Tropomyosin，GroEL和ATP synthase 在亚洲柑橘木虱传播黄龙病过程中的作用

Diaphorina citri (Hemiptera: Psyllidae) is the major vector that transmits Huanglongbing（HLB）,which is a devastating disease of citrus caused by ‘Candidatus Liberibacter asiaticus’ (CaLas). We have found Tm and ATP synthase subunit of citrus psyllid produced changes under HLB pathogen infection in previous studies.Meanwile, vast GroEL of CaLas was found in subcell of citrus psyllid. Our project is aimed to study on the interaction protein between CaLas with Tm and ATP synthase subunit of host, and research the role of GroEL in the process CaLas infected psyllid. BN PAGE will used to identify interaction protein complex with Tm under CaLas infected.2-DE combined Far-western will used to screen out interaction protein with bacterial GroEL in psyllid subcell. Meanwhile, the project will carry out Immune pathology observation and verify gene in the transcription. Our perspective will reveal molecular mechanism of Tm、GroELand ATP synthase involved in the CaLas infection the citrus psyllid and inner transfer of subcell, and will find CLas combined targets to get a new strategy of control HLB. The results will also benefit to support for studying on interaction pathogen between insect hosts and control the transmission of the pathogen.

柑橘木虱是柑橘毁灭性病害-黄龙病（Huanglongbing, HLB）的主要媒介宿主，HLB病菌能侵染柑橘木虱并在其体内繁殖生长。项目组在前期研究中已经明确了HLB病菌侵染会影响柑橘木虱的原肌球蛋白（Tropomyosin, Tm）和ATP synthase亚基等蛋白变化，同时在带菌木虱中发现了HLB病菌GroEL蛋白大量表达。本研究拟进一步解析HLB病菌侵染柑橘木虱引起宿主Tm和ATP synthase亚基变化机制以及菌体GroEL介导的病菌在木虱体内的转运过程。项目拟采用亚细胞BN PAGE技术鉴定参与HLB病菌和Tm互作的蛋白复合体，采用2-D电泳结合Far-western技术在亚细胞水平上筛选与菌体GroEL互作的柑橘木虱蛋白，辅助以免疫病理学观察分析和转录水平上的验证。希望研究获得Tm、GroEL和ATP synthase在HLB病菌入侵柑橘木虱以及细胞内转运的信息，为进一步研究HLB防治新策略、控制媒介昆虫的病原传播提供理论依据。

柑橘木虱是柑橘毁灭性病害黄龙病（HLB）的媒介宿主，病原菌（CLas）能侵染木虱并在其体内增殖，目前防控HLB的主要措施是切断传播途径，然而CLas在木虱体内的传播机制还不明确。本项目研究克隆表达并纯化柑橘木虱Tm成功制备抗体，采用亚细胞BN-PAGE和质谱鉴定了柑橘木虱Tm互作的柠檬酸合酶（CS）和V型ATPase亚基（VAT）相互作用，通过RNAi沉默Tm基因可以使木虱的CLas感染率升高31-43%，结合CLas侵染下柑橘木虱中肠的CLas的荧光标记观测，确定木虱Tm和actin在利用宿主ATPase作用下参与了CLas在宿主组织间跨膜转运扩散过程。克隆表达CLas病菌groEL基因融合蛋白制备抗体，WB检测携带CLas的柑橘木虱GroEL高丰度表达，qPCR结果显示在CLas侵染过程木虱中肠和血淋巴中groEL与柑橘木虱Hsp60-1、Hsp70表达均呈现显著相关性，与宿主能量代谢相关基因CS、Prx 1、COX4和COX6A具有显著正相关性；在CLas侵染过程采用RNAi技术对groEL沉默可以导致木虱宿主细胞传毒能力下降，木虱血淋巴中病原菌含量显著减少，说明病原菌的GroEL参与能量代谢和免疫逃避等途径。进一步通过BN-PAGE对Tm复合体分离和质谱分析，与Tm互作多数为能量代谢相关的蛋白：ATP synthase，ATPase、柠檬酸合成酶、过氧化物酶等。测定病原菌的GroEL的ATP酶活性，结果显示GroEL具有ATPase活性，能够结合ATP水解成ADP释放能量。这些结果表明病原菌侵染木虱利用GroEL对木虱能量代谢调控，引发宿主Tm与能量代谢系列酶参与形成了介导通路的关键因子，保护病原菌不被降解从而实现病菌体内传播过程。项目研究结果为进一步防控HLB媒介柑橘木虱的病原传播提供理论依据。