柑橘黄龙病菌非自主转座子MCLas-A中串联重复与转座酶互作及其调节寄主症状的机制

Citrus Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. ‘Candidatus Liberibacter asiaticus’ is a putative pathogen of HLB. In our previous study, two miniature inverted repeat transposable elements, MCLas-A and MCLas-B, were identified in ‘Ca. L. asiaticus’. A unique feature of MCLas-A is the presence of a three units of tandem repeats in its terminal inverted repeats (TIRs). Based on the diverse transposon footprints, we hypothesized that the tandem repeats in TIRs might play important roles in recognizing and binding of transposase. In this study, a bacterial one-hybrid reporter system with MCLas-A, deleted MCLas-A or chimeric element will be constructed to evaluate excision efficiency of transposase. Electrophoretic mobility shift assay (EMSA) and site-directed mutagenesis will be performed to examined the interaction of purified transposase with different parts of TIRs and central region of MCLas-A. To investigate the interaction between MCLas-A and the upstream transposase in vivo, transposase and MCLas-A(deleted or chimeric MCLas-A) will be cloned into citrus tristeza virus (CTV)-based vector under the control of specific CTV promoter and then inoculated onto host plants. Meanwhile, representative ‘Ca. L. asiaticus’ strains will be inoculated onto citrus plants under temperature stress to observe the transposition activity of MCLas-A. Real-time PCR and comparative transcriptiome approaches will be used to determine global gene expression changes in ‘Ca. L. asiaticus’ and genes and transcription factors related with dwarfish phenotype in citrus host. Information provided in the study will dissect the mechanisms of interactions between tandem repeat in MCLas-A and transposase, determine the molecular mechanisms of its impact on host, and built the theoretical basis for potential applications of the MCLas-A-based transposition system in HLB control.

柑橘黄龙病是当前柑橘生产上最具毁灭性的病害。研究发现黄龙病菌中存在两种非自主转座子MCLas-A和MCLas-B,其中具有转座活性的MCLas-A的末端反向重复区（TIR）具有串联重复的特殊结构。根据转座足迹，推测该串联重复结构在转座酶识别和切割中扮演重要角色。本研究拟构建细菌单杂交系统，评估转座酶对MCLas-A及其嵌合体、缺失体的切割效率，观察转座酶蛋白与TIR及中央区的互作；基于柑橘衰退病毒表达载体介导转座酶与MCLas-A互作和代表性黄龙病菌株系直接接种，分析MCLas-A在植株韧皮部组织中的转座活性；通过实时PCR和转录组测序，明确MCLas-A的转座介导黄龙病菌及柑桔寄主的基因差异表达情况。本研究有望解析黄龙病菌MCLas-A中串联重复与转座酶的互作机制，明确病原与寄主对转座的响应，为进一步研究MCLas-A在病原-寄主互作中的功能和该转座系统在黄龙病防控中的潜在应用奠定基础。

柑橘黄龙病是制约我国柑橘生产的重大病害。前期研究中从黄龙病菌中鉴定了一个活性的微型反向重复转座元件MCLas-A。为进一步探索MCLas-A的转座活性及转座对寄主产生的影响，本研究结合运用特异性引物扩增和代表性样品下一代测序分析，发现MCLas-A检出率仅为1.80%，其转座产物检出率达到81.23%，表明近5年收集的黄龙病样品中MCLas-A发生了高频转座；对中国江西、广西、贵州、四川、云南以及巴基斯坦旁遮普省收集的49个黄龙病菌进行全基因组测序分析，对其余70个样品进行侧翼区域扩增测序，表明MCLas-A及其转座产物主要存在于type2噬菌体中，而MCLas-B主要存在于type1和type3噬菌体，系统发育分析表明上游推断的转座酶存在两种高度保守的序列类型，分别命名为Tra1和Tra2，两者间序列相似度为66.08%；开展了柑橘衰退病毒（Citrus tristeza virus, CTV）表达载体介导转座酶和MCLas-A的体内表达研究，对来源于贵州罗甸的含MCLas-A黄龙病菌样品GZLD40进行了转座前后的转录组分析，表明转座与非转座相比差异表达基因可富集到多个相关代谢途径，其中细胞壁代谢、激素代谢等相关代谢途径呈上调表达，而生物胁迫病程相关蛋白等代谢途径呈下调表达，结合转座后寄主表现轻微矮化的生物学特征，推测MCLas-A转座对细胞壁相关通路和植物激素相关通路产生重要影响。本研究解析了MCLas-A的转座特征，初步明确了对寄主产生的影响，对MCLas-A高频转座的驱动因子和分子机制的深入研究将会加深对黄龙病菌生物学的认知。