Infectious TYMV RNA from cloned cDNA: effects in vitro and in vivo of point substitutions in the initiation codons of two extensively overlapping ORFs

Abstract

Full-length cDNA of the 6.3 kb turnip yellow mosaic virus (TYMV) genome was placed between a T7 promoter and a unique Hind III site. In vitro transcription of Hind III-linearized DNA of clone pTYMC yielded full-length RNA transcripts. In inoculations of Chinese cabbage protoplasts and plants, capped transcripts and virion RNA had similar specific infectivities and produced similar systemic symptoms. We have used the pTYMC clone in studies of the expression of two overlapping open reading frames (1.9 kb and 5.5 kb ORFs) by making mutants with alterations in the initiation codons. Evidence is presented from in vitro translations of mutant and wild type RNAs that both ORFs are expressed from TYMV RNA. A mutant in the initiation codon of the 5.5 kb ORF did not replicate in protoplasts, while mutants in the initiation codon of the 1.9 kb ORF replicated at low levels. The two groups of mutants were not able to complement each other.