Infectious cDNA clones of two grapevine viruses.

Abstract

Full-length cDNA copies of the genomes of Grapevine virus A (GVA) and Grapevine 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 Nicotiana plants. A full-length cDNA copy of GVB was engineered in pCass2, a plasmid containing a partially duplicated copy of the Ca35S promoter, but was rather unstable in Escherichia coli. No infection of Nicotiana plants was obtained following mechanical inoculation but detached Nicotiana leaves, inoculated by particle bombardment, supported the multiplication of a GVB isolate seemingly identical to the wild-type used for cloning. Nicotiana seedling inoculated with sap expressed from these leaves became infected showing typical GVB symptoms. Transient transcription of Ca35S driven cDNA clones was also detected by RT-PCR in leaves of the grapevine hybrid LN33 following inoculation by particle bombardment. The availability of infectious cDNA clones of GVA and GVB constitutes a tool for the study of genome expression and pathogenesis, and for the ultimate establishment of the aetiological role of these viruses.