Generation of Zucchini Tigre Mosaic Virus Mild Strains for Application in Cross-Protection

Zucchini tigre mosaic virus (ZTMV; Potyvirus pepotigris), which infects wax gourd (Benincasa hispida), was first identified in Taiwan in 2017 and designated ZTMV-TW. In this study, mild strains of ZTMV-TW were generated by modifying the pathogenicity factor HC-Pro to develop cross-protection strategies for cucurbit crops. A full-length infectious cDNA clone of ZTMV-TW was cloned in pCAMBIA1304 under the control of the CaMV 35S promoter (ZTMV-TWic). ZTMV-TWic induced typical potyvirus particles, cytoplasmic inclusion bodies, and severe symptoms in wax gourd, pumpkin, and zucchini plants. Conserved motifs of HC-Pro were mutated to generate four single mutants (F7I, R181I, F206L, and D397N) and three double mutants (F7I+F206L, R181I+D397N, and F206L+D397N). Mutants R181I and R181I+D397N caused mild or no symptoms in zucchini, while D397N and F206L+D397N were mild in wax gourd. Cross-protection assays showed that R181I and R181I+D397N provided complete protection against ZTMV-GFP in zucchini, whereas D397N and F206L+D397N conferred high protection in wax gourd. These results demonstrate the feasibility of host-specific mild strain selection for effective ZTMV cross-protection.