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Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 ( NbSABP2 ) and  NbNAC1  in  Nicotiana benthamiana  to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length  NbNAC1  gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of  NbSABP2  and  NbNAC1  was successfully amplified. Bacteria liquid PCR confirmed that the combination of  NbSABP2  and  NbNAC1  was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the  NbSABP2  and  NbNAC1  gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of  NbSABP2  and  NbNAC1  were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana