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 (Car ica papaya L.). Based on observations that viral replicase (RP) gene confer s resistance to virus in other plants, we designed a pair of primers and cl oned the RP gene from PRSV by RT-PCR. The 3'-truncated and 5'-extended RP g ene fragment was then oriented under the control of the CaMV35 S promoter a nd 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 LBA 4404 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 offsprin g carrying the RP gene. The consistency of the presence of the RP gene and PRSV resistance indicates that replicase-mediated resistance against PRSV w as attained in papaya. Possible mechanisms include RNA-mediated resistance and protein-mediated resistance, as well as others, although further studie s are required.