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A genetic complementation system was developed in which tobacco etch virus (TEV) polymerase (NIb)-expressing transgenic plants or protoplasts were inoculated with NIb-defective TEV mutants. A beta-glucuronidase (GUS) reporter gene integrated into the genomes of parental and four mutant viruses was used to assay RNA amplification. Two mutants (termed VNN and EDE) contained substitutions affecting the conserved "GDD" polymerase motif or a nuclear localization signal sequence, respectively; one (aD/b) contained a mutation debilitating the NIb N-terminal cleavage site, whereas the other (delta b) lacked the entire NIb sequence. Each mutant was unable to amplify in nontransformed tobacco protoplasts. In contrast, the VNN, EDE, and delta b mutants were complemented to various degrees in NIb-expressing cells, whereas the aD/b mutant was not complemented. The VNN mutant was complemented most efficiently, reaching an average of 11-12% the level of parental TEV-GUS, although in some experiments the level was near 100%. This mutant also replicated in, and spread through, whole transgenic plants to the same level as parental virus. The EDE mutant was complemented relatively poorly, reaching 1% or less of the level of parental TEV-GUS. Despite the close proximity of the EDE substitution to the N-terminal cleavage site, proteolytic processing of NIb was unaffected in an in vitro assay. The delta b mutant was complemented to an intermediate degree in protoplasts, reaching 3.5% the level of parental virus, and replicated and moved systemically in transgenic plants. These data indicate that free NIb supplied entirely in trans can provide all NIb functions essential for RNA amplification. The relative inefficient complementation of the EDE mutant suggests that the resulting mutant protein was transinhibitory.

Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells.