Functional Analysis of Cotton Leaf Curl Kokhran Virus/Cotton Leaf Curl Multan Betasatellite RNA Silencing Suppressors

Abstract

In South Asia, Cotton leaf curl disease (CLCuD) is caused by a complex of phylogenetically-related begomovirus species and a specific betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). The post-transcriptional gene silencing (PTGS) suppression activities of the transcriptional activator protein (TrAP), C4, V2 and βC1 proteins encoded by Cotton leaf curl Kokhran virus (CLCuKoV)/CLCuMuB were assessed in Nicotiana benthamiana. A variable degree of local silencing suppression was observed for each viral protein tested, with V2 protein exhibiting the strongest suppression activity and only the C4 protein preventing the spread of systemic silencing. The CLCuKoV-encoded TrAP, C4, V2 and CLCuMuB-encoded βC1 proteins were expressed in Escherichia coli and purified. TrAP was shown to bind various small and long nucleic acids including single-stranded (ss) and double-stranded (ds) RNA and DNA molecules. C4, V2, and βC1 bound ssDNA and dsDNA with varying affinities. Transgenic expression of C4 under the constitutive 35S Cauliflower mosaic virus promoter and βC1 under a dexamethasone inducible promoter induced severe developmental abnormalities in N. benthamiana. The results indicate that homologous proteins from even quite closely related begomoviruses may differ in their suppressor activity and mechanism of action. The significance of these findings is discussed.

Keywords: RNA silencing suppressor; begomovirus; betasatellite; post-transcriptional gene silencing; siRNA.

Figure 1

Suppression of gene silencing by proteins encoded by Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). Leaves of N. benthamiana 16c plants (16c) in which silencing was induced by infiltration with a construct for the expression of the (sense) green fluorescence protein (S-GFP) gene (A) or co-agroinfiltrated with S-GFP and a construct for the expression of an inverted-repeat of the GFP gene (IR-GFP) (B); In each case the plants were co-infiltrated with constructs for the expression of either β-glucuronidase (GUS), the p19 protein of TBSV (p19), the βC1 protein of CLCuMuB (βC1), or the transcriptional activator protein (TrAP), C4 protein (C4), or V2 protein (V2) encoded by CLCuKoV, respectively. Plants were photographed under UV illumination at 4 dpi; (C) Northern blot of total RNAs extracted from the infiltrated leaf patches of plants described for panels A hybridized with full-length GFP probe for the presence of GFP mRNA (upper panel) or siRNAs (lower panel); (D) Northern blot of total RNAs extracted from the infiltrated leaf patches described for panel B probed for the presence of GFP mRNA (GFP probe, upper panel), primary siRNAs (F probe; middle panel) or secondary siRNAs (G/P probe; lower panel). For the Northern blot analyses total RNA extracted from non-infiltrated N. benthamiana wild type plants (WT) and a non-infiltrated 16c plants (16c) are shown as controls. Samples were extracted at 4 dpi, the ethidium bromide stained ribosomal RNA (rRNA) band on the gel is shown below the blot to confirm equal loading. The numbers below the blots indicate the relative amounts of GFP mRNA and siRNA after normalization using 16S rRNA as a loading control. The values are given relative to untreated 16c, for GFP mRNA, or 16c co-inoculated with GUS, for GFP-derived siRNA. Image J software was used to estimate image intensities from the images of the blots.

Figure 2

Suppression of systemic silencing. The bar graph shows the percentages of N. benthamiana 16c plants exhibiting systemic silencing at 14 dpi with silencing inducer and constructs for the expression of β-glucuronidase (GUS), the p19 protein of TBSV (p19), the βC1 protein encoded by Cotton leaf curl Multan betasatellite, or the transcriptional activator protein (TrAP), C4 protein (C4), or V2 protein (V2) encoded by Cotton leaf curl Kokhran virus. The raw data is shown in tabulated form below the graph. The results are of five independent experiments. Plants were examined for systemic silencing at 14 dpi. Error bars represent significant differences in suppression efficiency between the individual constructs and the empty vector in Chi-square tests (p < 0.05). A,B,C: Indicates statistically significant differences at the 95% level

Figure 3

Electrophoretic mobility-shift assays of recombinant Trx-His tagged proteins. In each case, the nucleic acids used in the mobility-shift assays are indicated over the respective blots.