Fig. 5
Detection of viral CP and genomic RNA (gRNA) in tomato plants inoculated with PVYN. a For the detection of CP, total protein was extracted from the upper leaves of RDR6-, AGO2-, and DCL2.4-knockdown tomato plants (hpRDR6, hpAGO2.3, and hpDCL4.2) at 15 dpi. The CBB-stained gel was used as a loading control. b Semi-quantitative RT-PCR for the levels of PVYN genomic RNA in non-inoculated upper leaves of hpRDR6, hpAGO2.3, and hpDCL4.2 at 15 dpi. Their PCR products at 20, 25, and 30 cycles were fractionated using agarose gels. The actin gene was used as a control. c RT-qPCR to compare the DCL2s, DCL4, AGO2, AGO3, and RDR6 mRNA in the upper leaves of hpDCL2.4, hpAGO2.3, hpRDR6, and empty vector-transformed plants infected with PVYN (Empty-PVY) with the levels detected in healthy empty vector-transformed plants (Empty-healthy) at 15 dpi. 18S ribosome RNA was used as control. Error bars represent SE. Student’s t-test was applied to analyze the data. Each analysis was performed with three biological replicates; data were collected from three plants of each knockdown and empty vector-transformed transgenic lines. The values with the double asterisk (**p < 0.01) and single asterisk (*p < 0.05) were statistically significant at the 1% and 5% levels, respectively, compared with those of healthy empty vector-transformed plants