Abstract:
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 β-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 (Δb) lacked the entire NIb sequence.
Each mutant was unable to amplify in nontransformed tobacco
protoplasts. In contrast, the VNN, EDE, and Ab 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 Δb 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.
