To confirm the silencing of the EF1Bc gene in N. benthamiana, we performed RT-PCR amplification with primer pairs: #2952 (CGCGGATCCGGAAAGGTTCCTGTGCTTGA) and #2992 (C G C CT C G A G GTCCAGAAGTATCTCTCTACA TGTGG) on total RNA extract of pTRV2-EF1BcNt and pTRV2 empty agroinfiltrated N benthamiana plants. PCR conditions were as follows: 27 cycles of 94uC 20sec, 60uC 30sec, 68uC 30 sec with HiFi Taq polymerase. Tubulin mRNA control from the same total RNA samples was detected by RT-PCR using primers #2859 (TAATACGACTCACTATAGgaACCA AA TC AT T CATGTT-GCTCTC) and #2860 (TAGTGTATGTGATATCCCACCAA) [65] . The leaves of VIGS-treated plants were sap inoculated with TBSV, or TMV on the 9 th day after silencing [65] . Total RNA was extracted 3 or 5 days post inoculation [65] . For Northern blot analysis of the viral RNA level, we prepared 32 P-labeled complementary RNA probes specific for the 39-ends of the viral genomic RNAs based on T7 transcription. To obtain the PCR templates for the probes, we used the following primers for TBSV: #1165 (AGCGAGTAAGACAGACTCTTCA) and #22; for TMV: #2890 (TCTGGTTTGGTTTGGACCTC) and #2889 (GTAATACGACTCACTATAGGGATTCGAACCCC-TCGCTTTAT).