Complete nucleotide sequence and synthesis of infectious in vitro transcripts from a full-length cDNA clone of a rakkyo strain of tobacco mosaic virus.
The complete nucleotide sequence of the genome of a rakkyo strain of tobacco mosaic virus (TMV-R), which exhibits distinct host range differences from the common strain of TMV, was determined. The overall nucleotide sequence homology with TMV-U1 (a common strain of TMV) is 94.2%. The amino acid sequence homologies of the four encoded proteins (180K, 130K, 30K, coat protein) are from 95.9% to 98.0% compared with TMV-U1. To facilitate the analysis of the novel host range of TMV-R, a full-length clone of the genome containing a bacteriophage T7 RNA polymerase promoter was assembled from two cDNA clones and designated pRF3. In vitro transcripts derived from pRF3 were highly infectious. The infections of RF3, wild-type TMV-R, and U3/12-4 (derived from pU3/12-4, an infectious clone of TMV-U1) were compared on Nicotiana tabacum cv. Bright Yellow (BY) plants. No systemic mosaic symptoms were observed on plants inoculated with RF3 and TMV-R, while BY plants inoculated with U3/12-4 developed distinct mosaic symptoms on the upper leaves 8-9 days post-inoculation. The green fluorescent protein (GFP) gene was introduced into pRF3 and pU3/12-4 by replacing the coat protein gene to get two GFP expressing chimeric virus clones: pR-GFP or pU1-GFP. Transcripts from pU1-GFP produced strong fluorescence when inoculated onto BY leaves, while those from pR-GFP produced only very faint fluorescence.
Chen
J
J
Laboratory of Plant Pathology, Faculty of Agriculture, Saga University, Japan.
Watanabe
Y
Y
Sako
N
N
Ohshima
K
K
Okada
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D016679:Genome, Viral; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry*; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry*; D014027:Tobacco Mosaic Virus / Q000235:genetics*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D049452:Green Fluorescent Proteins