HopN1 Binds Plant VAP12 and a Rho-GTPase, Suggesting a Role in Membrane-Associated Processes.

Many Gram-negative bacterial pathogens deploy type III effector proteins (T3Es) to manipulate host cellular processes and suppress immune responses. Increasing evidence suggests that certain T3Es mimic eukaryotic FFAT (two phenylalanines in an acidic tract) motifs, enabling interaction with vesicle-associated membrane protein (VAMP)-associated proteins (VAPs). These interactions likely help pathogens target and exploit host membrane contact sites. However, the significance and distribution of FFAT mimicry across different bacterial pathogens remain poorly understood, which is crucial to uncovering its role in pathogenic strategies. In this study, we analyzed the T3E repertoire of the model plant pathogenic bacterium pv. (Pst) DC3000 to identify potential FFAT motifs. Our preliminary data reveal that HopN1, a Pst T3E belonging to the YopT/AvrPphB family of cysteine proteases, contains at least one functional FFAT motif. Yeast two-hybrid and co-immunoprecipitation assays confirmed that HopN1 interacts with plant VAP proteins. This interaction suggests that VAP binding may facilitate its localization to specific membrane compartments. Furthermore, HopN1 was shown to interact with a plant RHO-GTPase, hinting at a functional parallel to YopT in mammals. Our findings demonstrate that HopN1 interacts with VAP12 and a plant RHO-GTPase, suggesting a potential role in membrane-associated processes. However, whether HopN1 actively exploits VAP proteins for subcellular localization remains to be determined. While FFAT motif mimicry may contribute to effector targeting in plant-pathogenic bacteria, further studies are required to establish its functional significance in HopN1 virulence.