Identification of an RNA silencing suppressor encoded by an Indian citrus ringspot virus

Plant viruses encode RNA silencing suppressor (RSS) proteins to counter the induced antiviral defense, an RNAi silencing mechanism of the host. Indian citrus ringspot virus (ICRSV) causes the ringspot disease, which leads to significant yield loss of kinnow orange. The ICRSV genome contains six open reading frames (ORFs), however, the ORF encoding the potential RSS is not yet known. In this study, we have attempted to identify the RSS protein of ICRSV. To this end, ORF 2,3,4,5 and 6 were cloned into pCAMBIA1302 (35s-GFP) vector, followed by transformation of Agrobacterium tumefaciens and agro-infiltration into leaves of Nicotiana benthamiana 16c line. Only the leaves infiltrated with 35s-GFP/ORF5 showed a GFP fluorescence signal similar to 35s-GFP/P19, a well-studied positive RSS. Usually, the induced host RNAi silencing is supposed to cleave the expressed GFP-RNA. However, it is suspected that ORF5-encoded protein was able to suppress the host silencing mechanism, leading to the retention of the GFP fluorescence signal. This finding was further supported by beta-glucuronidase (GUS) histochemical assays by infiltrating the construct expressing ORF5-GUS under 35s promoter in the leaves of N. benthamiana. Leaves infiltrated with 35s-GUS/ORF5 formed diX-indigo precipitate similar to leaves infiltrated with, indicating the RSS activity of ICRSV. Later, semi-quantitative PCR and quantitative reverse transcription PCR (qRT-PCR) assays showed a higher expression of GFP and GUS in ORF5 agro-infiltrated leaves. Together, these results suggest that ORF5 encoded protein has the potential RSS function of ICRSV which successfully suppresses host RNAi silencing mechanism.