Identification of an RNA silencing suppressor encoded by citrus vein enation virus

Abstract

In order to know if any protein encoded by citrus vein enation virus (CVEV) might act as an RNA silencing suppressor (RSS), five open reading frames (ORF) of CVEV, including the coat protein (CP) gene, were individually cloned into the binary expression vector pBI121. The silencing suppression ability of the five ORFs was tested by ‘viral minireplicon infectivity assays’ based on a beet yellows virus (BYV) minireplicon carrying a reporter gene (BYVminiR-GFP). GFP imaging and microscopic analyses were used to observe whether RNA silencing suppression occurred or not. In addition, real-time quantitative RT-PCR was used to quantify the mRNA expression of GFP in the injected region. The fluorescence intensity of leaves with CP expression was stronger than that of the empty vector negative control, but it was weaker than that of the positive control HC-Pro of tobacco etch virus (TEV), indicating that the CP of CVEV partly inhibited the GFP gene silencing. Co-expression of each of the other four ORFs with BYVminiR-GFP showed an almost complete quenching of GFP fluorescence in infiltrated areas, similar to that of the negative control. The amount of GFP mRNA in the leaves co-expressed with CP was nearly twice as much as that of either the negative control or other ORFs. These results suggest that CP of CVEV is a weak gene silencing suppressor.