Mechnism Of Pathogenesis Mediated By Key Amino Acid Of Cucumber Mosaic Virus 2b Protein

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.