含LYSM结构域的棉花Gh-LYK2受体激酶抗黄萎病机制解析

Verticillium wilt is the most destructive disease in cotton production. Members of the family such as LYK1, LYK4 and LYK5 have been found played important roles in the resistance to fungal diseases in Arabidopsis, but not LYK2. The function of cotton LysM proteins have not been reported yet. We have isolated genes of LYK family of cotton and found Gh-LYK2 was vital to Verticillium wilt resistance. Its kinase region has been proved not active by phosphorylation analysis. In addition, the homologous genes of susceptible cultivars have been deprived resistant function due to the missing of transmembrane domain. This project intends to separate the interactional proteins of Gh-LYK2 by some kinds of methods, and characterize their function by transient silence and gene overexpression. At the same time, the mechanism of the effect of transmembrane region deletion to resistance will also be analyzed. In addition, the Gh-LYK2 would be transformed to susceptible cultivars to verify its resistant function and the transformed plants would be used for RNA-seq analysis to clarify the gene regulatory network. The implementation of this project will separate the Gh-LYK2 interactional proteins and characterize their function, explain the resistant mechanism of Gh-LYK2 and create new germplasm of broad-spectrum resistance to Verticillium wilt.

棉花黄萎病是中国棉花生产最主要的病害。已发现具有LysM结构域的受体激酶（LYK）家族成员LYK1、LYK4和LYK 5在拟南芥抵抗真菌病害中发挥关键作用，但未发现LYK2有类似功能。而棉花LysM蛋白的功能研究未见报道。申请人前期克隆了棉花的LYK家族，发现Gh-LYK2在抗黄萎病过程中起重要作用，磷酸化分析显示其激酶区没有活性，另外，感病材料来源的该基因缺失了跨膜区，不具有抗病性。本项目拟利用多种技术分离与Gh-LYK2互作蛋白，通过瞬时沉默和过表达明确互作蛋白的功能，分析跨膜区缺失影响抗病性的机制，并将Gh-LYK2转入缺失跨膜区的感病材料中，明确其抗性并开展RNA-seq分析，研究其基因调控网络。本项目的实施将明确Gh-LYK2的互作蛋白及功能，阐明Gh-LYK2的抗病机制，获得广谱抗病新种质。

棉花黄萎病是中国棉花生产最主要的病害。具有LysM结构域的受体激酶在抵抗真菌病害中发挥关键作用。本研究对包括对50个棉花品种的Gh-LYK2序列进行分析，证明了感病材料Gh-LYK2缺失跨膜区是一种普遍现象并开发了对应的分子标记；获得了Gh-LYK2过表达棉花并进行分子鉴定，筛选到了单拷贝高表达的株系，其黄萎病抗性较野生型显著提高，田间鉴定表现为R抗性水平；利用CO-IP筛选出与Gh-LYK2互作的蛋白Gh-CDPK2，该基因编码一个钙依赖性蛋白激酶，该类蛋白在调控植物抗病方面具有重要作用。同时前期研究发现Gh-LYK2的胞外区能够引起细胞凋亡，进一步鉴定出诱导坏死的最小区域为胞外的70个氨基酸序列，而Ca2+通道抑制剂能够明显抑制Gh-LYK2诱导产生的坏死。进一步证明Ca2+通道参与了Gh-LYK2引导的抗性防卫反应。通过本项目的执行，初步阐明了LYK2类基因的抗性机制，促进该类基因在抗病育种中的应用。