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

Abstract

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.

Glutathione contributes to resistance responses to TMV through a differential modulation of salicylic acid and reactive oxygen species

“…No obvious difference of the phenotype was observed between NbECS ‐ or NbGS ‐silenced plants and control plants (TRV:00) (Figure S2 a). To investigate the gene silencing efficiency of NbECS and NbGS genes, quantitative reverse transcription PCR (RT‐qPCR) was used to examine the expression levels of NbECS and NbGS in 12 days after silencing plants by VIGS using primers that anneal to NbECS and NbGS outside the region targeted for silencing (Wangdi, et al, 2010 ; Zhu et al, 2019 ). The results showed that the transcript levels of NbECS and NbGS were significantly reduced in the NbECS ‐ or NbGS ‐silenced plants as compared to the control plants (Figure S2 b,c).…”

“…For RT‐PCR, the first‐strand cDNA was prepared using M‐MLV reverse transcriptase (Takara). To test the effectiveness of VIGS in the gene‐silenced plants of NbECS and NbGS , RT‐qPCR (Wangdi et al, 2010 ; Zhu et al, 2019 ) was used to detect the expression levels of NbECS and NbGS using the unique 5′ region of NbECS and NbGS with specific primers. Primers that anneal outside the region targeted for silencing were used to ensure that only the endogenous gene could be tested.…”