Cloning And Preliminary Function Analysis Of TOM Orthologues From Tobacco And Tomato

A variety of host factors have been identified to be involved in the intracellular multiplication of viruses. The AtTOM1 gene of Arabidopsis thaliana has previously been shown to be essential for the efficient multiplication of Tobacco mosaic virus (TMV) in A. thaliana. In this study, AtTOM1-like genes named as NbTOM1 and TT0M1 were amplified by RT-PCR from Nicotiana benthamiana and Lycopersicon esculentum using degenerate primers designed based on TOM1 homologue sequences of Lycopersicon esculentum, A. thaliana and Oryza sativa. Sequence alignment show the amplified freagments are closely related to the TOM1 homologues. NbTOM1 and TT0M1 were introduced into Tomato yellow leaf curl China virus (TYLCCNV) DNAβ-based silencing vector to obtain recombinant vectors DNAmβ:NbTOM1 and DNAmP:TTOM1. Northern blot analysis indicated co-agroinoculation of DNAmβ: NbTOM1 with TYLCCNV, induced silencing of NbTOM1 and resulted in completely inhibition of the multiplication of TMV in N. benthamiana.. Semi-quantitative RT-PCR assay demonstrated that NbTOM1 mRNA accumulation was inhibited in N. benthamiana. These results suggest that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants and virus induced gene silencing is is a useful method for identification of gene function.