A Virulence Essential CRN Effector of Phytophthora capsici Suppresses Host Defense and Induces Cell Death in Plant Nucleus

Phytophthora capsici is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in P. capsici. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of PcCRN4 gene induced cell death in N. benthamiana, N. tabacum and Solanum lycopersicum. Overexpression of the gene in N. benthamiana enhanced susceptibility to P. capsici. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing PcCRN4 gene in P. capsici significantly reduced pathogen virulence. The expression of the pathogenesis-related gene PR1b in N. benthamiana was significantly induced when plants were inoculated with PcCRN4-silenced P. capsici transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with PcCRN4-silenced transformant, indicating that silencing of PcCRN4 in P. capsici reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when PcCRN4-silenced line were inoculated on Arabidopsis thaliana, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.

辣椒疫霉（Phytophthora capsici）是一类寄主范围广泛的土传植物病原菌。该病原菌在侵染寄主植物的过程中会分泌大量效应因子（effector），其中包括CRN效应因子（Crinkler, CRN）。然而，目前对于这类效应因子在病原菌致病过程中的功能，尤其是在辣椒疫霉中的具体作用，仍鲜有研究阐明。本研究通过农杆菌浸润法（agroinfiltration）筛选得到了一个可诱导细胞死亡的CRN效应因子PcCRN4。将PcCRN4基因在植物中进行瞬时表达，可在本氏烟草（Nicotiana benthamiana）、普通烟草（Nicotiana tabacum）以及番茄（Solanum lycopersicum）中诱导细胞死亡。在本氏烟草中过表达该基因，能够显著增强植株对辣椒疫霉的感病性。亚细胞定位实验结果显示，PcCRN4定位于植物细胞核中，且该定位特性是其兼具诱导细胞死亡活性与致病功能的必要条件。在辣椒疫霉中沉默PcCRN4基因，可显著降低病原菌的致病力。与野生型（原文笔误为wilt-type）对照相比，用PcCRN4基因沉默的辣椒疫霉转化子接种本氏烟草后，植株体内病程相关基因PR1b的表达量显著上调。同时，接种该沉默转化子的植物位点会出现大量胼胝质沉积，这表明在辣椒疫霉中沉默PcCRN4会削弱病原菌抑制植物防御反应的能力。当用PcCRN4沉默菌株接种拟南芥（Arabidopsis thaliana）时，植株体内细胞死亡相关基因的转录水平发生明显改变，这提示PcCRN4可能通过调控细胞死亡相关基因的表达来诱导细胞死亡。综上，本研究结果证实PcCRN4是一类对病原菌致病力至关重要的效应因子，且其需要靶向植物细胞核才能有效抑制植物的免疫反应。