Bacterially Expressed Double-stranded RNAs Targeting Three Viral Genes Of Papaya Ringspot Virus (PRSV)Interferes With PRSV Infection

Papaya ringspot virus (PRSV) is a plant virus belonging to the genus Potyvirus, family Potyviridae, with a positive sense RNA genome. PRSV causes severe economic losses in papaya throughout the tropics and subtropics. PRSV disease management practices include quarantine, eradication, avoidance by planting papaya in areas isolated from the virus, continual rogueing of infected plants, use of tolerant lines to lower the economic losses caused by PRSV, crossprotection and transgenic resistance. Development of PRSV-resistant cultivars through conventional breeding met with limited success because of difficulties in overcoming intergeneric reproductive barriers of wild, related species of papaya. In addition, partial loss of tolerance in back-crosses with the commercial papaya parent also limits the usefulness of this approach. Development of PRSV-resistant transgenic plants faces a major hurdle in achieving resistance against geographically distinct isolates. We provide a strategy to develop effective and stable PRSV-resistant plants via dsRNAs expressed in Escherichia coli.In this study,9different dsRNA prokaryotic expression vectors carrying different sizes of PRSV-NIb(312,501,809bp), PRSV-HC-Pro(315,489,824bp) and PRSV-CP(279,432,867bp)cDNA were constructed, which were named as, pSP73-RNAi-N312, pSP73-RNAi-N501, pSP73-RNAi-N809, pSP73-RNAi-H315, pSP73-RNAi-H489, pSP73-RNAi-H824, pSP73-RNAi-C279, pSP73-RNAi-C432, pSP73-RNAi-C867respectively. These prokaryotic expression vectors were transformed into the RNaseâ…¢-deficient strain M-Jm109LacY, and then were induced with IPTG, respectively. All these E.coli strains deficient for RNaseâ…¢ could express the predicted sizes of PRSV-NIb-dsRNA, PRSV-HC-Pro-dsRNA and PRSV-CP-dsRNA, respectively.To monitor the silencing efficiency of these9different bacterially expressed dsRNAs derived from PRSV-Nib, PRSV-HC-Pro and PRSV-CP, we developed an effective transient gene silencing system for using the bacterially expressed dsRNA in papaya leaf protoplasts. Three plant transient expression vectors encoding the PRSV-NIb, PRSV-HC-Pro and PRSV-CP with an N-terminal GFP fusion were constructed respectively, which were named as pNIb-GFP, pHC-GFP and pCP-GFP respectively. Protoplasts were co-transfected using the transient GFP-fusion expression vector and the corresponding bacterially expressed dsRNA. The results of GFP fluorescence intensity using a confocal microscope and semi-quantitative RT-PCR analysis of the PRSV-Nib, PRSV-HC-Pro and PRSV-CP mRNA expression showed that the effective bacterially expressed dsRNA-triggered gene silencing was detected. To prove whether the bacterial-produced dsRNA could interfere with PRSV infection, we carry out a protective resistance assay and a therapeutic resistance assay. In the protective resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surface before inoculation of papaya leaves with PRSV. Protective treatment experimental results showed dsRNAs derived from the different functional genes of PRSV could all protect papaya plants from virus infection, and the resistance was obviously different due to different vectors. ELISA analysis and Real-time PCR results confirmed that the virus accumulation could be inhibited in different degree by dsRNA. Northern blot showed that the small RNA fragments specifically complementary to the PRSV-Nib, PRSV-HC-Pro and PRSV-CP antisense probe were detected, which suggested that the resistance is an RNA-mediated virus resistance. In the therapeutic resistance assay, the bacterially expressed dsRNA was used to spray onto the plant surfaces after inoculation of papaya leaves with PRSV. ELISA analysis and Real-time PCR results showed that the virus accumulation declined slightly after spraying dsRNA for three days. Field protective experimental results showed that regular spraying the crude product of the bacterially expressed N809-dsRNA could obtain stable resistance to PRSV.Our work indicated that bacterially expressed double-stranded RNA targeting three viral genes of PRSV could interfere with PRSV infection, which is a potential green and effective approach protecting plants from virus infections compared with the requirements for regenerating PRSV-resistant transgenic plants.