Complementation and Disruption of Viral Processes in Transgenic Plants

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode the replicase genes P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the viral coat protein (CP). To investigate the mechanism of cross-protection, tobacco plants were transformed with wild-type and mutant DNA copies of the AIMV CP gene and the two replicase genes P1 and P2. Expression of wild-type CP at relatively low levels resulted in a resistance against infection with AIMV virus particles whereas at higher expression levels CP protected against infection with either AIMV particles or RNAs. Plants transformed with a mutant AIMV CP gene were not resistant to the wild-type virus but were resistant to AIMV with the same mutation in the CP gene. Transformation of plants with the wild-type P1 gene (P1 plants), P2 gene (P2 plants) or both these genes (P12 plants) did not result in resistance to AIMV. Instead, these plants could be infected with an inoculum lacking the gene(s) that was (were) integrated in the plant genome. Infection of non-transgenic plants, P1 plants or P2 plants with a mixture of AIMV genomic RNAs requires the presence of CP in the inoculum but P12 plants could be infected with RNA3 without any requirement for CP in the inoculum. Infection conditions in which 35S promoter/AIMV cDNA fusions were present in the inoculum instead of in the plant genome were used to shed light on the early function of CP. Finally, plants were transformed with P2 genes with mutations in the GDD-motif. A number of these transgenic lines showed a high level of resistance to AIMV.