Grapevine red blotch virus C2 and V2 are suppressors of post-transcriptional gene silencing

Abstract

Grapevine red blotch virus (GRBV) is the causative agent of grapevine red blotch disease (GRBD) which is one of the major threats faced by grapevine industry in the United States. Since its initial identification in 2011, the disease has rapidly spread in the major US grape-growing regions of the Pacific Northwest, causing major economic impacts. Geminiviruses, the largest family of plant viruses, can induce and be targeted by host post-transcriptional gene-silencing (PTGS) anti-viral mechanisms. As a counter-defense mechanism, viruses have evolved viral silencing suppressor proteins to combat PTGS mechanisms and establish a successful infection in host plants. Here we provide characterization of two ORFs of GRBV, C2 and V2 as viral silencing suppressors. In Nicotiana benthamiana line 16c GFP marker plants, synergism or additive effects of C2 and V2 suppressors was observed at the mRNA level when they are expressed together transiently. Additionally, we showed there is no evidence by yeast two-hybrid of self-interaction (dimerization) of C2 or V2 proteins, and no evidence of physical interaction between these two suppressors.

Keywords: Geminivirus; Grablovirus; Grapevine red blotch virus; Post-transcriptional gene silencing; Silencing suppressors; Viral ORF.

Fig. 1

Characterization of GRBV C2 and V2 as silencing suppressors. (A) Agrobacterium-mediated transient expression of GRBV genes C2 and V2 in Nicotiana benthamiana line 16c leaves. a: Mock-agroinfiltration with infiltration medium. b: Agroinfiltration with A. tumefaciens strain harboring pBI-mgfp5-ER (positive control for silencing). Co-agroinfiltration with A. tumefaciens strains harboring c: pBI-mgfp5-ER + pCAMBIA2301. d: pBI-mgfp5-ER + potyvirus HcPro (positive control for silencing suppression). e: pBI-mgfp5-ER + pCAM-C2-gus. f: pBI-mgfp5-ER + pCAM-V2-gus. g: pBI-mgfp5-ER + pCAM-C2-gus + pCAM-V2-gus. (B) Quantification of green fluorescence of agroinfiltrated zones in N. benthamiana 16c leaves. Different letters (a and b) indicate significant difference between samples, p < 0.05 (Student's t-test, equal variance). Average green fluorescence is calculated by averaging the mean fluorescence of different photographic images taken for each particular test sample. Biological replicates are denoted in parentheses in the X-axis. Error bars are s.e.m. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Validation of GRBV C2 and V2 silencing suppressors. (A) Quantification of green fluorescent protein (gfp) messenger RNA (mRNA) by RT-qPCR. Error bars are standard error. (B) RNA blot hybridization with gfp probe (gfp transcript 2 kb). Total RNA extracted from infiltrated areas of mock infiltrated leaves (IM), pBI-mgfp5-ER (trigger) infiltrated leaves (mgfp), pBI-mgfp5-ER (trigger) along with pCAMBIA2301 vector infiltrated leaves (pCAM2301), C2 co-infiltrated leaves (C2), V2 co-infiltrated leaves (V2), C2 and V2 co-infiltrated leaves (C2+V2) and HcPro co-infiltrated leaves (HcPro) were probed with the gfp coding sequence. To account for RNA loading, the 18S rRNA portion of the ethidium bromide-stained gel is placed at the bottom. (C) Normalized C2 and V2 silencing-suppression, from three experimental biological reps of quantified gfp mRNA full length signal strengths by RNA blot. Error bars represent s.e.m. * denotes statistical significance (p < 0.05) of two-tailed t-test when compared to mgfp and mgfp + pCAM. Different letters (a and b) indicate significant difference between C2 and V2 sample, p < 0.05) (Student's two-tailed t-test). Uncropped version of figure [2B] is provided as Supplement Fig. 2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Yeast Two-hybrid Assay. GRBV C2 and V2 cloned in bait and prey vectors were tested on (a) SD/–Leu/–Trp-double drop out (DDO) media for screening transformants. (b) SD/–Leu/–Trp/–His-triple drop out (TDO) media for screening interaction. (c) SD/–Leu/–Trp/–His/–Ade quadruple dropout (QDO) media for ruling out false positive interaction. (d) SD/–Leu/–Trp + X-α-Gal (DDO + X) (e) SD/–Leu/–Trp + X-α-Gal + Aureobasidin A (DDO + X + A) for ruling out false positive interaction. p53-T antigen in bait and prey vectors respectively served as positive control; Lam-T antigen in bait and prey vectors respectively served as negative control.

figs1

figs2