Construction of in vitro and in vivo infectious transcripts of a Taiwan strain of Zucchini yellow mosaic virus

The full-length cDNA of a Taiwan strain of Zucchini yellow mosaic virus (ZYMV TW-TN3) was constructed from five overlapping cDNA clones downstream from the bacteriophage T7 promoter in plasmid pT7ZYMV2-5. The plasmid was able to generate an in vitro transcript corresponding to TW-TN3 (9591 nt) with one extra guanosine residue at the 5' terminus and a poly(A)(95) tract at the 3' end. In addition, pT7ZYMV2-5 was used for the construction of p35SZYMV2-26 that contained the full-length cDNA of TW-TN3 with a Cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase (nos) terminator. The capped in vitro transcript generated from pT7ZYMV2-5 and the purified DNA of p35SZYMV2-26 were introduced into zucchini squash plants by mechanical inoculation and particle bombardment, respectively. Both in vitro and in vivo transcripts induced systemic symptoms on zucchini squash 4 to 6 days after inoculation. In addition, both transcripts also induced local lesions on plants of Chenopodium quinoa by mechanical inoculation. The results of infectivity assay, symptomatology, and serologically specific electron microscopy indicated that the in vitro and in vivo TW-TN3 transcripts derived from pT7ZYMV2-5 and p35SZYMV2-26, respectively, are infectious. The ability to generate biologically functional transcripts from the constructed cDNA clones is a significant step for molecular analyses of TW-TN3