Han Juan, Liu Chang-Xin, Liu Jian, Wang Cheng-Run, Wang Shun-Chang, Miao Guopeng
Department of Bioengineering, Huainan Normal University, Huainan, Anhui Province 232038, China.
Institute of Digital Ecology and Health, Huainan Normal University, Huainan, Anhui Province 232038, China.
Plant Physiol. 2024 Jun 28;195(3):1835-1850. doi: 10.1093/plphys/kiae186.
Plant transporters regulating the distribution of secondary metabolites play critical roles in defending against pathogens, insects, and interacting with beneficial microbes. The phosphorylation of these transporters can alter their activity, stability, and intracellular protein trafficking. However, the regulatory mechanism underlying this modification remains elusive. In this study, we discovered two orthologs of mammalian PKA, PKG, and PKC (AGC) kinases, oxidative signal-inducible 1 (OXI1) and its closest homologue, AGC subclass 2 member 2 (AGC2-2; 75% amino acid sequence identity with OXI1), associated with the extracellular secretion of camalexin and Arabidopsis (Arabidopsis thaliana) resistance to Pseudomonas syringae, and Botrytis cinerea. These kinases can undergo in vitro kinase reactions with three pleiotropic drug resistance (PDR) transporters: PDR6, PDR8, and PDR12. Moreover, our investigation confirmed PDR6 interaction with OXI1 and AGC2-2. By performing LC-MS/MS and parallel reaction monitoring, we identified the phosphorylation sites on PDR6 targeted by these kinases. Notably, chitin-induced PDR6 phosphorylation at specific residues, namely S31, S33, S827, and T832. Additional insights emerged by expressing dephosphorylated PDR6 variants in a pdr6 mutant background, revealing that the target residues S31, S33, and S827 promote PDR6 efflux activity, while T832 potentially contributes to PDR6 stability within the plasma membrane. The findings of this study elucidate partial mechanisms involved in the activity regulation of PDR-type transporters, providing valuable insights for their potential application in future plant breeding endeavors.
调节次生代谢物分布的植物转运蛋白在抵御病原体、昆虫以及与有益微生物相互作用中发挥着关键作用。这些转运蛋白的磷酸化可改变其活性、稳定性及细胞内蛋白质运输。然而，这种修饰背后的调控机制仍不清楚。在本研究中，我们发现了哺乳动物PKA、PKG和PKC（AGC）激酶的两个直系同源物，即氧化信号诱导1（OXI1）及其最接近的同源物AGC亚类2成员2（AGC2-2；与OXI1的氨基酸序列同一性为75%），它们与拟南芥中植保素的细胞外分泌以及对丁香假单胞菌和灰霉病菌的抗性有关。这些激酶可与三种多药耐药（PDR）转运蛋白PDR6、PDR8和PDR12进行体外激酶反应。此外，我们的研究证实了PDR6与OXI1和AGC2-2的相互作用。通过液相色谱-串联质谱（LC-MS/MS）和平行反应监测，我们确定了这些激酶靶向的PDR6上的磷酸化位点。值得注意的是，几丁质诱导PDR6在特定残基（即S31、S33、S827和T832）处发生磷酸化。通过在pdr6突变体背景中表达去磷酸化的PDR6变体获得了更多见解，结果表明靶残基S31、S33和S827促进PDR6外排活性，而T832可能有助于PDR6在质膜内的稳定性。本研究结果阐明了PDR型转运蛋白活性调节的部分机制，为其在未来植物育种中的潜在应用提供了有价值的见解。
