Engineering a Non-Aphid-Transmissible Mild Mutant of Zucchini Yellow Mosaic Virus for Cross Protection

Zucchini yellow mosaic virus (ZYMV; genus Potyvirus), an aphid-transmitted pathogen, has become a major threat to melon (Cucumis melo) production in China, severely reducing both crop yield and fruit quality. Cross protection using attenuated virus strains is a key strategy for controlling plant viral diseases. However, few ZYMV vaccine candidates possess both multiple attenuating mutations and impaired aphid transmissibility. In this study, we identified that the conserved residues in helper component-protease (HC-Pro), glycine at position 322 (G 322 ) and lysine at position 360 (K 360 ), played essential roles in RNA silencing suppression and ZYMV virulence. To reduce the risk of mild strains mutating to virulent ones and eliminate aphid transmission of an attenuated ZYMV strain, we substituted G 322 and K 360 with alanine (A) and aspartic acid (D), respectively, and further replaced A in the conserved DAG motif of the coat protein (CP) with threonine (T), generating a triple-mutant designated ZYMV-ADT. ZYMV-ADT maintained genetic stability through three serial passages and caused infection without apparent mosaic symptoms at 45 days post-agroinfiltration. Moreover, ZYMV-ADT provided effective protection in melon plants against severe ZYMV infection with a 10-day protection period. This study provides an attenuated mutant out of a non-aphid transmissible ZYMV strain that offers a promising approach for managing ZYMV through cross protection.