Bacterially Expressed Double-stranded RNAs Targeting PRSV-NIb Gene Inducing RNA Silencing in Papaya Protoplasts
To explore the use of PRSV-NIb gene prokaryotic expression of homologous dsRNA product feasibility for controlling papaya ringspot virus in vitro,three hairpin RNA coding structures with PDK intron were built with 3′-end 312 bp,501 bp and 809 bp sections of a more conservative PRSV-NIb gene.After inserted into the plasmid pSP73,the structures were transformed into M-Jm109LacY of the RNaseIII-deficient strain and induced with IPTG,respectively.The Escherichia coli strains could express N312-dsRNA,N501-dsRNA and N809-dsRNA,respectively.The plant transient expression vector encoding the PRSV-NIb with an N-terminal GFP fusion was constructed.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 mRNA expression showed that the effectively bacterially expressed dsRNA-triggered gene silencing was detected.Our work laid a foundation for a potential green and effective approach protecting papaya from PRSV.