黄瓜花叶病毒2b蛋白在RNA沉默通路中的作用方式研究
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摘要
黄瓜花叶病毒(Cucumber mosaic virus, CMV)是目前对农业经济危害最为严重的植物病毒之一。CMV 2b蛋白在CMV的致病过程中扮演着致病因子,RNA沉默抑制子和病毒长距离移动决定因子等多个重要的角色。因此,阐明CMV 2b蛋白的致病机理对于发展控制CMV新方法和新策略具有重要的理论指导意义。
FRad35~(2b)-CMV重组病毒是用Rad35-CMV株系的2b基因精确替换Fny-CMV株系的2b基因构建而成,该重组病毒在多种烟草寄主上均诱导轻花叶症状。其突变株FRad35~(2bLeu)-CMV病毒,和FRad35~(2b)-CMV仅有一个氨基酸之差,即第55位的Leu是由FRad35~(2b)-CMV的Pro突变而来。FRad35~(2bLeu) -CMV在本生烟上则引起严重卷曲叶、植株矮化、甚至植株坏死。Northern杂交分析FRad35~(2b)-CMV和FRad35~(2bLeu)-CMV在本生烟寄主中的子代RNA积累量,接种后第14天,结果显示: FRad35~(2bLeu)-CMV的子代RNA积累量显著高于FRad35~(2b)-CMV。由此可见,FRad35~(2bLeu)-CMV的强致病性与其子代RNA在寄主体内的高积累量有关。通过荧光定量PCR检测基因沉默通路中的相关蛋白因子—AGO蛋白家族和RDR蛋白,结果显示:具有高致病性的FRad35~(2bLeu)-CMV显著抑制寄主本生烟AGO家族中AGO1-1、AGO4-1的表达,而弱致病性的FRad35~(2b)-CMV侵染的本生烟中,这些沉默因子的表达量和Mock对照相比都有所升高。基于实验室前期的研究,利用构建好的含有FRad35~(2b)-CMV和FRad35~(2b) Leu-CMV的2b基因的农杆菌瞬时表达载体35S-2b~(Pro)和35S-2b~(Leu)分析2b蛋白的瞬时表达对外源GFP转录后基因沉默的影响。通过电转化获得含有不同载体的农杆菌,并将表达不同2b蛋白的农杆菌与35S-GFP的农杆菌共浸润,在转GFP本生烟16c叶组织中分别瞬时表达GFP和不同的2b蛋白。共浸润第3天,35S-2b~(Leu)和35S-Fny2b的浸润区出现亮绿斑,而35S-2b~(Pro)的浸润区变暗,GFP mRNA的Northern杂交分析确定GFP mRNA在35S-2b~(Leu)和35S-Fny2b的浸润区大量积累;共浸润后第7天,尽管35S-Fny2b和35S-2b~(Leu)仍表现亮荧光信号,但是,二者浸润区的GFPmRNA积累水平显著低于Mock对照,且与35S-2b~(Pro)相当,这些结果说明2b~(Leu)虽然在初期对GFP的局部沉默具有明显的抑制作用,但随着时间的推移,2b~(Leu)并不能抑制浸润区siRNA的产生,从而导致局部GFP mRNA的降解。在共浸润第14天,浸润35S-2b~(Pro)植株的顶端系统叶表现GFP系统沉默,而浸润35S-2b~(Leu)的植株则依旧显绿光,说明其顶端的系统叶GFP没有被诱导沉默。Northern杂交结果也显示:在浸润35S-2b~(Pro)的植株顶端系统叶中检测到少量的GFP mRNA积累,显著低于在35S-2b~(Leu)或35S-Fny2b浸润的植株顶端系统叶中的积累。通过荧光定量PCR检测第3天浸润区基因沉默通路中的AGO4-1和RDR6,结果显示:与Mock相比,出现亮绿斑的35S-2b~(Leu)的浸润区中AGO4-1和RDR6的积累受到抑制,而GFP发生降解的35S-2b~(Pro)的浸润区中AGO4-1和RDR6的表达量反而有一定程度的升高。因此,我们推测第55位的Leu对2b蛋白的高级结构有一定的影响,从而对AGO4-1和RDR6起到抑制作用,从而达到抑制系统沉默和DNA甲基化的目的。
综上所述,我们推测2b~(Leu)介导的高致病性是通过其阻止siRNA的系统移动,并抑制AGO4-1和RDR6等沉默因子的表达,来实现病毒高效的系统移动和复制,从而导致寄主表现出严重的病毒症状。
Cucumber mosaic virus (CMV) is one of the most economically important viruses to agriculture. CMV 2b protein was shown to be a multifunctional protein involved in sysptom induction, viral movement, suppressin of RNA silencing and so on. It is very important for developing new strategies of controlling CMV to investigate mechanism of pathogenicity of CMV 2b protein.
CMV recombinant FRad35~(2b)-CMV is generated by replacing 2b gene in Fny-CMV with 2b gene of Rad35-CMV. Our previous work has showed that the recombinant always induces mild mosaic in the most tested tobacco species. The mutant FRad35~(2bLeu) -CMV is different from FRad35~(2b)-CMV by only one amino acid, namely from Pro to Leu at the 55 position. The mutant caused leaf distortion, obvious stunting, even whole plant death on Nitociana benthamiana. When we inoculated FRad35~(2bLeu)-CMV in N. benthamiana as well as FRad35~(2b)-CMV,at 14 days post-inoculation (dpi), Northern blotting analysis showed that the accumulation of the viral RNAs in FRad35~(2bLeu)-CMV-infected plants was significantly higher than that in the FRad35~(2b)-CMV-infected plants. It is concluded that the high virulence of FRad35~(2bLeu)-CMV was associated with the high level of viral progeny RNA accumulation in the systemic infected leaves. By RT-qPCR, we analyzed effect of CMV 2b protein on expression of NbAGO and NbRDR. At 14 dpi, the transctritional levels of the NbAGO1-1 and NbAGO4-1 in N.benthamiana leaves infected with FRad35~(2bLeu)-CMV and FRad35~(2b)-CMV were determined by RT-qPCR, and compared with that in mock-inoculated leaves. The above results showed that FRad35~(2bLeu)-CMV significantly decreased the levels of the NbAGO1-1 and NbAGO4-1.
On the basis of preparatory work,we have constructed agrobacteria transient expression vectors of 2b genes from FRad35~(2b)-CMV and FRad35~(2bLeu)-CMV, namely 35S-2bPro and 35S-2bLeu respecively. When these vectors were co-infiltrated with 35S-GFP on N. benthamiana plant (line 16c), at 3 dpi, we found that zones infiltrated with 35S-2bLeu appeared bright green while zones infiltrated with 35S-2b~(pro) became dark. And northern blot also showed high level of GFP mRNA in zones infiltrated with 35S-2bLeu. At 7 dpi, those zones remained bright green, but the level of GFP mRNA became decreased, much less than mock. Thus, it is considered that 2bLeu could only suppress gene local silencing in early period, but it could not suppress the production of siRNA. The effect of Leu55 on systemic gene silencing was also investigated. It was found that at 14dpi, the top leaf of the plant infiltrated by 35S-2b~(Pro) turned red. While the top leaf of the plant infiltrated by 35S-2b~(Leu) have no effect. It seems that only 2b~(Leu) suppressed GFP systemic silencing. Northern blotting analysis showed that there was low level of GFP mRNA in the top leaf of plant infiltrated by 35S-2b~(Pro), and GFP mRNA accumulated highly in the top leaf of plant infiltrated by 35S-2bleu. By RT-qPCR, we analyzed effect of CMV 2b proteinon on the NbAGO4-1 and NbRDR6. At 3 dpi, the transcriptional levels of the NbAGO 4-1, NbRDR6 were determined in N.benthamiana leaves inoculated with 35S-2b~(Leu) and 35S-2b~(pro) by RT-qPCR and compared with that in mock-inoculated leaves. The results showed 35S-2b~(Leu) induced a decrease in the level of the AGO4-1, RDR6. It is predicted that the Leu55 can enhance the suppression to AGO and RDR6, then suppress the systemic silencing and RNA-directed DNA methylation (RdDM).
In summary, the results indicate that the 2b~(Leu)-mediated high pathogenic is achieved by suppressing systemic movement of siRNA, and inhii\MV can move and replicate more efficiently, inducing severe viral symptom on hosts.
FRad35~(2b)-CMV重组病毒是用Rad35-CMV株系的2b基因精确替换Fny-CMV株系的2b基因构建而成,该重组病毒在多种烟草寄主上均诱导轻花叶症状。其突变株FRad35~(2bLeu)-CMV病毒,和FRad35~(2b)-CMV仅有一个氨基酸之差,即第55位的Leu是由FRad35~(2b)-CMV的Pro突变而来。FRad35~(2bLeu) -CMV在本生烟上则引起严重卷曲叶、植株矮化、甚至植株坏死。Northern杂交分析FRad35~(2b)-CMV和FRad35~(2bLeu)-CMV在本生烟寄主中的子代RNA积累量,接种后第14天,结果显示: FRad35~(2bLeu)-CMV的子代RNA积累量显著高于FRad35~(2b)-CMV。由此可见,FRad35~(2bLeu)-CMV的强致病性与其子代RNA在寄主体内的高积累量有关。通过荧光定量PCR检测基因沉默通路中的相关蛋白因子—AGO蛋白家族和RDR蛋白,结果显示:具有高致病性的FRad35~(2bLeu)-CMV显著抑制寄主本生烟AGO家族中AGO1-1、AGO4-1的表达,而弱致病性的FRad35~(2b)-CMV侵染的本生烟中,这些沉默因子的表达量和Mock对照相比都有所升高。基于实验室前期的研究,利用构建好的含有FRad35~(2b)-CMV和FRad35~(2b) Leu-CMV的2b基因的农杆菌瞬时表达载体35S-2b~(Pro)和35S-2b~(Leu)分析2b蛋白的瞬时表达对外源GFP转录后基因沉默的影响。通过电转化获得含有不同载体的农杆菌,并将表达不同2b蛋白的农杆菌与35S-GFP的农杆菌共浸润,在转GFP本生烟16c叶组织中分别瞬时表达GFP和不同的2b蛋白。共浸润第3天,35S-2b~(Leu)和35S-Fny2b的浸润区出现亮绿斑,而35S-2b~(Pro)的浸润区变暗,GFP mRNA的Northern杂交分析确定GFP mRNA在35S-2b~(Leu)和35S-Fny2b的浸润区大量积累;共浸润后第7天,尽管35S-Fny2b和35S-2b~(Leu)仍表现亮荧光信号,但是,二者浸润区的GFPmRNA积累水平显著低于Mock对照,且与35S-2b~(Pro)相当,这些结果说明2b~(Leu)虽然在初期对GFP的局部沉默具有明显的抑制作用,但随着时间的推移,2b~(Leu)并不能抑制浸润区siRNA的产生,从而导致局部GFP mRNA的降解。在共浸润第14天,浸润35S-2b~(Pro)植株的顶端系统叶表现GFP系统沉默,而浸润35S-2b~(Leu)的植株则依旧显绿光,说明其顶端的系统叶GFP没有被诱导沉默。Northern杂交结果也显示:在浸润35S-2b~(Pro)的植株顶端系统叶中检测到少量的GFP mRNA积累,显著低于在35S-2b~(Leu)或35S-Fny2b浸润的植株顶端系统叶中的积累。通过荧光定量PCR检测第3天浸润区基因沉默通路中的AGO4-1和RDR6,结果显示:与Mock相比,出现亮绿斑的35S-2b~(Leu)的浸润区中AGO4-1和RDR6的积累受到抑制,而GFP发生降解的35S-2b~(Pro)的浸润区中AGO4-1和RDR6的表达量反而有一定程度的升高。因此,我们推测第55位的Leu对2b蛋白的高级结构有一定的影响,从而对AGO4-1和RDR6起到抑制作用,从而达到抑制系统沉默和DNA甲基化的目的。
综上所述,我们推测2b~(Leu)介导的高致病性是通过其阻止siRNA的系统移动,并抑制AGO4-1和RDR6等沉默因子的表达,来实现病毒高效的系统移动和复制,从而导致寄主表现出严重的病毒症状。
Cucumber mosaic virus (CMV) is one of the most economically important viruses to agriculture. CMV 2b protein was shown to be a multifunctional protein involved in sysptom induction, viral movement, suppressin of RNA silencing and so on. It is very important for developing new strategies of controlling CMV to investigate mechanism of pathogenicity of CMV 2b protein.
CMV recombinant FRad35~(2b)-CMV is generated by replacing 2b gene in Fny-CMV with 2b gene of Rad35-CMV. Our previous work has showed that the recombinant always induces mild mosaic in the most tested tobacco species. The mutant FRad35~(2bLeu) -CMV is different from FRad35~(2b)-CMV by only one amino acid, namely from Pro to Leu at the 55 position. The mutant caused leaf distortion, obvious stunting, even whole plant death on Nitociana benthamiana. When we inoculated FRad35~(2bLeu)-CMV in N. benthamiana as well as FRad35~(2b)-CMV,at 14 days post-inoculation (dpi), Northern blotting analysis showed that the accumulation of the viral RNAs in FRad35~(2bLeu)-CMV-infected plants was significantly higher than that in the FRad35~(2b)-CMV-infected plants. It is concluded that the high virulence of FRad35~(2bLeu)-CMV was associated with the high level of viral progeny RNA accumulation in the systemic infected leaves. By RT-qPCR, we analyzed effect of CMV 2b protein on expression of NbAGO and NbRDR. At 14 dpi, the transctritional levels of the NbAGO1-1 and NbAGO4-1 in N.benthamiana leaves infected with FRad35~(2bLeu)-CMV and FRad35~(2b)-CMV were determined by RT-qPCR, and compared with that in mock-inoculated leaves. The above results showed that FRad35~(2bLeu)-CMV significantly decreased the levels of the NbAGO1-1 and NbAGO4-1.
On the basis of preparatory work,we have constructed agrobacteria transient expression vectors of 2b genes from FRad35~(2b)-CMV and FRad35~(2bLeu)-CMV, namely 35S-2bPro and 35S-2bLeu respecively. When these vectors were co-infiltrated with 35S-GFP on N. benthamiana plant (line 16c), at 3 dpi, we found that zones infiltrated with 35S-2bLeu appeared bright green while zones infiltrated with 35S-2b~(pro) became dark. And northern blot also showed high level of GFP mRNA in zones infiltrated with 35S-2bLeu. At 7 dpi, those zones remained bright green, but the level of GFP mRNA became decreased, much less than mock. Thus, it is considered that 2bLeu could only suppress gene local silencing in early period, but it could not suppress the production of siRNA. The effect of Leu55 on systemic gene silencing was also investigated. It was found that at 14dpi, the top leaf of the plant infiltrated by 35S-2b~(Pro) turned red. While the top leaf of the plant infiltrated by 35S-2b~(Leu) have no effect. It seems that only 2b~(Leu) suppressed GFP systemic silencing. Northern blotting analysis showed that there was low level of GFP mRNA in the top leaf of plant infiltrated by 35S-2b~(Pro), and GFP mRNA accumulated highly in the top leaf of plant infiltrated by 35S-2bleu. By RT-qPCR, we analyzed effect of CMV 2b proteinon on the NbAGO4-1 and NbRDR6. At 3 dpi, the transcriptional levels of the NbAGO 4-1, NbRDR6 were determined in N.benthamiana leaves inoculated with 35S-2b~(Leu) and 35S-2b~(pro) by RT-qPCR and compared with that in mock-inoculated leaves. The results showed 35S-2b~(Leu) induced a decrease in the level of the AGO4-1, RDR6. It is predicted that the Leu55 can enhance the suppression to AGO and RDR6, then suppress the systemic silencing and RNA-directed DNA methylation (RdDM).
In summary, the results indicate that the 2b~(Leu)-mediated high pathogenic is achieved by suppressing systemic movement of siRNA, and inhii\MV can move and replicate more efficiently, inducing severe viral symptom on hosts.
引文
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