Candidatus Liberibacter asiaticus Minimally Alters Expression of Immunity and Metabolism Proteins in Hemolymph of Diaphorina citri, the Insect Vector of Huanglongbing

Huanglongbing (HLB), also known as citrus greening disease, is the most serious disease of citrus plants. It is associated with the Gram-negative bacterium ‘Candidatus Liberibacter asiaticus’ (CLas), which is transmitted between host plants by the hemipteran insect vector Diaphorina citri in a circulative, propagative manner involving specific interactions with various insect tissues including the hemolymph, fluid that occupies the body cavity akin to insect blood. High resolution quantitative mass spectrometry was performed to investigate the effect of CLas exposure on D. citri hemolymph at the proteome level. In contrast to the broad proteome effects on hundreds of proteins and a diverse array of metabolic pathways previously reported in gut and whole insect proteome analyses, the effect of CLas on the hemolymph was observed to be highly specific, restricted to key immunity and metabolism pathways, and lower in magnitude than that previously observed in the whole insect body and gut. Vitellogenins were abundantly expressed and CLas-responsive. Gene-specific RNA expression analysis suggests that these proteins are expressed in both male and female insects and may have roles outside of reproductive vitellogenesis. Proteins for fatty acid synthesis were found to be up-regulated, along with metabolic proteins associated with energy production, supported at the organismal level by the previously published observation that D. citri individuals experience a higher level of hunger when reared on CLas-infected plants. Prediction of post-translational modifications identified hemolymph proteins with phosphorylation and acetylation upon CLas exposure. Proteins derived from the three most prominent bacterial endosymbionts of the psyllid were also detected in the hemolymph, and several of these have predicted secretion signals. A DNAK protein, the bacterial HSP70, detected in the hemolymph expressed from Wolbachia pipientis was predicted to encode a eukaryotic nuclear localization signal. Taken together, these data show specific changes to immunity and metabolism in D. citri hemolymph involving host and endosymbiont proteins. These data provide a novel context for proteomic changes seen in other D. citri tissues in response to CLas and align with organismal data on the effects of CLas on D. citri metabolism and reproduction.

柑橘黄龙病（Huanglongbing, HLB）又称柑橘绿化病，是危害柑橘类植物最严重的病害。该病害与革兰氏阴性菌亚洲韧皮部杆菌（‘Candidatus Liberibacter asiaticus’, CLas）密切相关，该病原菌可通过半翅目昆虫媒介柑橘木虱（Diaphorina citri）在寄主植物间传播，传播方式为循回增殖型，且需与包括血淋巴（hemolymph，昆虫体腔内类似血液的体液）在内的多种昆虫组织发生特异性相互作用。本研究采用高分辨率定量质谱（mass spectrometry）技术，从蛋白质组（proteome）层面探究了CLas侵染对柑橘木虱血淋巴的影响。此前针对昆虫肠道及全虫体蛋白质组的分析报道显示，CLas侵染会对数百种蛋白质及多种代谢通路产生广泛的蛋白质组学影响；与之形成鲜明对比的是，CLas对木虱血淋巴的影响具有高度特异性，仅局限于关键免疫与代谢通路，且影响幅度远低于此前在全虫体及肠道中观察到的水平。卵黄原蛋白（vitellogenins）在血淋巴中大量表达且响应CLas侵染。针对特定基因的RNA表达（RNA expression）分析显示，这类蛋白质在雌雄昆虫体内均有表达，其功能可能不限于生殖过程中的卵黄发生（vitellogenesis）。研究发现脂肪酸合成（fatty acid synthesis）相关蛋白质与能量代谢相关蛋白质的表达量均显著上调，这一结果在个体水平上得到了此前已发表研究的佐证：取食CLas侵染植株的柑橘木虱个体表现出更高的饥饿程度。翻译后修饰（post-translational modifications）预测分析显示，CLas侵染后，血淋巴蛋白质发生了磷酸化（phosphorylation）与乙酰化（acetylation）修饰。研究同时在血淋巴中检测到了来自该木虱三种主要细菌内共生菌（endosymbionts）的蛋白质，其中部分蛋白质被预测带有分泌信号肽。在检测到的内共生菌蛋白质中，来自沃尔巴克氏体（Wolbachia pipientis）的DNAK蛋白，即细菌热休克蛋白70（HSP70），被预测携带真核生物核定位信号（nuclear localization signal）。综上，本研究数据揭示了CLas侵染后柑橘木虱血淋巴中免疫与代谢通路的特异性变化，此类变化涉及宿主与内共生菌来源的蛋白质。该研究为解析CLas侵染后柑橘木虱其他组织的蛋白质组学变化提供了全新视角，同时也与已报道的CLas对木虱代谢与生殖功能影响的个体水平研究结果相契合。