liu, C.-x., Liu, J., Wang, C.-r., Wang, S.-c.. 2022-02-10. PDR6-mediated camalexin efflux and disease resistance are regulated through direct phosphorylation by the kinases OXI1 and AGC2-2. https://doi.org/10.1101/2022.02.08.479400

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