Factors affecting efficient infection of tobacco with in vitro RNA transcripts from cloned cDNAs of satellite tobacco mosaic virus

Abstract

Recombinant cDNA clones of the complete satellite tobacco mosaic virus (STMV) genome (1059 ribonucleotides) were constructed with unique Xbal and HindllI or Pstl restriction sites engineered at the 5′ and 3′ termini, respectively. The genome-length cDNAs were positioned downstream of T7 or SP6 phage promoters. Genome-sense RNAs transcribed in vitro from the T7 promoter were biologically active, while negative-sense RNAs transcribed in vitro from the SP6 promoter were not. Constructs that were identical to STMV and two other constructs in which there were two or six specific nucleotide differences in the 3′ noncoding region yielded RNAs that were infectious. Sequence analysis of the progeny RNA derived from infections with transcripts containing nucleotide differences between nucleotides 682 and 753 revealed that these changes in sequence were maintained. In contrast, differences in the nucleotide sequence between nucleotides 989 and 1059 were not maintained in progeny RNA; one mutant reverted to the wild-type sequence, and the other generated a new sequence during infection.