Cloning And Preliminary Function Analysis Of AtTOM-like Genes From Nicotiana Benthamiana And Tomato

Plant viruses encoded only few genes,which are not sufficient for viruses to finish their life circle in plants,so a variety of host factors were require for virus infection in plants.Some host factors were identified to be related with virus replication.For example,the AtTOM1 gene of Arabidopsis thaliana has been shown to be essential for the efficient multiplication of tobacco mosaic virus(TMV)in A. thaliana.In Nicotiana benthamiana and N.tabacum cv.Samsun,homologues of AtTOM1 gene,NbTOM1,NtTOM1,and NtTOM3 were also shown to have similar functions.In this study,two AtTOM1-like genes,NbTOM1 and NbTOM3 were amplified by RT-PCR from N.benthamiana using specific primers designed based on NtTOM1 and NtTOM3 sequences of N.tabacum cv.Samsun(GenBank accession numbers AB193039,AB193040)and then sequenced.Sequence alignment shows NbTOM1 and NbTOM3 are closely related to NtTOM1 and NtTOM3 with 95.9%and 98.2% sequence identity,respectively.The two genes were inserted into tobacco curly shoot virus(TbCSV)DNA1-based silencing vector,respectively,to obtain recombinant vectors DNA1:TOM1,DNA1:TOM3 and DNA1:TOM1-TOM3.The recombinant vectors were introduced into Agrobacterium tumefaciens strain EHA105 by triparental mating,then co-inoculated with tomato yellow leaf curl China virus (TYLCCNV)into N.benthamiana plants.14 days after inoculation,semi-quantitative RT-PCR assay demonstrated that NbTOM1 mRNA accumulation was inhibited in plants co-inoculated with TYLCCNV and DNA1:TOM1,but NbTOM3 mRNA accumulation was not inhibited.In N.benthamiana plants co-inoculated with TYLCCNV and DNA1:TOM3,NbTOM3 but not NbTOM1 mRNA accumulation was inhibited.The two gene were silenced simultaneously in the plants co-inoculated with TYLCCNV and DNA1:TOM1-TOM3.The silenced plants were challenge-inoculated with TMV:GFP,GFP visualization and Northern blot analysis indicated that TMV multiplication was inhibited in NbTOM1-silenced,NbTOM3-silenced,or NbTOM1 and NbTOM3-silenced N.benthamiana plants.The above results suggest that NbTOM3 may have similar function as NbTOM1 in inhibition of TMV replication.AtTOM1-like genes were also amplified in Lycopersicon esculentum by RT-PCR using specific primers designed based on reported AtTOM1-like sequences(GenBank accession numbers AB193041,AB193042,AB193043).The three fragments (LeTH1,LeTH2 and LeTH3)were then introduced into the same TbCSV DNA1-based silencing vector to obtain recombinant vector DNA1:LeTH1-LeTH2-LeTH3.The recombinant vector was introduced into Agrobacterium tumefaciens strain EHA105 by triparental mating,then co-inoculated with TYLCCNV into tomato.20 days after inoculation,semi-quantitative RT-PCR assay demonstrated that all of the LeTH1, LeTH2 and LeTH3 mRNA accumulation was inhibited.GFP visualization and Northern blot analysis of the silenced tomato plants challenge-inoculated with TMV:GFP indicated that TMV multiplication was inhibited.The function of LeTH1, LeTH2 or LeTH3 and their redundancy need be further investigated.The TbCSV DNA1-based silencing system is an effective and symptomless silencing system.It can be used for rapid identification of gene function.Inhibition of AtTOM1-like genes via RNA silencing is a potentially useful method for generating TMV-resistant plants...