番茄抗病膜蛋白TARK1稳定性的调控机理研究

Tomato Atypical Receptor-Like Kinase 1 (TARK1), as "dead" kinase, is indispensible to suppress the proliferation of Xanthomonas campestris pv. vesicatoria (Xcv) through PAMP trigger immunity (PTI). TARK1 is the tomato receptor of the effector XopN from Xcv, as the first report on "dead" kinase associated with PTI. The reasearch on stability and regulation of TARK1, a "dead" kinase involved in PTI in tomato, will advance the rational utilization of the tomato resistance genes, and will promote the discovery and functional analysis of other "dead" kinases. In this study, we will screen the elicitor which can specially induce TARK1 expression in TARK1-HA transgenic tomato treated by different elicitors including Flg22, Harpin, SA, JA, Xcv; and we will produce TARK1 transgenic line and pub12/13 X TARK1、fls2 X TARK1 crossing lines of Arabidopsis and analyze the regulation between Fls2, Pub12/13 and TARK1. Further, the interactors of TARK1 will be screened using yeast two hybrid system and Pull-down Assay, and the stability of TARK1 will be investigated by transient expression system in Nicotiana benthimiana. The phosphorylation assay and unbiquitinlation assay of associated proteins will be performed and the site of phosphorylation will be identified. The contribution of phosphorylation and ubiquitinlation of TARK1-associated proteins to PTI will be verified. Eventually, the model will be set up of regulation of TARK1 expression.

TARK1是诱导番茄PTI所必需的蛋白，是Xcv效应蛋白XopN的受体蛋白，也是第一个被发现的参与PTI作用的"死"激酶。通过对番茄TARK1稳定性调控机理解析，可为番茄相关抗病基因的合理利用提供依据；同时促进其他"死"激酶的发现与功能解析。本项目中，我们将首先利用Flg22、Harpin等不同的激发子处理TARK1转基因番茄，找出可以专化性诱导TARK1表达的激发子；同时产生TARK1拟南芥转基因系及其与pub12/13、fls2的杂交系，解析Fls2的积累以及Pub12/13泛素化作用与TARK1稳定性的关系；并通过酵母双杂交、Pull-down Assay筛选TARK1互作因子，利用瞬时表达体系分析互作因子对TARK1稳定性的影响，同时进行蛋白泛素化、磷酸化分析，鉴定蛋白磷酸化位点，验证相关蛋白泛素化、磷酸化与TARK1依赖的PTI功能之间的关系。最终提出TARK1表达调控模式。

TARK1是诱导番茄PTI所必需的蛋白，是Xcv效应蛋白XopN的受体蛋白，也是第一个被发现的参与PTI作用的“死”激酶。通过对番茄TARK1稳定性调控机理解析，可为番茄相关抗病基因的合理利用提供依据；同时促进其他“死”激酶的发现与功能解析。本项目中，我们首先研究发现PopW激发子可诱导TARK1稳定表达，PopW可诱导番茄上的PAMP-triggered immunity，而降低细菌性疮痂病菌的病情指数。产生TARK1拟南芥转基因系及其与pub12/13、fls2的杂交系，研究发现在fls2XTARK1、pub12/13XTARK1 (F0)杂交系上，激发子flg22等诱导的TARK1表达量降低。通过酵母双杂交、Pull-down Assay筛选TARK1互作因子，Western Blot 结果显示TARK1的稳定性受“候选互作因子1”的正向调控。泛素化实验结果显示，TARK1泛素化可能与效应蛋白XopN有关。TARK1与其互作因子的磷酸化结果显示，TARK1的功能与其磷酸化有关，TARK1的磷酸化不依赖于效应蛋白 XopN，也与筛选到的其他互作因子A1、A2无关。相关研究结果明确了 PopW 在诱导植物抗性，帮助植物抵御逆境中的 primming 的功能。同时发现，相关膜蛋白在 PopW 抗旱抵抗病原菌侵染过程中发挥重要作用。以上结果为后续研究 PopW 调控的植物抗性信号传导奠定了坚实的基础。