【作者】 陈静；

【导师】 陶小荣；

【作者基本信息】 南京农业大学 ， 植物病理学， 2022， 博士

【摘要】 植物免疫系统对植物的健康和生存至关重要,NLR(Nucleotide-binding leucine-rich repeat)免疫受体在植物对抗病原菌的攻击中发挥重要作用。这类受体蛋白通过直接或者间接的方式识别病原菌的效应因子(Effector)进而激活免疫反应。NLR蛋白也是植物中最大的一类抗病蛋白,其蛋白结构主要由核苷酸结合位点(Nucleotide binding site,NBS)和富亮氨酸重复序列(Leucine rich repeat,LRR)的结构域组成,也被称为NLR免疫受体。番茄斑萎病毒(Tomato spotted wilt virus,TSWV)是布尼亚病毒目(Bunyavirales)番茄斑萎病毒科(Tospoviridae)的重要成员,该病毒在全球范围内分布广泛,对辣椒、番茄等重要农业经济作物造成重大危害。Tsw是一个辣椒中对TSWV侵染具有免疫作用的NLR免疫受体,该受体特异性地识别TSWV编码的非结构蛋白NSs进而产生强烈过敏性坏死反应(Hypersensitive response,HR)。本研究中我们分析Tsw免疫受体的结构发现,Tsw的LRR结构域(1680个氨基酸)是常规NLR免疫受体LRR结构域(约600个氨基酸)的近三倍。经蛋白三维结构预测分析发现,Tsw的LRR结构域与茉莉酸受体COI1、生长素受体TIR1及独脚金内酯共受体MAX2的LRR结构域具有三维结构上的同源性,这暗示着病毒可能攻击植物的这三大类激素受体,Tsw主要通过识别TSWV编码的NSs蛋白诱导抗性反应。因此我们进一步推测TSWV NSs蛋白攻击这三条激素通路,而Tsw进化出模拟这些激素受体的LRR结构域(Phytohormone receptor-like,PRL)来感知病毒的攻击进而诱导植物抗性。本研究以此为切入点,对TSWV攻击激素受体的行为机制进行了系统的探究,同时对免疫受体Tsw模拟激素受体监控TSWV攻击进而诱导抗性的机制进行了深入的解析。主要研究结果如下:1.TSWV编码的NSs蛋白靶向茉莉酸、生长素和独脚金内酯激素受体信号通路促进病毒侵染基于上述分析,我们首先测试了TSWV侵染是否能够抑制茉莉酸(Jasmonic acid,JA)、生长素(Auxin,AUX)和独脚金内酯(Strigolactone,SL)激素受体的响应通路。通过对TSWV侵染的拟南芥转录组测序结果分析,发现病毒侵染后拟南芥中的JA、AUX和SL激素响应基因均被下调。通过q RT-PCR的进一步验证,发现JA、AUX和SL等激素信号途径在病毒侵染中的确被抑制。通过测定这三类激素的水平发现,TSWV侵染的植株中茉莉酸和生长素水平明显下调,同时TSWV的侵染干扰了独脚金内酯激素的水平。外源喷施JA、AUX激素激活寄主植物相应信号通路能够较好的抗TSWV的侵染。为了进一步明确Tsw NLR识别的无毒因子TSWV NSs在其中的作用,将TSWV NSs转入到拟南芥中,发现转基因拟南芥的JA、AUX和SL激素响应基因同样被抑制,同时NSs转基因植物能够吸引更多的传毒介体蓟马,表明病毒NSs直接抑制这三条激素响应通路。免疫共沉淀(Co-IP)、双分子荧光互补(Bi FC)、荧光素酶互补(SLC)等实验表明NSs可以和COI1受体、TIR1受体、MAX2共受体蛋白在植物体内发生互作(互作主要发生在细胞核内),同时NSs也可以和Tsw-PRL在植物体内发生互作。这些结果表明,TSWV NSs可以靶向激素受体抑制JA、AUX等多种主要激素信号通路进而促进病毒侵染。2.TSWV NSs通过TCP21干扰COI1/TIR1/MAX2激素受体信号的分子机制为进一步解析TSWV NSs蛋白攻击COI1/TIR1/MAX2激素受体抑制激素信号的分子机制,用酵母双杂交(Y2H)进一步验证却发现NSs与COI1/TIR1/MAX2激素受体并无直接相互作用。为了寻找NSs靶向三种激素受体可能利用的中间寄主因子,研究进一步利用TSWV NSs为诱饵对拟南芥酵母双杂c DNA文库进行筛选,发现TCP转录因子家族成员TCP7、TCP14与NSs互作。TCPs是一类植物特有的具有保守TCP结构域的转录调控蛋白,广泛参与植物生长发育等过程。研究进一步筛选了全部24个TCP家族成员与NSs的互作,发现仅有TCP21可以既与NSs蛋白发生互作,也与COI1/TIR1/MAX2激素受体发生互作,同时还与Tsw-PRL结构域发生互作。转基因过表达TCP21的拟南芥植株能够同时抑制JA、AUX、SL激素响应基因的应答,而敲除TCP21基因则部分激活JA、AUX激素响应基因的应答。SLC、Y2H、GST Pull-down等实验进一步发现,TCP21可以直接结合激素受体COI1的LRR结构域,TCP21通过与激素受体的LRR结构域互作,阻碍激素受体COI1/TIR1/MAX2结合到转录抑制子JAZ/IAA/SMXL上,进而阻碍这些转录抑制子的降解和相关激素响应通路的激活。进一步研究发现,再加入TSWV NSs在体内和体外都能够进一步增强TCP21与COI1/TIR1/MAX2激素受体的互作,进而进一步阻碍激素受体COI1/TIR1/MAX2结合到转录抑制子JAZ/IAA/SMXL上,并且TSWV NSs阻碍转录抑制子JAZ/IAA/SMXL的降解依赖TCP21。过表达TCP21能够促进TSWV的侵染,而敲除/沉默TCP21的植株则更抗TSWV的侵染。过表达TCP21同样能够促进灰霉菌(Botrytis cinerea)的侵染,而敲除TCP21的植株更抗灰霉菌。这些研究结果表明TCP21可以直接结合三种激素受体进而调控激素响应通路,而TSWV NSs通过靶向TCP21进而干扰激素受体信号通路来促进TSWV的侵染。3.NLR免疫受体Tsw监控TSWV攻击激素受体进而诱导植物免疫的分子机制上述研究结果表明,NSs通过TCP21攻击三个激素受体蛋白抑制激素响应信号通路促进病毒侵染,并且深入解析了其分子机制。本章重点解析TSWV NSs攻击三个激素受体蛋白抑制激素响应信号的行为被Tsw监控识别进而诱导植物免疫的分子机制。SLC、Y2H、GST Pull-down等试验结果发现,免疫受体Tsw模拟激素受体的LRR结构域Tsw-PRL与辣椒CaTCP21直接互作。进一步研究发现,病毒侵染或表达NSs均能显著增强Tsw-PRL与CaTCP21的互作。并且,在病毒侵染或NSs存在的情况下,Tsw-PRL与CaTCP21的互作相对COI1/TIR1/MAX2激素受体与CaTCP21的互作具有更强的亲和力。在本氏烟中沉默TCP21,NSs与Tsw共表达诱导的HR死亡反应显著减弱,而过表达TCP21则显著增强NSs与Tsw诱导的HR反应。另外,沉默TCP21后明显减弱NSs与Tsw在植物体内的互作,表明TCP21介导了Tsw对NSs的免疫识别。在含有Tsw的抗性辣椒(PI152225)中沉默TCP21,接种TSWV后,病毒可以系统突破植株的抗性,表明TCP21介导了Tsw对病毒的抗性。综上所述,本研究阐明了番茄斑萎病毒通过TCP21这一新型的寄主因子抑制多种主要激素受体信号进而促进病毒侵染的机制,同时明确了TCP21在免疫受体Tsw识别病毒效应子NSs中发挥的关键作用。研究系统探究了病毒攻击激素途径促进侵染和植物进化出的NLR蛋白Tsw监控病毒攻击激素受体行为而诱导植物免疫的“军备竞赛”的分子机制,研究结果增进了我们对于病毒TSWV和植物R基因Tsw的“攻击”与“防御”的新的认识。更多还原

【Abstract】 Plant immune system is crucial for plant health and survival.Nucleotide-binding leucine-rich repeat(NLR)immune receptors play an important role in plant resistance to pathogen attack.These receptor proteins can recognize the effector of pathogen directly or indirectly,which activates the immune response.NLR protein,also known as NLR immune receptors,is also the largest class of disease-resistant proteins in plants,and its protein structure is mainly composed of nucleotide binding sites(NBS)and leucine rich repeat(LRR)domains.Tomato spotted wilt virus(TSWV)is an important member of the family Tospoviridae(Bunyavirales),which has a wide global distribution and causes significant damage to important agro-economic crops such as peppers and tomatoes.Tsw is an NLR immune receptor in pepper that has an immune effect on TSWV infection.This receptor specifically recognizes the non-structural protein NSs encoded by TSWV and then produces a strong hypersensitive response(HR).In this study,we analyzed the structure of Tsw immune receptor and found that the LRR domain of Tsw(1680 amino acids)was nearly three times larger than the LRR domain of conventional NLR immune receptor(about 600 amino acids).The three-dimensional structure prediction analysis showed that the LRR domain of Tsw has three-dimensional structural homology with the LRR domain of jasmonic acid receptor COI1,auxin receptor TIR1 and strigolactone coreceptor MAX2,suggesting that the virus may attack these three kinds of hormone receptors in plants and that Tsw induces resistance responses mainly by recognizing TSWV-encoded NSs proteins.Therefore,we further hypothesized that TSWV NSs proteins attack these three hormone pathways,and Tsw evolved a phytohormone receptor-like LRR domain that mimics these hormone receptors PRL to sense virus attack and induce plant resistance.In this study,the behavioral mechanism of TSWV attack on hormone receptors was systematically explored,and the mechanism of immune receptor Tsw mimicking hormone receptors to monitor TSWV attack and induce resistance was also deeply analyzed.The main findings are as follows:1.TSWV NSs promotes virus infection by targeting jasmonate,auxin and strigolactone hormone receptor signaling pathwaysBased on the above analyses,we first tested whether TSWV infection could inhibit the response pathways of Jasmonic acid(JA),Auxin(AUX)and Strigolactone(SL)hormone receptors.Transcriptome sequencing of TSWV-infected Arabidopsis revealed that JA,AUX and SL hormone-responsive genes were down-regulated after infection.Further verification by q RT-PCR showed that JA,AUX,SL and other hormone signaling pathways were indeed inhibited in virus infection.By measuring the levels of these three hormones,it was found that the levels of jasmonic acid and auxin were significantly down-regulated in TSWV-infected plants,and the levels of strigolactone hormone were interfered by TSWV infection.Exogenous spraying of JA and AUX hormones could activate the corresponding signaling pathways of host plants and better resist TSWV infection.In order to further clarify the role of the non-toxic factor TSWV NSs recognized by Tsw NLR,TSWV NSs was transferred into Arabidopsis thaliana,and it was found that JA,AUX and SL hormone-responsive genes of transgenic Arabidopsis thaliana were also inhibited,and the NSs transgenic plants could attract more of the virulent mediator thrips.These results indicated that viral NSs directly inhibited the three hormone response pathways.Co-immunoprecipitation(Co-IP),bimolecular fluorescence complementation(Bi FC),split-luciferase complementation(SLC)and other experiments showed that NSs could interact with COI1 receptor,TIR1 receptor,and MAX2 co-receptor proteins in plants(the interactions occur mainly in the nucleus).At the same time,NSs can also interact with TSW-PRL in plants.These results suggest that TSWV NSs can target hormone receptors to inhibit JA,AUX and other major hormone signaling pathways to promote virus infection.2.Molecular mechanism of TSWV NSs interfering with COI1/TIR1/MAX2 hormone receptor signal through TCP21To further elucidate the molecular mechanism of TSWV NSs attacking COI1/TIR1/MAX2 hormone receptors and inhibiting hormone signaling,further validation by yeast two-hybrid(Y2H)revealed that that NSs did not directly interact with COI1/TIR1/MAX2 hormone receptors.In order to find the intermediate host factors that might be utilized by NSs targeting the three hormone receptors,the study further used TSWV NSs as bait to screen the Arabidopsis yeast two hybrid c DNA library,and found that TCP transcription factor family members TCP7 and TCP14 interacted with NSs.TCPs are a class of plant-specific transcriptional regulatory proteins with conserved TCP domains,which are widely involved in plant growth and development.The interactions between all 24 TCP family members and NSs were further screened.Only TCP21 could interact with both NSs protein and COI1/TIR1/MAX2 hormone receptor,as well as with Tsw-PRL domain.Transgenic Arabidopsis overexpressing TCP21 could inhibit the responses of JA,AUX and SL hormone-responsive genes simultaneously,while TCP21 knockdown partially activated the responses of JA and AUX hormone-responsive genes.SLC,Y2H,and GST pull-down experiments further found that TCP21 could directly bind to the LRR domain of hormone receptor COI1,and that TCP21,by interacting with the LRR structural domain of the hormone receptor,could prevent the hormone receptor COI1/TIR1/MAX2 from binding to the transcriptional repressors JAZ/IAA/SMXL,which inhibits the degradation of these transcriptional repressors and the activation of related hormone response pathways.Further studies showed that the addition of TSWV NSs could further enhance the interaction between TCP21 and COI1/TIR1/MAX2 hormone receptor both in vivo and in vitro,which further blocked the binding of COI1/TIR1/MAX2 hormone receptor to the transcriptional repressor JAZ/IAA/SMXL.Moreover,TSWV NSs prevented the degradation of the transcriptional repressor JAZ/IAA/SMXL dependent on TCP21.TCP21 overexpression could promote TSWV infection,while TCP21 knockout/silencing plants were more resistant to TSWV infection.TCP21 overexpression could also promote the infection of Botrytis cinerea,while TCP21 knockout plants were more resistant to Botrytis cinerea.These findings suggest that TCP21 could directly bind to three hormone receptors to regulate hormone response pathways,and TSWV NSs could promote TSWV infection by targeting TCP21 and interfering with hormone receptor signaling pathways.3.Tsw NLR detects TSWV’s disabling hormone receptors induces plant immunityThese results indicated that NSs inhibit the hormone response signaling pathway by attacking three hormone receptor proteins through TCP21 to promote virus infection,and the molecular mechanism was further analyzed.This chapter focuses on the molecular mechanism by which TSWV NSs attacks three hormone receptor proteins to inhibit hormone response signals and is monitored and recognized by Tsw to induce plant immunity.SLC,Y2H and GST pull-down tests showed that the LRR domain of immune receptor Tsw mimics hormone receptor Tsw-PRL directly interacts with CaTCP21.Further studies showed that both virus infection and NSs expression significantly enhanced the interaction between Tsw-PRL and CaTCP21.Moreover,in the presence of virus infection or NSs,the interaction between Tsw-PRL and CaTCP21 has a stronger affinity than the interaction between COI1/TIR1/MAX2 hormone receptor and CaTCP21.When TCP21 was silenced in tobacco,the HR death response induced by co-expression of NSs and Tsw was significantly attenuated,while overexpression of TCP21 significantly enhanced the HR response induced by NSs and Tsw.In addition,TCP21 silencing significantly attenuated the interaction between NSs and Tsw in plants,indicating that TCP21 mediated the immune recognition of NSs by Tsw.Silencing of TCP21 in Tsw-containing resistant peppers(PI152225)and inoculation with TSWV allowed the virus to systematically break through the resistance of the plants,indicating that TCP21 mediated the resistance of Tsw to virus.In conclusion,this study clarified the mechanism by which tomato spotted wilt virus promotes virus infection by inhibiting various major hormone receptor signals through TCP21,a novel host factor,and also clarified the key role of TCP21 in the recognition of viral effector NSs by the immune receptor Tsw.This study systematically explored the molecular mechanism of the"arms race"in which the viral attack hormone pathway promotes infection and the plant evolved NLR protein Tsw monitors the behavior of viral attack hormone receptor and induces plant immunity.The results will improve our understanding of the"attack"and"defense"of viral TSWV and plant R gene Tsw.更多还原