The Cloning And Functional Identification Of A RNA Virus Host Factor Gene ToTOM3 From Tomato

A homologue of AtTOM3 of Arabdopsis thaliania, designed ToTOM3, was isolated from tomato (Lycopersicum esculentum) and shown to encode a host factor that supports the replication of both Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV).ToTOM3 cDNA was isolated by methods of RT-PCR and 5′- and 3′-rapid amplication of cDNA ends (RACE). The 1267 nt full-length cDNA of ToTOM3 contains an ORF of 888 nt encoding a 295 aa protein with a calculated molecular mass of 34.4 kDa, a 72 nt 5′- and a 307 nt 3′-untranslated regions. The deduced amino acid sequences of the ToTOM3 protein contains several highly hydrophobic regions and is predicted to have seven transmembrane domains. ToTOM3 shares 60.2% identity at nucleotide level and 76.9% identity at amino acid level with that of AtTOM3, which supports the replication of TMV in Arabdopsis. ToTOM3 also shares significant homology (58.6% at amino acid level) with ToTOM1, a gene previously shown to support the replication of CMV in tomato.A genomic fragment of 6885 bp in length that contains the full-length transcribed region of ToTOM3 was isolated by PCR and mini-library screening. As revealed by comparison with full-length cDNA, the ToTOM3 is composed of 11 exons and 10 introns. The size of exons ranges from 46 to 400 bp and the introns from 75 to 2031 bp. All of the exon / intron junction sequences conform to the GT/AG rule. The expression patterns of ToTOM3 were examined by RT-PCR, using tubulin RNA as internal control. It was found that there was not significant difference in mRNA level in different tissues and at different growing stages of tomato.To identify the function of ToTOM3 during the viral infection of the plant host, transgenic tomato plants expressing ToTOM3 antisense RNA and RNAi fragments were generated using Agrobacterium-mediated transformation methods. In the antisense transgenic plants, the full-length transcribed region of the genomic ToTOM3 fragment in reverse orientation was under the control of Cauliflower mosaic virus (CaMV) 35S promoter. And in the RNAi transgenic plants, selected sequences of ToTOM3 cDNA were used to construct a head-to-head inverted repeat that was under the control of CaMV 35S promoter. All of the 26 transgenic tomato plants obtained appeared to grow normally and were able to fruit.The nontransgenic and transgenic tomato plants were inoculated with TMV and CMV. At day 10 post inoculation of TMV, typical mosaic symptoms appeared in the nontransgenic plants. In contrast, no visible symptom was observed in transgenic ones. By day 90 post inoculation, while the nontransgenic plants showing leaf mottling, the transgenic lines remained symptomless. As revealed by both local lesion assay on Chenopodium amaranticolor or DAS-ELISA with TMV-specific antibody, accumulation of TMV in the transgenic plants was significantly lower than that of nontransgenic plants. Similar results were obtained from experiments with CMV infection. The transgenic plants only showed mild symptom 90 days post inoculation, while the control nontransgenic plants showed typical mosaic. The accumulation of CMV in transgenic plants was 12.4 to 58.1% of that of the nontransgenic plants. Transgenic lines that were highly resistant to TMV were also highly resistant to CMV.The above results demonstrate that ToTOM3 is a host factor involved in the replication of TMV and CMV in tomato. This also provided a successful example that resistance to viral infection can be engineered by suppression of a host factor that supports the replication of virus in a major crop.