Completion of nucleotide sequence and generation of highly infectious transcripts to cucurbits from full-length cDNA clone of Kyuri green mottle mosaic virus

The nucleotide sequence of the genome of the type strain of Kyuri green mot tle mosaic virus (KGMMV-C1) has been completely determined. The genome stru cture and sequence of the virus were compared to those of Yodo strain of KG MMV (KGMMV-Y). The genome of KGMMV-C1 is 6,514 nucleotides long consisting of 5' and 3' nontranslated regions (NTRs) and four open reading frames codi ng for 131 kDa and 189 kDa viral replicases, 28 kDa movement protein and 17 kDa coat protein. The nucleotide and amino acid sequences identities of th e four encoded proteins and two NTRs between KGMMV-C1 and KGMMV-Y were 85.6 % to 93.9% and 87.6% to 95.5%, respectively. Full-length cDNA of KGMMV-C1 w as directly amplified by reverse-transcription polymerase chain reaction (R T-PCR) with a set of 5'-end primer anchoring T7 RNA promoter sequence and 3 '-end primer. This full-length RT-PCR product allowed RNA to be transcribed in vitro. The T7 promoter-anchored RT-PCR product was cloned and used as t emplates for transcription for plant inoculation test. Capped transcript RN As transcribed from the full-length cDNA clone as well as capped transcript RNAs from the uncloned RT-PCR products were infectious and caused symptoms characteristic of KGMMV when mechanically inoculated to systemic host plan ts such as zucchini squash, cucumber and Nicotiana benthamiana. Transcript- derived progeny virus was indistinguishable from the wild-type virus with t he same biological and biochemical properties. To our knowledge, this is th e first report of the generation of a biologically active KGMMV clone, driv en by the T7 promoter, that is highly infectious to cucurbitaceous plants.