柑橘黄龙病菌分泌蛋白ES40抑制寄主细胞坏死的分子机制

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. Due to the lack of pure culture, the pathogenesis of “Candidatus Liberibacter asiaticus” is poorly understood. In our previous study, secretion protein ES40 identified from 69 predicted secretion proteins can suppress plant cell death triggered by BAX and INF1 and can promote the infection of Nicotiana benthamiana by PVX. We hypothesized that ES40 might play important roles in pathogenic process of the pathogen in citrus hosts. In this study, we will elucidate its contribution to the pathogenicity of “Candidatus Liberibacter asiaticus” by Citrus tristeza virus (CTV)-based ES40 expression system; identify its functional domains and key residues based on its sequence polymorphism in the isolates from different geographical locations and different virulence, combined with alanine scanning; specify its regulating effect on the plant immunity by transcriptomic and proteomic studies and the functional analysis of important genes; identify its interacting targets in the Nicotiana benthamiana and citrus hosts using yeast two-hybrid, bimolecular fluorescence complementation, co-immunoprecipitation, pull-down coupled with LC-MS/MS methods, and dissect the functions of plant targets through virus induced gene silencing and transgenosis. Information provided in the study will dissect the mechanism that ES40 is involved in the “Candidatus Liberibacter asiaticus” pathogenesis. It will provide effective theoretical basis and practical targets to design novel prevention and control strategies against citrus HLB.

柑橘黄龙病是全球柑橘生产上毁灭性病害，由于黄龙病菌难培养，极大制约了对病原致病机制的研究。我们前期从69个预测的黄龙病菌分泌蛋白中鉴定了一个可抑制BAX、INF1激发的本氏烟细胞坏死的分泌蛋白ES40，该蛋白亦可促进PVX侵染本氏烟，因此推测其在黄龙病菌致病进程中扮演重要角色。本研究拟基于柑橘衰退病毒（CTV）载体表达ES40，明确该分泌蛋白是否促进黄龙病菌侵染；比较不同株系中该蛋白的序列，结合丙氨酸突变体分析，鉴定抑制坏死的功能域和关键位点；以功能缺失型点突变为对照，对瞬时表达该蛋白的本氏烟进行转录组和蛋白组分析并验证重要基因功能，明确其对植物免疫反应的调控；筛选该分泌蛋白在本氏烟和柑橘中的作用靶标，通过VIGS和转基因，明确靶标在植物抗病性的作用。本研究有望解析分泌蛋白ES40参与致病的作用机制，为进一步阐明黄龙病菌致病机理奠定基础，并为基于靶标基因编辑等黄龙病防控新策略提供理论支撑。

柑橘是全球最大的水果。由韧皮部杆菌亚洲种（“Candidatus Liberibacter asiaticus”, CLas）引起的柑橘黄龙病（Citrus Huanglongbing, HLB）是柑橘生产上的毁灭性病害。受限于黄龙病菌定殖于寄主韧皮部，至今未能成功纯培养，传统的分子和遗传操作难以进行，故该病致病机理研究滞后。与许多胞外植物病原细菌不同，黄龙病菌缺乏III型和IV型分泌系统，但其具有Sec分泌系统，可分泌效应蛋白到宿主韧皮部筛管细胞和伴胞并抑制寄主免疫。我们前期研究表明黄龙病菌效应蛋白ES40（CLIBASIA_04405，后续称为SDE4405）可抑制INF1和BAX引起的细胞坏死。为明确SDE4405对柑橘寄主的响应，本研究构建了SDE4405转基因柑橘，并进行了黄龙病菌接种和接种前后比较转录组分析，结果表明SDE4405通过抑制寄主免疫促进黄龙病菌定殖和增殖。进而通过酵母双杂、BiFC、GST pull down和Co-IP证明SDE4405与甜橙自噬相关蛋白ATG8c（CsATG8c）在体外和体内互作，且SDE4405的ATG8结合位点（AIM）是互作关键区域。在本氏烟中瞬时表达SDE4405和CsATG8c并用E64d处理，表明两者互作激活了本氏烟的细胞自噬。电镜观察E64d处理转基因柑橘植株表明转基因植株自噬小体数量显著高于对照植株，且NBR1蛋白水平显著低于对照植株，表明SDE4405转基因柑橘植株亦诱导了细胞自噬。基于TRV介导的ATG8c沉默本氏烟和柑橘，以及瞬时表达ATG8c和SDE4405并接种假单胞菌和溃疡病菌，表明ATG8c在SDE4405抑制寄主免疫过程中发挥重要作用。综上所述，本研究发现SDE4405与自噬元件互作并负调控寄主免疫，首次解析了黄龙病菌利用分泌蛋白调控寄主自噬的机制，加深了对黄龙病菌与寄主互作机制的认知。