Nonsense and missense translational suppression in plant cells mediated by tRNALys

A nuclear tRNA(Lys) gene from Arabidopsis thaliana was cloned and mutated so as to express tRNAs with altered anticodons which bind to a UAG nonsense (amber) codon and to the Arg (AGG), Asn (AAC,AAT), Gln (GAG) or Glu (GAG) codons. Concomitantly, a codon in the firefly luciferase gene for a functionally important Lys was altered to an amber codon, or to Arg, Asn, Gin, Glu, Thr and Trp codons, so as to construct reporter genes reliant upon incorporation of Lys. The altered tRNA(Lys) and luciferase genes were introduced into Nicotiana benthamiana protoplasts and expression of the mutated tRNAs was verified by translational suppression of the mutant firefly luciferase genes. Expression of the amber suppressor tRNA(CUA)(Lys) from non-replicative vectors promoted 10-40% suppression of the luciferase nonsense reporters while expression of the amber and missense tRNA(Lys) suppressor genes from a geminivirus vector capable of replication promoted 30-80% suppression of the luciferase nonsense reporter and up to 10% suppression of the luciferase missense reporters with Arg, Asn, Gin and Glu codons.