翻译控制肿瘤蛋白CsTCTP1介导TOR信号通路调控黄瓜防卫反应的分子机制

Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, growth factors, and stress signals to promote survival and growth in all eukaryotes. It is important to understand how TOR is regulated. In yeast and animal, studies have shown that translationally controlled tumour protein (TCTP) is involved in TOR signalling. TCTP is required for organ growth by promoting Rheb GTPase (Ras superfamily member) function for TOR signalling as a guanine nucleotide exchange factor. However, the role of TCTP in TOR signalling has not been reported in plants. In the previous study, we know that CsTCTP1 is a conserved and ubiquity protein involved in plant growth and stress responses in Cucumis sativus. In this proposal, we hypothesize that CsTCTP1 maybe interact with CsRab GTPase (Ras superfamily member) to activate TOR, manipulating TOR kinase activity or TOR expression levels leads to changes in biotic stress responses. We are planning to use Yeast two hybrid assay, Pull-down, BiFC to study how CsTCTP1 interacting with CsRab at protein level, and use 10µM rapamycin effectively inactivates endogenous TOR activity of cucumber seedlings to study the effects of TOR in biotic stress process, further use RNA-seq and RT-PCR technology to clarify signalling regulators, and stress-related proteins regulated by TOR signalling. The predicted results will be helpful to explore the regulation of plant growth and stress resistance, and will be valuable for developing cucumber genetic improvement of resistance.

翻译控制肿瘤蛋白（TCTP）和雷帕霉素靶蛋白（TOR）普遍存在于真核生物中，具有调节疾病发生与免疫应答等生物学功能。前期研究表明，CsTCTP1与黄瓜应答白粉病菌侵染密切相关，但作用机制还不清楚，CsTCTP1能否如动物TCTP一样，通过结合Ras-型GTPase激活TOR信号，进而调控防卫反应，尚缺乏直接、有力的证据。本项目拟利用酵母双杂交、Co-IP、Pull-down、BiFC等方法，鉴定与CsTCTP1互作的CsRab（Ras-型GTPase）；利用Rapamycin抑制黄瓜幼苗TOR活性，分析黄瓜-病原物互作表面超微结构和生理变化，并结合RNA-seq及遗传转化等技术解析CsTCTP1介导TOR信号通路调控黄瓜幼苗抗病性的作用机制。本研究将有助于揭示CsTCTP1应答黄瓜白粉病菌侵染的作用机制，对于深入探究植物抗病性的调控有着重要的理论和实践意义。

白粉病[Sphaerotheca fuliginea (Schlecht) Poll.]是瓜类作物广泛发生的一种世界性病害，深入研究黄瓜响应白粉病菌侵染的分子机制并寻找新的抗性基因资源具有重要意义。项目前期研究发现，TCTP（translationally controlled tumor protein）在黄瓜响应白粉病菌侵染过程中起到重要的调节作用，但其作用机理还不清楚。TCTP在真核生物中普遍存在且高度保守，推测它们可能具有相似的功能。动物和酵母中，TCTP能够激活Rheb（Ras-型小GTPase）的活性，进而激活TOR（target of rapamycin）信号通路，调控细胞和器官生长、疾病发生与免疫反应等。黄瓜CsTCTPs能否如其它生物中一样，通过结合Ras-型GTPase激活TOR信号，进而调控防卫反应，尚缺乏直接、有力的证据。本项目从探究CsTCTP与TOR信号途径上游关键调控因子CsRab（Ras superfamily member）间的互作入手，解析CsTCTP介导TOR信号通路调节黄瓜幼苗的抗病性及其机制。首先利用黄瓜子叶瞬时遗传转化及拟南芥突变体互补实验等，明确CsTCTP基因可以负调控黄瓜抗病性，并可能通过TOR信号和ABA信号途径调控黄瓜防卫反应。利用酵母双杂交、LUC、BiFC等方法，鉴定到与CsTCTP互作的CsRab11A，并初步明确CsRab11A能负调控黄瓜幼苗的抗病性。采用5μM的TOR活性抑制剂AZD8055预处理黄瓜幼苗，能一定程度上抑制幼苗的生长，但可以显著提高黄瓜幼苗的抗病性。转录组分析表明，在黄瓜幼苗TOR活性被抑制的情况下，上调差异表达基因涉及JA、ABA、ETH和SA等信号途径，下调表达基因主要与Auxin、BR和GA等信号途径相关，推测TOR可能通过调节这些激素信号途径来平衡植物的生长发育与抗逆性。植物的生长、抗病和抗逆很大程度上是TOR偶联在一起的，对其或通路中特定目标过程的微调，有望增加植物对胁迫容忍度，提高作物质量和产量。进一步深入探究CsTCTP/CsRab/CsTOR中关键基因之间的调控关系，揭示TOR信号通路调控黄瓜生长发育与防卫反应的上下游信号，对于深入探究黄瓜“胁迫刺激-生长发育”的精细调控有着重要的理论和实践意义。