Infectious cDNA clones of two grapevine viruses

Full-length cDNA copies of the genomes of Grapevine virus A (GVA) and Grape vine virus B (GVB) under the control of bacteriophage T7 promoter have been synthesized, which were refractory to cloning in Escherichia coli. However , both transcribed cDNAs were infectious when mechanically inoculated to Ni cotiana plants. A full-length cDNA copy of GVB was engineered in pCass2, a plasmid containing a partially duplicated copy of the Ca35S promoter, but w as rather unstable in Escherichia coli. No infection of Nicotiana plants wa s obtained following mechanical inoculation but detached Nicotiana leaves, inoculated by particle bombardment, supported the multiplication of a GVB i solate seemingly identical to the wild-type used for cloning. Nicotiana see dling inoculated with sap expressed from these leaves became infected showi ng typical GVB symptoms. Transient transcription of Ca35S driven cDNA clone s was also detected by RT-PCR in leaves of the grapevine hybrid LN33 follow ing inoculation by particle bombardment. The availability of infectious cDN A clones of GVA and GVB constitutes a tool for the study of genome expressi on and pathogenesis, and for the ultimate establishment of the aetiological role of these viruses.