Identification and functional characterization of chemoreceptors for phenolic acids in Agrobacterium tumefaciens

Phenolic acids influence host-pathogen interactions and function as key signals in Agrobacterium-mediated transformation or plant-microbe symbiosis. Agrobacterium tumefaciens uses chemotaxis to detect plant-secreted phenolic compounds and migrates to infection sites, though the chemotactic mechanism remains unclear. In this study, starting with structurally simple phenolic acids, the chemotactic response of A. tumefaciens C58 was investigated. The chemotaxis of A. tumefaciens toward 4-hydroxybenzoate and protocatechuate is not impacted by the methyl-accepting chemotaxis proteins (MCPs) Atu0387 and Atu0738, which share a four-helix bundle domain with previously discovered phenolic-sensing MCPs. To identify chemoreceptors for phenolic acids, a heterologous expression and functional screening system was constructed in Escherichia coli. Among the 13 MCPs, Atu0872 could respond to both 4-hydroxybenzoate and protocatechuate. Furthermore, atu0872 deletion weakened chemotaxis toward vanillin, acetosyringone, guaiacol, caffeic, vanillic, salicylic, gallic, p-coumaric, syringic, and sinapinic acids. Although the ligand-binding domain of Atu0872 was predicted to be a nitrate- and nitrite-sensing domain, the A. tumefaciens deletion mutant Δatu0872 did not affect chemotaxis toward nitrate and nitrite. In addition to chemotaxis, atu0872 deletion decreased the tumor weight on Daucus carota roots, Kalanchoe daigremontiana leaves, and the number of bacterial colonies per 0.1 g of tumor, implying that atu0872 affects bacterial colonization on the host by regulating chemotactic behavior. To our knowledge, this is for the first study identifying Atu0872 as a core chemoreceptor in A. tumefaciens for phenolic compounds, providing a theoretical foundation for elucidating the chemotaxis-pathogenicity relationship in A. tumefaciens and optimizing its use in genetic transformations.