Electroporetic transfection of pepper protoplasts with plant potyviruses

Potyviruses are a persistent threat to bell pepper (Capsicum annuum L.) production worldwide. Much effort has been expended to study the resistance response of pepper cultivars at whole plant levels but with only limited effort at the cellular level using protoplasts. A pepper protoplast isolation procedure is available but an inoculation procedure is needed that provides consistent and highly efficient infection. An electroporation-based procedure for inoculation of potyviruses was developed using a base procedure developed for Cucumber mosaic virus (CMV). The final parameters identified for efficient potyvirus infection of pepper protoplasts involves two 25 ms pulses, 200 V each pulse with a 10 s interval between pulses. Depending on the method of detection, e.g., ELISA versus RT-PCR, potyvirus RNA inoculum ranged from 10 to 40 μg with infection detection occurring with samples of 50,000-100,000 protoplasts.

► To study plant virus infection at the cellular level requires an efficient plant protoplast inoculation system. ► Such a system will allow comparative studies for virus replication at the cellular level and evaluation of sources of genetic resistance. ► An efficient electroporation system for inoculation of plant potyviruses in pepper protoplasts was developed. ► A systematic approach evaluated numerous parameters important to the electroporation process.