Cloning of the papaya ringspot virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene

Papaya ringspot virus (PRSV) can cause a destructive disease in papaya (Carica papaya L.). Based on observations that viral replicase (RP) gene confers resistance to virus in other plants, we designed a pair of primers and cloned the RP gene from PRSV by RT-PCR. The 3'-truncated and 5'-extended RP gene fragment was then oriented under the control of the CaMV35 S promoter and nos termination sequence in the mini Ti plasmid vector pRok to construct a plant expression vector, designated pRPTW. Papaya (C. papaya L.) cv. Tai-nong-2 embryogenic calli were transformed by Agrobacterium tumefaciens LBA4404 harboring the pRPTW vector. After selection on 100 mg/ml kanamycin, 20 putative transgenic papayas were regenerated and confirmed by PCR-Southern blot and Southern blot analyses. PRSV inoculation tests showed that the RP gene conferred resistance to PRSV in transgenic papayas and those offspring carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV was attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studies are required.