小麦类枯草杆菌蛋白酶TaSBT1调控条锈菌免疫应答的分子基础

Wheat stripe rust, caused by Puccinia stiiformis f. sp. tritici (Pst), is one of the most destructive diseases that threaten wheat production and food security. Our previous study showed that the wheat subtilisin-like protease TaSBT1 was highly induced during the early stage of incompatible interaction between wheat and Pst. Our recent genetic studies further demonstrate that TaSBT1 likely plays a positive role in pathogen-induced programmed cell death and wheat basal resistance against Pst. However, possible target proteins of TaSBT1 have yet to be identified, and nothing is known about the nature of TaSBT1-target protein interaction, and the signal transduction pathway(s) TaSBT1 might regulate. To understand how TaSBT1 regulates immune response in wheat, candidate genes that either encode TaSBT1-target proteins and/or regulatory components of the TaSBT1-regulated defense signaling pathway(s) will be identified by RNA-seq analysis of wild-type and TaSBT1-silenced wheat plants prior to and after Pst infection. Next, TaSBT1-interacting proteins will be identified through co-immunoprecipitation and Pull-down followed by mass spectrometry, such interactions will be further confirmed by yeast-two-hybrid and bimolecular fluorescent complementation assays, and the interacting proteins will be assessed to see if they are direct substrates of TaSBT1 using bacterially expressed recombinant proteins. Finally, if time permits, the possible roles of TaSBT1 target proteins in wheat basal resistance against Pst will be investigated using gene-editing tools. Results from the proposed research will not only elucidate the molecular mechanisms by which TaSBT1 regulates wheat immune response but should also provide new gene information and novel strategy for enhancing wheat disease resistance through molecular breeding.

由条形柄锈菌（Puccinia striiformis f. sp. tritici, Pst）引起的小麦条锈病是威胁小麦生产和粮食安全的重要病害。前期研究表明，小麦类枯草杆菌蛋白酶TaSBT1基因在小麦抵御条锈菌侵染的早期阶段具有显著作用，但TaSBT1的靶标蛋白尚未鉴定，它们之间的互作及信号传递通路与调控机理当然亦不清楚。本研究在明确TaSBT1抗Pst侵染和诱导细胞坏死功能的基础上，拟采用RNA-seq技术分析TaSBT1参与的信号通路，进一步综合运用免疫共沉淀、Pull-down和蛋白质谱分析，以及基因定点失活等技术以期获得TaSBT1在小麦-Pst互作体系中的作用靶标，并采用酵母双杂交、双分子荧光互补和TaSBT1重组蛋白的酶活分析来验证其与靶标蛋白的关系，从而对TaSBT1及其靶标蛋白在小麦抗条锈病过程中的作用机理进行探索研究，为小麦抗病育种提供新的思路及基因资源。

小麦条锈病是由专性寄生真菌——条形柄锈菌小麦专化型（Puccinia striiformis f. sp. tritici）引起的一种真菌病害，对全球小麦安全生产造成了巨大威胁。类枯草杆菌蛋白酶（subtilases, SBTs）可以在植物—病原互作过程中诱导寄主的过敏性坏死反应（HR）和系统获得抗性（SAR）。本研究通过全基因组挖掘发现一个小麦类枯草杆菌蛋白酶基因TaSBT1参与小麦-Pst非亲和互作过程中的PCD反应。对小麦中的TaSBT1基因进行沉默，接种条锈菌非亲和小种CYR20的对照小麦叶片观察到明显的过敏性坏死，但在目的基因沉默植株上产生大量的夏孢子堆。TaSBT1的3个同源序列均在SA处理0.5 h后被诱导上调表达，而在MeJA和ETH诱导后无显著变化，由此推测TaSBT1基因可能参与水杨酸信号转导途径。亚细胞定位结果显示TaSBT1蛋白可以被分泌到细胞质膜外侧，但是在激素SA、MeJA和ETH诱导条件下TaSBT1均可移动到细胞质中，暗示其作用靶标可能位于胞内。随后通过筛选酵母双杂交文库的方式获得了TaSBT1的候选互作蛋白TaGDSL，进而通过Y2H和荧光素酶实验明确了TaGDSL脂肪酶可以作为TaSBT1蛋白酶的互作底物。目前对TaGDSL的后续功能研究已经展开。以上研究结果为丰富小麦抗病信号传递途径，揭示小麦抗条锈病分子机理奠定了理论基础。