(A) Schematic diagrams of pBY-agMR, pGD-NLP, pGD-VSRs, pMD-HvCCR4, and derivative plasmids. Mixtures of these plasmids transiently express anti-genomic minireplicon RNA, BYSMV N, P, and L proteins, as well as suppressors of RNA silencing (VSRs) in N. benthamiana leaves. The HvCCR4, HvCCR4N, HvCCR4C and HvCCR4mEEP (N260A/E305A) ORFs were cloned into pMDC32 for transient overexpression. (B) RFP foci in N. benthamiana leaves agroinfiltrated with Agrobacterium mixture harboring BYSMV-agMR combinations containing an empty vector (EV), HvCCR4, HvCCR4N, HvCCR4C, or HvCCR4mEEP. Representative images were taken at five dpi. Bar = 1 mm. (C) Western blotting analysis showing accumulation of RFP, N, P, and HvCCR4 proteins in the leaves shown in panel (B) with rabbit α-RFP, α-N, α-P, or α-Flag protein antibodies, respectively. N. benthamiana leaves infiltrated with the pGD vector were mock controls. (D) qRT-PCR analysis of minigenome RNA replication in the samples shown in panel (B). (E) qRT-PCR analysis of normalized levels of RFP transcription by comparing the relative levels of mRNA versus minigenome RNA in the samples shown in panel (B). EF1A served as an internal control gene. The values of viral replication and transcription in leaf samples agroinfiltrated with the EV plasmid were set to 1. Error bars indicate standard errors of three independent experiments. Letters above the bars indicate statistical significance (p<0.01) evaluated by Turkey’s Multiple Comparison Test analysis.