Boyce Thompson Institute for Plant Research, Ithaca, NY 14853, U.S.A.
Section of Plant Pathology and Plant-Microbe Biology, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853, U.S.A.
Biochem J. 2019 Jun 11;476(11):1621-1635. doi: 10.1042/BCJ20190299.
Plant immune responses, including the production of reactive oxygen species (ROS), are triggered when pattern recognition receptors (PRRs) become activated upon detection of microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases are key components of PRR-dependent signaling pathways. In tomato, two such kinases, Pti1a and Pti1b, are important positive regulators of the plant immune response. However, it is unknown how these kinases control plant immunity at the molecular level and how their activity is regulated. To investigate these issues, we used mass spectrometry to search for interactors of Pti1b in leaves and identified a PP2C protein phosphatase, referred to as Pic1. An pull-down assay and split-luciferase complementation assay verified this interaction. Pti1b was found to autophosphorylate on threonine-233, and this phosphorylation was abolished in the presence of Pic1. An arginine-to-cysteine substitution at position 240 in the MARIS kinase was previously reported to convert it into a constitutive-active form. The analogous substitution in Pti1b made it resistant to Pic1 phosphatase activity, although it still interacted with Pic1. Treatment of leaves with the MAMP flg22 induced threonine phosphorylation of Pti1b. The expression of Pic1, but not a phosphatase-inactive variant of this protein, in leaves greatly reduced ROS production in response to treatment with MAMPs flg22 or csp22. The results indicate that Pic1 acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.
植物的免疫反应，包括活性氧（ROS）的产生，是在模式识别受体（PRRs）检测到微生物相关分子模式（MAMPs）时被激活而引发的。类受体细胞质激酶是 PRR 依赖的信号通路的关键组成部分。在番茄中，两种这样的激酶，Pti1a 和 Pti1b，是植物免疫反应的重要正调控因子。然而，这些激酶如何在分子水平上控制植物的免疫力以及它们的活性如何被调节尚不清楚。为了研究这些问题，我们使用质谱法在叶片中搜索 Pti1b 的相互作用物，并鉴定出一种称为 Pic1 的 PP2C 蛋白磷酸酶。下拉测定和分裂萤光素酶互补测定验证了这种相互作用。发现 Pti1b 在苏氨酸-233 上自身磷酸化，而在 Pic1 存在下，这种磷酸化被消除。先前报道在 MARIS 激酶的 240 位精氨酸突变为半胱氨酸，可将其转化为组成型激活形式。Pti1b 中的类似取代使其对 Pic1 磷酸酶活性具有抗性，尽管它仍然与 Pic1 相互作用。用 MAMP flg22 处理叶片诱导 Pti1b 的苏氨酸磷酸化。Pic1 的表达，而不是该蛋白的无磷酸酶活性变体的表达，在叶片中大大减少了对 MAMPs flg22 或 csp22 的 ROS 产生的反应。结果表明，Pic1 通过去磷酸化 Pti1b 激酶来作为负调节剂发挥作用，从而干扰其激活植物免疫反应的能力。
