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

Abstract

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 signifcant 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-infltration into leaves of Nicotiana benthamiana 16c line. Only the leaves infltrated with 35s-GFP/ORF5 showed a GFP fuorescence 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 fuorescence signal. This fnding was further supported by beta-glucuronidase (GUS) histochemical assays by infltrating the construct expressing ORF5-GUS under 35s promoter in the leaves of N. benthamiana. Leaves infltrated with 35s-GUS/ORF5 formed diX-indigo precipitate similar to leaves infltrated 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-infltrated leaves. Together, these results suggest that ORF5 encoded protein has the potential RSS function of ICRSV which successfully suppresses host RNAi silencing mechanism.