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

Abstract

Passiflora mottle virus (PaMoV), an aphid-borne potyvirus, is the primary causal virus of the devastating passionfruit woodiness disease in Vietnam. Here we generated a non-pathogenic, 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 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 plants, PaMoV-E53 elicited severe leaf mosaic, 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 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.

Mutating the arginine residue within the FRNK motif of HC-Pro yield highly attenuated strains that confer complete cross protection against telosma mosaic virus (TelMV) in passion fruit (Passiflora edulis)

“…It seems the FRNK mutant of HC-Pro often exhibited abolished or weak RSS activities. This is supported by the current study of TelMV as well as many other cases among different potyviruses, including WMV, SCMV, TuMV and PaMoV (Xu et al 2020;Kung et al 2014;Do et al 2023). Nevertheless, the FINK mutant of ZYMV HC-Pro maintains RSS activity, despite that the corresponding viral mutant caused attenuation of symptoms in cucurbits (Shiboleth et al 2007).…”

“…The highly conserved FRNK motif of potyviral HC-Pro is important for viral pathogenicity, mainly evidenced by the previous reports that mutating the Arginine (R) residue resulted in reduced pathogenicity of various potyviruses, including ZYMV, TuMV, PLDMV, EAPV, PaMoV and PRSV (Gal-On 2000; Lin et al 2007;Shiboleth et al 2007;Tran et al 2023;Kung et al 2014;Tuo et al 2020;Chong et al 2023;Do et al 2023). Consistently, in our comprehensive exam on the role of Arginine residue within the FRNK motif of HC-Pro in TelMV infection, three out of six mutants (R 181 K, R 181 D and R 181 E, but not R 181 H, R 181 I and R 181 A) exhibited highly attenuated infections that induced unnoticeable symptom in passion fruit plants (Fig 3A).…”

“…(Yang et al 2018;Do et al 2021;Ochwo-Ssemakula et al 2012). The most frequent cause of viral diseases in passion fruit belongs to the genus Potyvirus, including passion fruit woodiness virus (PWV), cowpea aphid-borne mosaic virus (CABMV), East Asian Passiflora virus (EAPV), Passiflora mottle virus (PaMoV), and telosma mosaic virus (TelMV) (Do et al 2021;Do et al 2023;Gou et al 2023). It is worth mentioning that TelMV is an emerging viral pathogen causing a major global threat to passion fruit plantations.…”

Development of Attenuated Viruses for Effective Protection against Pepper Veinal Mottle Virus in Tomato Crops

“…Local lesion formation is a defensive hypersensitive response (HR) triggered by virus infection to localize and eliminate invasive viruses [46]. Previous studies revealed that protective mild viruses can infect but do not produce macroscopically observable HR on inoculated C. quinoa leaves [31,[47][48][49], suggesting that viral virulence is associated with the induction of HR in C. quinoa leaves. To visualize invisible lesions, viruses were genetically engineered to carry the GFP reporter prior to mutagenesis [31,33].…”

Virus-induced gene silencing (VIGS) as a tool for functional genetic analysis in passion fruit plants

“…Unfortunately, it showed that TRV could not infect passion fruit plants using the agroinfiltration or rub-inoculation method in our current experimental setup (data not shown). Another reason is that the passion fruit infecting-virus infectious clone/vector has been reported recently, including TelMV, Passiflora mottle virus (PaMoV) and East Asian passiflora virus (EAPV) (Gou et al, 2023; Do et al, 2023; Chong et al, 2023). We expected that these newly constructed passion fruit infecting-virus infectious clones would be utilized as viral tools for protein expression and gene silencing in passion fruit plants.…”