Mutations in the coat protein gene of Plum pox virus suppress particle assembly, heterologous encapsidation and complementation in transgenic plants of Nicotiana benthamiana

Two different motifs in the coat protein (CP) of Plum pox virus (PPV) (R(30 15)Q(3016), D-3059) were mutated by replacing the respective amino acids wi th others possessing different chemical properties. The mutated CP genes we re introduced into an infectious full-length clone of PPV (p35PPV-NAT) to i nvestigate their influence on systemic infection of transgenic wild-type PP V CP-expressing and non-transgenic plants of Nicotiana benthamiana, All mut ants failed to establish systemic infections in non-transgenic N. benthamia na plants, but were complemented by intact CP in transgenic plants. Moreove r, the CP-RQ-D mutant (carrying mutations in both the RQ and D motifs) was introduced into p35PPV-NAT engineered to express beta-glucuronidase (GUS) f or direct observation of systemic movement and particle assembly in N. bent hamiana leaves. GUS-staining revealed that the CP mutant (RQ-D) was restric ted to initially infected cells without forming virions. Systemic movement and particle assembly were restored in CP-transgenic N, benthamiana plants. Finally, transgenic N, benthamiana plants were generated that expressed ea ch of the three mutated CP genes. Homozygous T-2 lines were selected and te sted for resistance to PPV, Immunogold labelling and electron microscopy re vealed that heterologous encapsidation with challenging Chilli veinal mottl e virus and Potato virus Y was suppressed in these lines. In addition, asse mbly mutants did not complement CP-defective p35PPV-NAT, The possible use o f modified viral CP genes for the production of virus-resistant transgenic plants, thereby reducing the putative risks of heterologous encapsidation a nd complementation, is discussed.