Mechanism of signal transduction pathways for the host response to virus infection

黄瓜花叶病毒(CMV)和烟草花叶病毒(TMV)是目前研究最多的正链RNA病毒。本课题对小麦抗CMV和TMV的信号转导途径进行了研究。CMV-拟南芥系统：拟南芥生态型C24对CMV黄色株系[CMV(Y)]具有抗性。这种对CMV(Y)的抗性是由RPP8/HRT抗性基因家族中的一个显性基因RCY1决定的。我们对缺乏SA、JA和乙烯信号通路的拟南芥突变体的分析表明，SA和乙烯依赖的信号通路在RCY1基因对CMV(Y)的高水平抗性表达中起着重要作用，但在nahG etr1 RCY1双突变体中SA和乙烯信号的联合损伤并不能完全消除RCy1赋予的对CMV(Y)的抗性。因此我们提出，除了SA和乙烯信号外，还有一条新的未被鉴定的信号通路是Requu…对黄瓜花叶病毒(Y)的完全抗性是由RCY1基因赋予的。此外，SA和JA信号通路之间的串扰是RCy1对CMV(Y)抗性的重要调节器。对双突变体的分析表明，依赖CO/1的机制负调控了RCy1介导的SA信号通路的激活和对CMV(Y)的抗性。Di-17生态型HRT对芜菁花叶病毒(TCV)的基因对基因抗性依赖于SA介导的信号通路，而不依赖于JA和乙烯信号通路。虽然生态型C24中的RCy1与生态型Di-17中的HRT同源，但在抗CMV(Y)和TCV中对SA信号的需求程度在RCy1和含有HRT的植株中有所不同。针对这两种病毒的防御机制要求的差异在拟南芥ssi2突变体的研究中得到了进一步的例证。虽然ssi2突变体表现出对CMV(Y)的增强抗性，但它并没有增强对TCV的抗性。综上所述，我们的结果表明，拟南芥的RCyI和HRT基因进化为招募不同的信号转导途径组合，以赋予对两种不同病毒的抗性。TMV-烟草系统：在携带温度敏感的N抗性基因的TMV侵染烟草植株中，利用一个同步的HR细胞-死亡诱导系统以及与HR-信号交叉对话的伤害-信号途径，研究了获得病原菌侵染的信号途径。我们研究了HR触发后3h表达被诱导或被抑制的基因的功能。WRK：HR触发和损伤后迅速诱导了受体样蛋白激酶基因的表达。WRK在不依赖N基因的30℃到20℃之间进行简单的温度转移而获得低水平的结构性表达。在WRK表达抑制的转基因烟草中，WRK和SIPK的激活、JA的积累和PI-II等伤害诱导基因的表达被抑制，这表明WRK至少参与了伤害信号转导。HSP90：在HSP90基因表达被抑制的转基因烟草植株中，HR诱导的电解质渗漏水平(细胞死亡的指标)显著受到抑制，对TMV的抗性减弱。用Hsp90的特异性抑制剂格尔达那霉素处理后，WIPK和SIPK的离子渗漏和创伤诱导的激活减少，表明HSP90参与了HR信号转导。CaM(钙调素)：我们分离了13个烟草基因，编码三种植物特有的CaM类型，其中一些是由HR触发或伤害诱导的。每种类型的CaM的数量在HR或伤害后动态变化，烟草CaM以钙依赖和类型特定的方式特异性地激活目标酶。作为一种新的CaM结合蛋白，我们分离到一种MAPK磷酸酶(MKP1)。该基因产物在体外使SIPK失活。在mkp1过表达中，创伤诱导的WIPK和SIPK的激活被抑制，提示Ca^2/CaM信号在MAPK的上游发挥作用，至少在创伤信号转导中起作用。WAF-1：WIPK是通过TMV感染对创伤和HR做出反应而被酶激活的。(11E，13E)-Labda-11，13-diene-8a，15-diol，命名为WAF-1，鉴定为侵染烟草花叶病毒的WIPK激活物质。WAF-1是一种新的二萜类化合物，其功能是作为内源信号介导烟草对TMV侵染和伤害的防御反应。较少

Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV) are the best-characterized positive-sense single-stranded RNA viruses. In this project, the signal transduction pathways for the resistance to CMV and TMV were studied. CMV-Arabidopsis system: A. thaliana ecotype C24 is resistant to a yellow strain of CMV [CMV(Y)]. This resistance to CMV(Y) was conferred by a single dominant gene, RCY1, which is a member of RPP8/HRT resistance gene family. Our analysis of Arabidopsis mutants deficient in SA, JA and ethylene signaling pathways indicated that an SA-and ethylene-dependent signaling pathway played an important role in the expression of the high level of resistance to CMV(Y) conferred by the RCY1 gene, However, the combined impairment of SA and ethylene signaling in the nahG etrl RCY1 double mutant did not completely abolish the RCY1-conferred resistance to CMV(Y). We therefore proposed that in addition to SA and ethylene signaling, a novel uncharacterized signaling pathway was requ … More ired for the full resistance to CMV(Y) conferred by the RCY1 gene. Moreover, cross-talk between the SA and JA signaling pathways is an important modulator of RCY1-conferred resistance to CMV(Y). The analysis of double Arabidopsis mutants indicated that a CO/1-dependent mechanism negatively modulated the RCY1-mediated activation of SA signaling pathway and resistance to CMV(Y). The gene-for-gene resistance to Turnip mosaic virus (TCV) conferred by HRT of ecotype Di-17 was dependent on the SA-mediated signaling pathway, but not on the JA and ethylene signaling pathways. Although RCY1 in ecotype C24 was an ortholog of HRT in the ecotype Di-17, the extent of the requirement for SA signaling in conferring resistance to CMV(Y) and TCV was different between RCY1-and HRT-containing plants. The difference in the requirement of defense mechanisms targeting these two viruses is further exemplified by studies with the Arabidopsis ssi2 mutant. While the ssi2 mutant exhibited enhanced resistance to CMV(Y), it did not confer enhanced resistance to TCV. In summary our results suggest that the Arabidopsis RCYI and HRT genes evolved to recruit different combinations of signal transduction pathways for conferring resistance to two different viruses. TMV-Tobacco system: The, signaling pathway for acquired resistance to pathogen infection has been examined using a synchronous HR cell-death-inducing system in TMV-infected tobacco plants carrying the temperature-sensitive N resistance gene, as well as the wound-signaling pathway which cross-talks with HR-signaling. We studied the function of genes whose expression were induced or repressed 3 h after HR triggering. WRK: A receptor-like protein kinase gene was induced rapidly after HR triggering and wounding. WRK was constitutively expressed at a low level, and induced by a simple temperature transfer from 30'C to 20'C independent of the N gene. In transgenic tobacco with suppressed WRK expression, wound-induced phenomena such as activation of defense-related MAPKS (WIPK and SIPK), JA accumulation, and expression of wound-inducible genes such as PI-II were inhibited, suggesting involvement of WRK at least in wound-signaling. HSP90 : In transgenic tobacco plants with suppressed HSP90 gene expression, the level of HR-induced electrolytic leakage, an indicator of cell death, was considerably inhibited with attenuated resistance to TMV. Treatment with geldanamycin, a specific inhibitor of Hsp90, reduced the ion leakage and wound-induced activation of WIPK and SIPK, indicating the involvement of HSP90 in HR-signaling. CaM (calmodulin) : We isolated 13 tobacco genes encoding three plant-specific types of CaMs, some of which were induced by HR-triggering or wounding. The amount of each type CaM dynamically changed after HR or wounding, Tobacco CaMs activates target enzymes specifically in a Ca^<2+>dependent and type-specific manner. As a new CaM-binding protein, a MAPK phosphatase (MKP1) was isolated. The gene product inactivated SIPK in vitro. In MKP1 overexpressors, wound-induced activation of WIPK and SIPK was inhibited, suggesting Ca^2/CaM signal functions upstream in MAPK-cascade at least in wound-signaling. WAF-1: WIPK is activated enzymatically in response to wounding and HR by TMV infection. (11E,13E)-labda-11,13-diene-8a,15-diol, designated WAF-1 was identified as WIPK-activating substance from TMV-infected tobacco leaves. WAF-1, a novel diterpene, functions as an endogenous signal mediating defense responses of tobacco plants to TMV infection and wounding. Less