TITLE	Infectivity assays of in vitro and in vivo transcripts of papaya ringspot potyvirus
AUTHOR	Chu-Hui Chiang Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, Republic of China Shyi-Dong Yeh Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, Republic of China
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ABSTRACT	A full-length cDNA with nucleotide sequence representing the genomic RNA of a Hawaii strain of papaya ringspot potyvirus (PRSV HA) was constructed downstream from a bacteriophage T3 promoter in an in vitro transcription vector. The plasmid was able to generate an in vitro transcript corresponding to PRSV RNA (10326 nt) with one extra guanosine residue at the 5' terminus and 12 nonviral nucleotides at the 3' end following a poly(A)36 tract. In vitro translation products and immunoprecipitation analysis with the antiserum to PRSV verified correctness of the gene expression of the transcript. When the capped transcript was mechanically introduced to the systemic host papaya and the local lesion host Chenopodium quinoa, typical symptoms of PRSV HA appeared at almost the same time as on those host plants inoculated with native PRSV RNA. Western blotting and serologically specific electron microscopy with PRSV antiserum confirmed the infection. The uncapped in vitro transcript and the transcript with longer nonviral nucleotides (64 nt) at the 3' end were not infectious. The full-length cDNA was also constructed with a cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase (NOS) terminator in an in vivo expression vector. Purified plasmids were applied directly onto host plants either mechanically or by bombardment with a particle delivery system to analyze their infectivity. The plasmid without extra nucleotides between the 35S promoter and the 5' end of PRSV sequence and with 10 nonviral residues and a NotI site at its 3' end was infectious, as evident from symptom development, and ELISA, immunodiffusion, and serologically specific electron microscopy analyses with PRSV antiserum. The construct with 33 nonviral nucleotides at the 5' end of the PRSV sequence and more than 64 nucleotides at the 3' end was not infectious. The infectious in vitro and in vivo transcripts derived from the full-length cDNA to PRSV RNA are the longest so far recorded for a plant virus.
KEYWORD	In vitro transcript; In vivo transcript; Papaya ringspot potyvirus;
ARTICLE INFO	Botanical Bulletin of Academia Sinica, Volume 38 Number 3 July 1997, page 153-163, 11 pages
PUBLISHER	Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan, Republic of China