寄生疫霉外泌相关基因PpSec24和Ppsec15调控效应分子外泌的机理研究

Phytophthora parasitica is one of the most widespread and destructive soilborne pathogens. It has a wide host range and has been responsible for major losses of vegetables production all over the world each year. It can secreted a large set of effectors that are divided into two categories: cytoplasmic effectors and apoplastic effectors. Apoplastic effectors PpCBEL and cytoplasmic effectors PpCRN63 will be overexpressed in P.parasitica strain with GFP fusion protein. Then these overexpressed strains will be treated with BFA that is an inhibitor of protein trafficking. We focus on investigating how phytophthora secretes effectors during host cell invasion. First we select two key sites in secereted pathway. Sec24p has been thought to be an essential component of the COPII coat and Sec15p mediate docking and fusion of vesicles with the plasma membrane target site. Then double-stranded RNA of PpSec24 and PpSec15 are synthesized in vitro, we use transformation with double-stranded RNA in overexpressed effector strains. We are specically interested in determining whether cytoplasmic effectors and apoplastic effectors destine for translocation to host cells follow the different secretory route. The content of effector proteins are analyzed with fluorescence observation and detection of extracellular effector using western blot. The inhibition of effector secretion maybe provide a novel molecular targets for fungicide，that can provide the basis for fungicide screen on pepper phytophthora blight.

寄生疫霉引起的绵疫病是世界茄子生产的最重要病害之一，该病原菌寄主范围广，每年给茄子产业造成巨大损失。寄生疫霉在侵染过程中分泌大量的胞质效应分子和质外体效应分子干扰植物的免疫反应，但对其分泌机制尚不清楚。本研究拟通过在寄生疫霉中过量表达融合GFP的质外体效应分子PpCBEL和胞质效应分子PpCRN63，应用外泌途径抑制剂BFA分析两个效应分子的外泌差异；为进一步研究两个效应分子的外泌机制，从外泌途径中筛选到内质网到高尔基体、参与COPII形成的sec24和参与外泌囊泡与质膜融合的Sec15两个关键阶段基因，在过表达效应分子PpCBEL和PpCRN63的菌株中分别瞬时沉默sec24和Sec15基因，通过荧光蛋白观察和western blot检测胞外蛋白的含量，分析效应分子可能的外泌机制。以期从阻断效应分子外泌途径关键基因中发掘新的杀菌剂分子靶标，从而为绵疫病防控药物的筛选提供理论依据。

蛋白乙酰化修饰是生物体表观遗传修饰的一种重要方式，通过调控基因表达参与生物生长发育过程和免疫等。大豆疫霉根腐病是大豆生产上的重要病害之一，严重威胁我国和其他国家的大豆产量和质量。大豆疫霉在侵染大豆过程中，通过分泌的效应蛋白抑制大豆细胞内组蛋白乙酰化，最终成功侵染大豆。病原真菌菌的乙酰基转移酶参与了病原菌的生长发育和致病性。而目前卵菌的乙酰化修饰研究非常少，其功能尚不清楚。本研究以大豆疫霉蛋白组乙酰化修饰为研究对象，对大豆疫霉赖氨酸乙酰化修饰进行系统分析。发现大豆疫霉共有1150个蛋白具有2197个赖氨酸乙酰化位点。通过分析发现，绝大部分的蛋白只含有一个赖氨酸乙酰化位点。而且这些赖氨酸乙酰化位点具有一定的保守性，如KY、KXF、LK、KXL等。这些蛋白参与多种代谢途径，如碳源代谢，氨基酸代谢等。在被乙酰化蛋白中我们发现已有蛋白被证实参与大豆疫霉的致病过程。如效应蛋白Avr1d和CBEL等，外泌相关蛋白PsVPS1和YKT6等，这说明大豆疫霉的蛋白乙酰化参与了病原菌的致病性，因此为疫霉的化学防治提供新的策略。项目资助发表SCI 2篇，专利1项，待发表论文2篇。项目投入经费13万元，各项支出与预算相符。剩余经费8万元，剩余经费计划用于本项目研究后续支出。