WRKY transcription factors are essential for mediating many developmental processes, such as fruit ripening, a highly controlled and intricate physiological phenomenon. In the current research, a novel transcription factor, MdWRKY9, was identified and categorized, which significantly promotes apple fruit ripening. Its role was validated through a combination of transient injection and stable overexpression-transformed tomato experiments. Notably, MdWRKY9 interacts with the fruit ripening suppressor MdERF5L at protein and DNA levels. This interaction counteracts MdERF5L-mediated MdACS1 repression. MdACS1 is an important ethylene biosynthesis enzyme; the mentioned process eventually allows fruit ripening. Furthermore, phosphorylation, a post-translational modification, regulates maturation and ethylene synthesis. Through liquid chromatography–tandem mass spectrometry, we identified phosphorylation sites within the MdWRKY9-GFP protein. MdMAPK6–MdWRKY9 interaction and MdMAPK6-mediated phosphorylation of MdWRKY9 were confirmed through protein–protein interaction assays, such as bimolecular fluorescence complementation (BIFC), yeast two-hybrid (Y2H), protein phosphorylation, luciferase complementation imaging (LCI), and pull-down assays. Specifically, MdMAPK6 phosphorylates MdWRKY9 at Tyr394 site, enhancing the stability and activity of MdWRKY9 and thus modulating its regulatory role in fruit maturation. These findings provide new directions and insights into the intricate regulatory network governing the ripening of apples.

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