Transgenic or plant expression vector-mediated recombination of Plum pox virus

Different mutants of an infectious full-length clone (p35PPV-NAT) of Plum p ox virus (PPV) were constructed: three mutants with mutations of the assemb ly motifs RQ and DF in the coat protein gene (CP) and two CP chimeras with exchanges in the CP core region of Zucchini yellow mosaic virus and Potato virus Y. The assembly mutants were restricted to single infected cells, whe reas the PPV chimeras were able to produce systemic infections in Nicotiana benthamiana plants. After passages in different transgenic N. benthamiana plants expressing the PPV CP gene with a complete (plant line 4.30.45.) or partially deleted 3'-nontranslated region (3'-NTR) (plant line 17.27.4.), c haracterization of the viral progeny of all mutants revealed restoration of wild-type virus by recombination with the transgenic CP RNA only in the pr esence of the complete 3'-NTR (4.30.45.). Reconstitution of wild-type virus was also observed following cobombardment of different assembly-defective p35PPV-NAT together with a movement-defective plant expression vector of Po tato virus X expressing the intact PPV-NAT CP gene transiently in nontransg enic N. benthamiana plants. Finally, a chimeric recombinant virus was detec ted after cobombardment of defective p35PPV-NAT with a plant expression vec tor-derived CP gene from the sour cherry isolate of PPV (PPV-SoC). This chi meric virus has been established by a double recombination event between th e CP-defective PPV mutant and the intact PPV-SoC CP gene. These results dem onstrate that viral sequences can be tested for recombination events withou t the necessity for producing transgenic plants.