马蜂柑响应黄龙病菌不同侵染时期的生物学和转录组学分析

Biologic and Transcriptomic Analysis of Citrus hystrix Responses to‘Candidatus Liberibacter asiaticus’at Different Infection Stages

【目的】柑橘黄龙病(Huanglongbing,HLB)是制约柑橘产业发展的重大病害,我国发生的黄龙病由韧皮部杆菌亚洲种(‘Candidatus Liberibacter asiaticus’,CLas)引起。本研究通过分析潜在耐黄龙病柑橘品种马蜂柑(Citrus hystrix)在感染黄龙病后不同时期的生物学症状、显微结构和转录组学差异,揭示马蜂柑不同时期对黄龙病菌具有耐性的分子机理,为进一步深入挖掘抗性基因及开展抗病育种打下基础。【方法】以嫁接在两年生卡里佐枳橙砧木上的马蜂柑作为供试材料,使用只感染CLas、其他常见柑橘病毒类病原均呈阴性的毒源为接毒材料对马蜂柑进行接种,并在接种毒源后每隔15 d进行定期的荧光定量PCR检测。将马蜂柑接种毒源4个月(最早检测到CLas阳性)作为感病前期处理,接种毒源14个月作为感病后期处理,以健康植株为对照,进行生物学症状和显微结构观察,分析其感病后不同时期的结构变化。结合比较转录组学分析与荧光定量PCR验证,解析其耐病相关机制。【结果】症状观察发现,马蜂柑在感病前期和感病后期,植株叶片几乎不显症;显微结构观察表明,马蜂柑在感病前期中脉组织结构清晰,细胞形态正常,无淀粉粒积累的现象,仅在后期出现韧皮部极少数的筛管被堵塞;对转录组测序结果进行筛选鉴定,马蜂柑感病前期共筛选鉴定到181个差异表达基因,感病后期共筛选鉴定到1384个差异表达基因;比较转录组分析表明,马蜂柑感病前期和后期的差异表达基因主要涉及细胞壁代谢、防御反应、淀粉与蔗糖代谢、胼胝质合成以及信号转导等方面,其中在感病前期,淀粉与蔗糖代谢、细胞壁代谢相关的调控基因下调表达,在感病后期,水杨酸代谢及其信号转导途径、病程相关蛋白和谷胱甘肽转移酶相关的调控基因上调表达。【结论】马蜂柑响应CLas早期侵染主要表现为物理结构稳定、淀粉合成和光合作用等途径不受干扰;而水杨酸介导的抗性信号转导、效应蛋白触发免疫反应(effector-triggered immunity,ETI)激活的病程相关蛋白和谷胱甘肽转移酶参与的解毒作用是感病后期的主要耐病机制。

【Objective】Citrus Huanglongbing(HLB),associated with phloem-colonized‘Candidatus Liberibacter asiaticus’(CLas),severely impedes worldwide citrus production.The objective of this study is to analyze the biological symptoms,microstructures and transcriptomes of Citrus hystrix in response to CLas infection at different stages,to reveal the tolerance mechanism of C.hystrix,and to provide a basis for further screening of resistance genes and HLB-tolerant/resistant citrus breeding.【Method】The budwoods of C.hystrix grafted on two-year-old Carrizo citrange rootstocks(C.sinensis×P.trifoliata)used in this study were graft-inoculated with budwoods from a CLas(strain GZBJT)-infected Guanximiyou pummelo maintained in a greenhouse at Citrus Research Institute.The budwoods used as inoculum were tested CLas positive and free of other potential phloem-limited pathogens,such as Citrus tristeza virus(CTV)or Citrus tatter leaf virus(CTLV)by PCR before grafting.Inoculated plants were kept in greenhouse along with mock-inoculated healthy control plants.Real-time quantitative PCR(qPCR)was performed every 15 days after inoculation.Four months after inoculation(the earliest establishment of CLas by qPCR)and 14 months after inoculation were defined as the early infection stage and the late infection stage,respectively.The biological symptoms and microstructures were observed to analyze the structural changes of different infection stages.Combined with comparative transcriptome and RT-qPCR validation,the response mechanism of C.hystrix against HLB was explored.【Result】No typical symptom was observed in C.hystrix at the early and late stages of infection.Light microscopy observation from the midribs of HLB-affected and uninfected C.hystrix revealed that no significant structure change was found at the early infection stage and only a few sieves in the phloem were blocked at the late stage.By comparing the RNA-seq data,181 and 1384 genes were found to be differentially expressed at the early stage and late stage,respectively.Differentially expressed genes(DEGs)mainly involved in cell wall metabolism,host defense response,starch and sucrose metabolism,callose synthesis and signal transduction.Comparative transcriptome analysis showed that the expression of related genes in starch and sucrose metabolism and cell wall metabolism was down-regulated at the early infection stage,and the expression of related genes in salicylic acid metabolism,salicylic acid signal transduction pathway,pathogenesis-related protein and glutathione-S-transferases was up-regulated at the late infection stage.【Conclusion】The early response of C.hystrix to CLas infection is mainly characterized by stable physical structure,undisturbed pathways such as starch synthesis and photosynthesis.Salicylic acid-mediated resistance signals,effector-triggered immunity(ETI),and glutathione-S-transferases mediated detoxification contribute to the tolerance of C.hystrix against CLas at the late infection stage.