A series of recombinant viruses were engineered by gene replacements or gene insertions into an infectious cDNA clone of Tobacco necrosis virus A Chinese isolate (TNV-A^C). TNV-A^C-based virus induced gene silencing (VIGS) containing different structural arrangements of sequences targeting specific genes and variations in the positions of exogenous fragment insertions were evaluated along with temperature effects on inoculated plants. Replacement of the coat protein (CP) gene by exogenous gene fragments could not be applied to TNV-A^C because the chimeric TNV-A^C constructs were unable to induce silencing of corresponding endogenous gene and derivatives lacking the CP could not move systematically in Nicotiana benthamiana. We found that the optimal position for insertion of exogenous gene fragments was the region immediately behind the stop codon of the CP gene, and the maximum lengths of fragments that could be accommodated within virions was around 120 nt. An NbPDS fragment inserted into TNV-A^C as an inverted repeat produced a VIGS derivative that could silence endogenous NbPDS in N. benthamiana with extremely high efficiency. We also found that temperature could significantly affect gene silencing efficiency of inoculated plants and the stability of heterologous gene fragments. Plants grown at 18℃ exhibited substantially higher gene silencing efficiency compared with 24℃, and the duration of silencing persisted for more than 110 days. The TNV-A^C-based VIGS vectors were also able to induce efficient silencing of endogenous Su and ChlH genes in N. benthamiana. Taken together, our results show that TNV-A^C has the potential for development into a novel VIGS vector suitable for functional genomics of N. benthamiana.