Concurrent Control of Two Aphid-Borne Potyviruses in Cucurbits by Two-in-One Vaccine

The application of attenuated viruses has been widely practiced for protecting crops from infection by related severe strains of the same species. Papaya ringspot virus W-type (PRSV W) and zucchini yellow mosaic virus (ZYMV) devastate cucurbits worldwide. However, the prevailing of these two viruses in cucurbits cannot be prevented by a single protective virus. In this study, we disclosed that co-infection of horn melon plants by two mild strains, PRSV P-type (PRSV P) HA5-1 and ZYMV-ZAC (a previously developed mild mutant of ZYMV) confers concurrent protection against PRSV P and ZYMV. Consequently, mild mutants of PRSV W were created by site-directed mutagenesis through modifications of the pathogenicity motifs FRNK and PD in helper component-protease (HC-Pro). A stable PRSV W mutant WAC (PRSV-WAC) with R181I and D397N mutations in HC-Pro was generated, inducing mild mottling, followed by symptomless recovery in cucurbits. Horn melon plants pre-infected by PRSV-WAC and ZYMV-ZAC showed no apparent interference on viral accumulation with no synergistic effects on symptoms. An agroinfiltration assay of mixed HC-Pros of WAC_HC-Pro + ZAC_HC-Pro revealed no additive effect of RNA silencing suppression. PRSV-WAC or ZYMV-ZAC alone only antagonized a severe strain of homologous virus, while co-infection with these two mild strains provided complete protection against both PRSV W and ZYMV. Similar results were reproduced in muskmelon and watermelon plants, indicating the feasibility of a two-in-one vaccine for concurrent control of PRSV W and ZYMV in cucurbits.