在小麦与叶锈菌互作过程中TaCRK2调控HR的作用机制研究

HR is an important defense response in the wheat against Puccinia triticina infection. TaCRK2 is a cysteine-rich receptor-like kinases cloned from wheat based on transcriptome analysis in our group. Using VIGS and RNAi, TaCRK2 has been shown to positively regulate the rate of cell death in HR. On this basis, the main task of this project is to systematically study the mechanism of action of TaCRK2 in the HR response induced by Puccinia triticina infecting wheat. Screening of target proteins interacting with TaCRK2 using IP and LC-MS; identification of target proteins by membrane proteins yeast two-hybrid, BiFC and Co-IP respectively; site-directed mutagenesis and yeast two-hybridization combined determination of active sites in TaCRK2 that interact with target proteins.The kinase activity of TaCRK2 was determined by phosphorylation assay after prokaryotic expression of TaKRK2 kinase domain and its target protein; The relationship between the expression of TaCRK2 and calcium signal, H2O2 and NO was investigated by combining pharmacological tests with gene silencing.This study will greatly promote the study of physiological functions of CRKs proteins in the process of plants resistant to live pathogen infections， and it is of great significance for improving the molecular mechanism of wheat resistance to Puccinia triticina infection.

HR是小麦抵抗叶锈菌侵染的重要防卫反应。TaCRK2是基于转录组分析从小麦中克隆的一个富含半胱氨酸的类受体激酶，利用VIGS和RNAi证明，TaCRK2对发生HR的细胞死亡速度有正调控作用。在此基础上，本项目的主要任务就是对TaCRK2在叶锈菌侵染小麦诱发的HR反应中的作用机制进行系统研究。利用IP和LC-MS结合筛选TaCRK2的互作靶蛋白；分别利用膜蛋白酵母双杂交、BiFC和Co-IP对互作靶蛋白进行鉴定；定点突变和酵母双杂交相结合确定TaCRK2中与靶蛋白作用的活性位点；通过原核表达TaCRK2激酶区和其互作靶蛋白后再经磷酸化试验明确TaCRK2的激酶活性；药物学试验和基因沉默相结合探讨TaCRK2的表达与钙信号、H2O2和NO的关系。该项研究对揭示CRKs类蛋白在植物抵抗活体病原菌侵染过程中的生理功能有促进作用，对完善小麦抵抗叶锈菌侵染的分子机理具有重要意义。

HR是小麦抵抗叶锈菌侵染的重要防卫反应，本课题组长期从事小麦与叶锈菌互作过程中诱发HR的分子机制研究，着力发掘抗叶锈病相关基因，为培育抗病种质提供基因储备。TaCRK2是基于转录组分析从小麦中克隆的一个富含半胱氨酸的类受体假激酶，利用VIGS和RNAi技术证明，TaCRK2对发生HR的细胞死亡速度有正调控作用。在此基础上，利用IP和LC-MS结合筛选TaCRK2的互作靶蛋白，分别利用膜蛋白酵母双杂交、BiFC和Co-IP对互作靶蛋白进行鉴定，证明了TaCRK2可以与抗性蛋白TaTCTP和Ta14-3-3互作；通过原核表达TaCRK2激酶区和其互作靶蛋白，再经磷酸化试验结合质谱分析明确了TaCRK2的假激酶性质；经药物学试验和基因沉默相结合揭示了TaCRK2的表达受钙信号和NO信号的调控，而TaCRK2的表达可以调节H2O2水平。该项研究首次揭示了CRKs类假激酶在植物抵抗活体病原菌侵染过程中的生理功能，对完善小麦抵抗叶锈菌侵染的分子机理具有重要意义。