Abstract: ATP binding is an essential event in diverse biological processes including plant immunity. The ATP‐binding domains in plant kinases share similar structural properties, providing a potential common target for pathogens. However, effectors targeting the ATP‐binding domains to modulate kinase activity have not been identified. In this study, we identified a conserved effector containing an Egh16‐like domain (Cee1) in Fusarium graminearum. As an in planta induced gene, CEE1 plays a stage‐specific role in infectious growth within wheat rachis. Upon translocation into plant cells, Cee1 interacts with the ATP‐binding domain of TaMPK3 via its Egh16‐like domain. This interaction interferes with the ATP binding and impairs the kinase activity of TaMPK3, leading to reduced phosphorylation levels of TaWRKY33 and subsequent inactivation of downstream resistance responses. F. graminearum1 harbors three paralogs of Cee1, each containing two adjacent motifs responsible for specific interaction with ATP‐binding pockets, all crucial for pathogenesis. The quadruple mutant lacking these four CEE genes shows drastically reduced pathogenicity, and CEE genes have been identified as silencing targets for improving wheat FHB resistance. Taken together, Cee1 and its paralogs act as core effectors in F. graminearum1 by targeting the ATP‐binding domains of plant kinases, demonstrating the representative mode of action of the Egh16‐like domain in fungal‐plant interactions.

Key words: ATP binding, egh16‐like domain, fungal effector, Fusarium graminearum, Fusarium head blight, kinase activity