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 t obacco mosaic virus (TMV-R), which exhibits distinct host range differ ences from the common strain of TMV, was determined. The overall nucle otide sequence homology with TMV-U1 (a common strain of TMV) is 94.2%. The amino acid sequence homologies of the four encoded proteins (180 K, 130 K, 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 fu ll-length clone of the genome containing a bacteriophage T7 RNA polyme rase promoter was assembled from two cDNA clones and designated pRF3. In vitro transcripts derived from pRF3 were highly infectious. The inf ections of RF3, wild-type TMV-R, and U3/12-4 (derived from pU3/12-4, a n infectious clone of TMV-U1) were compared on Nicotiana tabacum cv. B right 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 da ys post-inoculation. The green fluorescent protein (GFP) gene was intr oduced into pRF3 and pU3/12-4 by replacing the coat protein gene to ge t two GFP expressing chimeric virus clones: pR-GFP or pU1-GFP. Transcr ipts from pU1-GFP produced strong fluorescence when inoculated onto BY leaves, while those from pR-GFP produced only very faint fluorescence .