效应蛋白靶标GmLHP1招募GmBTB/POZ介导大豆应答疫霉根腐病菌的机制研究

Phytophthora sojae produces effector protein Avr1b that is transferred into the cytoplasm of host cells to suppress host defenses by the RXLR motifs binding to the phospholipid, phosphatidylinositol-3-phosphate (PI3P), but the mechanism of Avr1b acting on target genes to regulate resistance reaction to P. sojae infection in soybean is still unknown. In previous study, our research suggested that GmLHP1 interacted with Avr1b in yeast cells and in planta. We also demonstrated that GmLHP1 could form the homologous dimmers and interacted with GmBTB/POZ in yeast cells and in planta. We speculated that GmLHP1 and GmBTB/POZ form an inhibitory complex as the target of Avr1b, and meditate the resistance reaction by combination of H3K27me3 on chromosome structure to change the structure of chromosome. This research project aims to screen on interaction proteins by way of yeast two-hybird, BiFC and pull-down, identify the binding to H3K27me3, target the genes of GmLHP1and GmBTB/POZ inhibition complex by chromatin immunoprecipitation, which will lay basis on modulation mechanisms of Avr1b targets GmLHP1 recruiting GmBTB/POZ in response to Phytophthora sojae in soybean.

大豆疫霉菌效应蛋白Avr1b的RXLR结构域能够通过磷脂PI3P调控其通过內吞作用进入宿主细胞，从而抑制宿主细胞的抗病反应，但对于Avr1b通过作用于毒性靶标应答大豆防卫反应的机理尚不明晰。申请者及团队验证了GmLHP1与Avr1b及GmBTB/POZ互作，并且GmLHP1能够形成同源二聚体。我们推测GmLHP1作为Avr1b的毒性靶标，招募GmBTB/POZ形成一个抑制复合体，通过结合染色体上的H3K27me3改变染色体上的结构，应答大豆对疫霉菌的防卫反应。本项目将利用酵母双杂交、BiFC、Pull-down验证蛋白间的互作，ChIP-chip验证GmLHP1与组蛋白的H3K27me的互作，利用ChIP-Seq及ChIP-qPCR找到GmLHP1和GmBTB/POZ调控的靶标基因，可为解析效应蛋白靶标GmLHP1招募GmBTB/POZ介导大豆应答疫霉菌的侵染机制提供依据。

前期研究证实GmBTB/POZ是一种含有核蛋白的BTB/POZ结构域，通过依赖于水杨酸（SA）的过程增强大豆对大豆疫霉的抗性。在这里，我们证明GmBTB/POZ在体内外直接与大豆异染色质蛋白1（GmLHP1）结合，转基因株系的过表达和RNA干扰分析均表明，GmLHP1通过降低SA水平和抑制GmPR1的表达负调控大豆对大豆疫霉的反应。WRKY转录因子基因GmWRKY40是SA信号通路中SA诱导的基因，GmLHP1通过至少两种机制抑制其表达（直接结合其启动子并损害SA积累）。此外，GmLHP1的核定位是GmLHP1介导的免疫负调控、SA水平和GmWRKY40表达抑制所必需的。GmBTB/POZ的过度表达释放了GmLHP1调节的GmWRKY40抑制，并增加了GmLHP1对大豆疫霉的抗性。这些发现揭示了GmBTB/POZ-GmLHP1调控大豆对大豆疫霉抗性的调控机制，是通过调控下游靶基因GmWRKY40的表达。