Silencing of NbSKP1s enhances CLCuMuV DNA accumulation and results in typical disease symptoms.

(A1, A2 and A3) Six- to seven-week-old N. benthamiana plants were agroinoculated with CLCuMuV (CA) and βM2-SKP1F1 (A1), βM2-SKP1F2 (A2), βM2-SKP1F3 (A3) or βM2-βC1F (as the control). (B1, B2 and B3) Silencing of NbSKP1s enhanced CLCuMuV DNA accumulation. Each group contained 7 plants. At 14 dpi, total DNA was extracted from each plant respectively and subjected to quantitative real-time PCR (means±SEM, n = 7) to quantify viral DNA accumulation. The internal reference method was used to calculate the relative amount of viral DNA. (C1, C2 and C3) Real-time RT-PCR confirmed silencing of NbSKP1s. Total RNA was extracted from each plant respectively and subjected to quantitative RT-PCR (means±SEM, n = 4) to quantify NbSKP1s mRNA level. Actin was used as the internal reference. The raw data of (B1–B3) and (C1–C3) were analysed by two-sample t-test to show the significance level at 0.05 (*) and 0.01 (**). These experiments were repeated at least twice. (D1, D2 and D3) 50% plants infected with CA+βM2-SKP1F1 (D1), 50% plants infected with CA+βM2-SKP1F2 (D2) and 100% plants infected with CA+βM2-SKP1F3 (D3) show severe symptoms at 21 dpi. (E1, E2, E3 and E4) Apical leaves of plants infected with CA+βM2-βC1F (E1), CA+βM2-SKP1F1(E2), CA+βM2-SKP1F2 (E3) and CA+βM2-SKP1F3 (E4) at 21 dpi.