【作者】 王宁；

【导师】 温孚江； 朱常香；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2018， 博士

【摘要】 虫媒病害的发生与否和流行的严重程度,依赖于病毒-昆虫-植物三者互作关系。在植物与植食性昆虫防御与反防御的博弈中,当植物感受到植食性昆虫的信号时,能迅速激活植食性昆虫分子模式相关的免疫信号转导,继而调控众多抗性相关基因的转录表达,介导防御物质的合成,从而提高植物对昆虫侵害的抵御能力。为了应对植物的抗性免疫,植食性昆虫在取食过程中可以分泌特异性的唾液效应子来抑制植物的免疫激活,从而抑制植物防御基因的表达和抗性物质的合成,进而保证昆虫在寄主植物上正常的取食、生长与繁衍。关于昆虫分泌的效应子,已有的研究多集中在对效应子的筛选和其抑制抗性免疫的生物学现象的探究,然而对于效应子如何调控植物免疫途径的机理性研究少有报道。烟粉虱(Bemisia tabaci)是传播植物病毒的媒介昆虫,由烟粉虱传播的病毒高达200多种,是农业生产上的重要害虫。番茄黄化曲叶病毒(Tomato yellow leaf curl virus,TYLCV)是以烟粉虱作为媒介昆虫的双生病毒,每年在粮食及经济作物上引起重大经济损失。本课题以烟粉虱和番茄黄化曲叶病毒为研究对象,探究了烟粉虱诱导的植物早期免疫反应,进而筛选了TYLCV诱导的烟粉虱唾液效应子,并对唾液效应子在植物免疫调控的作用机制进行了研究,最终阐明了受双生病毒诱导的烟粉虱效应子如何影响植物抗性途径来促进病毒烟粉虱的互惠关系。本研究的主要结果如下:(1)烟粉虱可以诱导激活植物MAPKs级联途径。通过Western blot检测烟粉虱取食后拟南芥中MAPKs的激活水平,发现MPK3和MPK6能够被微弱的激活,并且短时间内激活水平并没有随取食时间的增加有明显的变化;这不同于咀嚼式昆虫处理的烟草中,MAPKs被迅速激活并达到较高的表达水平;说明刺吸式昆虫烟粉虱对MAPKs的激活有另一种作用模式。(2)拟南芥Pep/PEPR途径参与了烟粉虱介导的植物的免疫激活。通过qRT-PCR对烟粉虱处理的拟南芥中PEPR1受体的表达水平的定量分析,发现烟粉虱处理可以诱导PEPR1受体的表达;通过Western blot对Pep1处理后拟南芥中MAPKs激活进行检测,发现Pep1处理对拟南芥MAPKs的激活模式跟烟粉虱取食十分相似,即Pep1处理可以部分模拟烟粉虱取食对植物免疫的激活作用。(3)烟粉虱取食引起了植物胼胝质的积累。通过苯胺蓝染色检测烟粉虱取食和Pep1处理的拟南芥中胼胝质的分布和含量,发现烟粉虱和Pep1处理都能引发植物体中胼胝质的积累;胼胝质积累信号大多富集在烟粉虱取食部位筛管附近,说明胼胝质的积累是植物体一类重要的抗虫响应。(4)拟南芥MPK3和MPK6负调控了植物的抗虫免疫。通过对mpk3和mpk6突变体的胼胝质染色,发现拟南芥MPK3和MPK6在烟粉虱诱导的胼胝质积累的调控中发挥作用;通过烟粉虱的双向选择实验和产卵量的统计实验,发现MPK3和MPK6的激活有利于烟粉虱的选择并能提高其在植物上的适应力,说明MPK3和MPK6是抗虫反应的负调控因子;另外我们还发现MPK3和MPK6之间在抗性调控方面存在着功能的冗余,MPK6在其中发挥得作用更大。(5)MPK4作为植物抗虫免疫调控的正调控因子在烟粉虱诱导的免疫激活中发挥平衡作用。我们发现烟粉虱处理对MPK4与MPK3和MPK6有不同的激活模式,烟粉虱的双向选择实验和产卵量测定发现MPK4的激活提高了植物对烟粉虱的抗性,说明MPK4与MPK3/MPK3在抗虫免疫中作用模式不同。(6)筛选到了受TYLCV诱导的烟粉虱唾液蛋白。通过qRT-PCR对烟粉虱取食过的感染TYLCV的番茄中病毒滴度的检测,发现烟粉虱处理提高了感染TYLCV的番茄中病毒的积累量;取食过程中病毒和烟粉虱的信号交流与烟粉虱的唾液分泌相关,我们通过对感染TYLCV烟粉虱的转录组测序,与烟粉虱唾液腺转录组进行比对分析,通过筛选具有分泌蛋白的特征且在烟粉虱唾液腺中表达的蛋白,最终找到了28个受TYLCV诱导的唾液蛋白。(7)建立了针对烟粉虱唾液蛋白功能的新型筛选系统。我们利用荧光素酶报告系统,将PDF1.2基因的启动子区域连接萤光素酶作为报告系统,将候选的效应蛋白基因用35S启动子驱动作为驱动系统,共同在本生烟草中瞬时表达,用Pep1处理模拟烟粉虱行为对植物的免疫激发,通过萤光素酶检测定量分析抑制效率。如果候选蛋白可以抑制Pep1诱导的PDF1.2的表达,则可以观察到荧光强度的减弱。这套系统可以高通量有针对性的验证昆虫的效应蛋白的功能。(8)烟粉虱效应子Bep1可以有效抑制植物的抗虫反应。通过烟粉虱唾液蛋白功能的筛选,在候选唾液蛋白中我们筛选到Bep1可以有效抑制植物的抗虫反应。烟粉虱的双向选择实验和产卵量测定发现Bep1转基因植物提高了烟粉虱的适应力和繁殖能力。(9)Bep1与拟南芥转录因子WRKY33互作调控抗虫反应。通过酵母双杂交,双分子荧光互补的实验发现Bep1可以与拟南芥转录因子WRKY33互作。竞争性抑制实验的结果发现Bep1与WRKY33的互作竞争性抑制了MPK6与WRKY33的互作,干扰了MPK6的信号传递并且抑制了WRKY33的转录作用。对wrky33突变体的烟粉虱生测实验首次发现WRKY33不仅在调控病原菌免疫方面起重要作用,也参与了植物的抗虫免疫。烟粉虱效应子Bep1抑制了WRKY33介导的抗虫性,从而有助于烟粉虱更好的生长繁殖。本论文的研究丰富了双生病毒-烟粉虱-植物三者互作的内容,有助于揭示植物的抗虫免疫与抗病机制,对于从三者互作领域防治虫传病害的生物学策略的制定具有重要的参考价值。更多还原

【Abstract】 Around 80% of known plant viruses are vector-borned.Emergence of insect-transmitted plant viruses over the past years has caused reduced agricultural productivity.Plant insect-borne diseases are highly dependent on virus-insect-plant tripartite interaction,which is involved in arms race of signal recognition and counter recognition among them.Host immune systems mount insect-pathogen double challenges when pathogen-carrier insect infests.Plants have evolved a sophisticated defensive system,including immunity,to protect themselves from invaders.Plants percept pathogens or insects and consequently trigger immune responses to defend,and in turn,pathogens and insects have evolved mechanisms to overcome these immune defenses.Early response to herbivores begins with PRR perception of damage patterns.Danger-associated molecular patterns(DAMPs)and pathogen-associated molecular patterns(PAMPs)have been characterized as pattern-triggered plant immunity(PTI)in plants.Activation of several MAPKs are early signaling response following perception of PAMPs or DAMPs.Subsequently,MAPKs phosphorylate their substrates,and thus trigger downstream responses,including production of reactive oxygen species(ROS),callose deposition,and induction of defense genes.Several distinct MAPK cascades in plant and animal have been implicated in the regulation of defense against pathogens.To overcome plant immune defenses,insects have evolved strategies including effector salivation to inhibit insects triggered immunity.However,what’s biological roles of MAPK in plant response agaisnt hemipterans such as whitefly and the mechanisms of insect effector in immune system are still elusive.Here we first found that infestation of whitefly triggers plant MAPKs(mitogen-activated protein kinase)activation in unique mode differ from chewing insects.The activation of MAPKs then trigger defense pathway including callose deposition.Upon whitefly infestation,the expression level of AtPEPR1 that encoded a receptor to recognize DAMP was heavily induced within half a hour.It raised the possibility of PEPR signaling are involved in host immune response against whitefly infestation.The activation of MAPKs and callose deposition induced by whitefly are similar with Pep1 treatment raise a propose that Pep1 treatment can mimic the whitefly infestation in the defense response.Then we found that MPK3 and MPK6 are negative regulator in the defense response to whitefly.The mpk3 and mpk6 mutants are more attractive to whitefly and showed lower resistance level.MPK4 are positive regulator to fine-tune the immune response.From transcriptome analysis of TYLCV infected whitefly,we found 28 candiated effectors in the screening of whitefly saliva protein.The Bep1 protein can effective inhibit the defense gene PDF1.2 expression induced by whitefly infestation.The interation between Bep1 and WRKY33 competitive inhibit the combine of WRKY33 and MPK6.The whitefly effector Bep1 effected MAPK3 and MAPK6 therefore inactivates early immune signaling.This immune alternation by Bep1 affects whitefly feeding behavious thereby leading a possible enhancing of viral transmission.This discovery represents the first evidence of MAPK cascade in defense against whitefly infestation and a novel mechanism of immune suppression on targeting host MAPK components by a effector protein.Our data present a novel biochemical activity for a whitefly and counter-host defense mechanism of whitefly effector.This study enrichs our understanding on virus-plant-whitefly tripartite interaction.Our research will be helpful to making effective strategies in the protection of crops from both insects and disease infection.更多还原