|Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. We have expended much effort to study the resistance response of pepper cultivars at cellular and whole plant levels. To evaluate the resistance response at the cellular level, mesophyll protoplasts are isolated and inoculated with viral RNA. An efficient isolation procedure was available but an inoculation procedure was needed that provided consistent and highly efficient inoculation. An electroporation inoculation procedure was developed by evaluation of key parameters that included voltage, number of pulses, time interval between pulses, viral RNA concentration and number of evaluated protoplasts. Consistent infection with the highest virus titer and protoplast viability resulted when 40 μg of virus RNA was used to inoculate 500,000 protoplasts using two 25-msec pulses of 200 volts each with a 10-sec time interval between pulses.
Two important sources of resistance were evaluated for their response to inoculation with four strains of Tobacco etch virus (TEV, genus Potyvirus). The resistant cultivars were CA4 and Dempsey which contain the pvr1 and pvr12 resistance genes, respectively. Both cultivars resisted infection by TEV strain NW; however, two CA4 plants inoculated with NW maintained in pepper became infected. When the infected CA4 plants were used as inoculum of additional CA4 plants, the newly inoculated plants developed systemic symptoms and accumulated virus in non-inoculated leaves more quickly than the originally infected CA4 plants. This new NW isolate, referred to as NW-CA4 was tested extensively and shown to overcome the resistances expressed by both CA4 and Dempsey. The potyviral VPg is believed to be the determinant for pvr1 and pvr12 resistance genes, both of which are eIF4E encoding genes. The VPg amino acid sequence for NW-CA4 was determined and compared with that of NW isolates; two substitutions were found within regions of the VPg that were shown to be associated with overcoming eIF4E related resistances by other potyviruses.