Intron hairpin RNA derived from the HC-Pro gene confers resistance to papaya ringspot virus in transgenic Nicotiana benthamiana

In the present study, cDNA from a papaya leaf sample infected with Papaya Ringspot Virus (PRSV) was used for the amplification of the HC-Pro gene, which yielded an expected amplicon size of ~1350 bp. The partial coding sequence of HC-Pro was used to design a hairpin RNA (hpRNA) construct, which included a cowpea spliceosomal intron (148 bp) inserted between the hpRNA arms (ihpRNA HC-Pro). The potential siRNAs and Virus Induced Gene Silencing (VIGS) sequence predicted by integrated bioinformatics tools (siDirect 2.0 and pssRNAit) identified the best region of 201 bp (557-757 bp). The designed intron hairpin RNA (ihpRNA) HC-Pro was synthesized and cloned into the plant expression vector (pBI121) and the resulting recombinant plasmids (pBI121::CaMV35s::ihpRNA-HC-Pro::NOS) were subsequently transformed into Agrobacterium tumefaciens (LBA4404) followed by co-cultivation in N. benthamiana, a model plant. Molecular confirmation of the T0 lines via PCR indicated that three out of four plants were contaminated with Agrobacterium in the apoplast. A single line that was positive for ihpRNA HC-Pro amplification and negative for VirG, was forwarded to the T1 generation. Six out of ten T1 lines were amplified for ihpRNA HC-Pro. Single copy insertions were confirmed by southern blotting. Bioassay and gene expression tests in T1 transgenic plants showed lower levels of NbPOD, NbAPX, NbCAT and HcPro, like healthy plants, suggesting that PRSV resistance was successfully achieved through ihpRNA-mediated gene silencing.