Intron–hairpin RNA Derived from Helper Component proteinase (HC-Pro) Gene Confers Immunity to Papaya Ringspot Virus Infection in Transgenic Tobacco Plants

Abstract

Abstract Papaya ringspot virus (PRSV) is the most destructive disease of papaya which limits its production and cultivation worldwide. Pathogen-derived resistance (PDR) approach as a popular strategy has been utilized previously to develop resistant cultivars in papaya. However, the pathogen has already overcome the resistance which necessitates an alternative strategy for PRSV disease management. RNAi is an effective method of obtaining resistant transgenic plants against viruses by inducing RNA silencing via expressing virus-derived double-stranded RNA in plants. The current study deals with the isolation and characterization of the HC-Pro gene from PRSV, the development of intron hairpin RNA construct followed by transformation and confirmation in the T1 generation of Nicotiana benthamiana. The partial coding region of the helper component (HC-Pro) gene of PRSV was used to design hairpin RNA which includes a spliceosomal intron inserted between the hairpin RNA arms (HC-Pro ihpRNA). Designed HC-Pro ihpRNA was introduced into the plant expression vector (pBI121) and the recombinant plasmids were transformed into Agrobacterium tumefaciens (LBA4404) followed by transformation into Nicotiana bennthamiana as a model plant. Gene expression analysis of transgenic T1 plants infected with PRSV showed reduced NbPOD, NbAPX and NbCAT compared to inoculated control plants. Results revealed that the suppression of the HC-Pro gene has an effect similar to that of non-infected control plants and hence confers resistance to PRSV infection. We have demonstrated that transgenic tobacco plants expressing partial PRSV HC-pro gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to PRSV infection.