Preparation of an Infectious cDNA Clone of the Plum Pox Virus Strain PPV-Rec

Plum pox virus (PPV) is a devastating pathogen of stone fruit trees. Three PPV strains have a heavy economic impact in Europe: PPV-D, PPV-M and PPV-Rec. While infectious clones of the first two strains have been prepared, the full-length infectious cDNA of PPV-Rec was not for disposal. We describe two strategies for construction of a cDNA clone of the isolate BOR-3, a representative of PPV-Rec. De novo genome assembling from overlapping PCR products using unique restriction sites and cloning in a constructed vector failed due to selection of noninfectious clones bearing nonsense mutations. The second approach used the infectious clone pIC-PPV (strain PPV-D) containing a plant intron in the P3 cistron to avoid potential toxicity of the P3 protein for bacteria. pIC-PPV served as a backbone where particular segments were step by step exchanged by homologous parts of the BOR-3 genome. The infectivity of intermediate products was controlled after each cloning step by biolistic bombardment of plants. Six obtained chimeric cDNA were infectious in Nicotiana benthamiana. Replacement of the P3 genomic segment by the BOR-3 sequence without the intron lead to non-infectious clones. Preservation of the intron in the BOR-3 sequence by a hybrid PCR gave rise to noninfectious clones, too. Further attempts to complete the BOR-3 cDNA are going on. Analyses of intermediate constructs showed the key role of N-terminal part of PPV capsid protein in its immunochemical and electrophoretic properties and implicated the influence of the middle part of PPV genome on the symptoms manifested in Nicotiana benthamiana.