A cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain

Little is currently known regarding the specific interactions that govern transmission of plant viruses by their vectors. A cucumber necrosis virus (CNV) variant (LL5) deficient in fungal transmissibility has been isolated from mechanically passaged CNV and characterized. Although LL5 accumulates to wild-type (WT) levels, is capable of rapid systemic infection, and produces stable, highly infectious particles, it is only inefficiently transmitted by Olpidium bornovanus zoospores. The LL5 coat protein (CP) gene was amplified by RT-PCR and cloned in place of the WT CNV CP gene in an infectious CNV cDNA clone. Particles derived from this construct also failed to be efficiently transmitted. The LL5 CP gene was sequenced and found to contain two amino acid substitutions relative to WT CNV CP. One substitution (Phe to Cys) occurred in the arm region and another (Glu to Lys) in the shell domain. These amino acid changes were separately introduced into the WT CNV genome through in vitro mutagenesis and it was found that the Glu to Lys change in the LL5 CP shell domain is largely responsible for the loss of transmissibility. In vitro binding assays were developed to determine if the defect in transmissibility was due to a defect in binding zoospores. LL5 particles were found to bind less efficiently than WT CNV. Furthermore, the nontransmissible tomato bushy stunt virus did not detectably bind zoospores. These binding studies suggest that the specificity of CNV transmission by O. bornovanus occurs through specific recognition of a putative zoospore receptor.