Generation of Attenuated Passiflora Mottle Virus Through Modification of the Helper Component Protease for Cross Protection

Passiflora mottle virus (PaMoV), an aphid-borne potyvirus, is the primary causal virus of devastating passionfruit woodiness disease in Vietnam. Here we generated a nonpathogenic, attenuated PaMoV strain for disease control by cross protection. A full-length genomic cDNA of PaMoV strain DN4 from Vietnam was constructed to generate an infectious clone. The green fluorescent protein was tagged at the N-terminal region of the coat protein gene to monitor in planta the severe PaMoV-DN4. Two amino acids within the conserved motifs of helper component protease (HC-Pro) of PaMoV-DN4 were mutated individually or in combination as K53E or/and R181I. Mutants PaMoV-E53 and PaMoV-I181 induced local lesions in Chenopodium quinoa plants, while PaMoV-E53I181 caused infection without apparent symptoms. In passionfruit (Passiflora edulis) plants, PaMoV-E53 elicited severe leaf mosaic and PaMoV-I181 induced leaf mottling, while PaMoV-E53I181 caused transient mottling followed by symptomless recovery. PaMoV-E53I181 was stable after six serial passages in yellow passionfruit (Passiflora edulis f. flavicarpa) plants. Its temporal accumulation levels were lower than those of the wild type, with a zigzag accumulation pattern, typical of a beneficial protective virus. An RNA silencing suppression (RSS) assay revealed that all three mutated HC-Pros are defective in RSS. Triplicated cross-protection experiments with a total of 45 plants showed that the attenuated mutant PaMoV-E53I181 provided a high protection rate (91%) against the homologous wild-type virus in passionfruit plants. This work revealed that PaMoV-E53I181 can be used as a protective virus to control PaMoV by cross protection.