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

Abstract

The nucleotide sequence of the genome of the type strain of Kyuri green mottle mosaic virus (KGMMV-C1) has been completely determined. The genome structure and sequence of the virus were compared to those of Yodo strain of KGMMV (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 coding 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 the 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 was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) with a set of S'-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 templates for transcription for plant inoculation test. Capped transcript RNAs 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 plants such as zucchini squash, cucumber and Nicotiana benthamiana. Transcript-derived progeny virus was indistinguishable from the wild-type virus with the same biological and biochemical properties. To our knowledge, this is the first report of the generation of a biologically active KGMMV clone, driven by the T7 promoter, that is highly infectious to cucurbitaceous plants.