Deciphering the proximal proteome of two YopJ family acetyltransferases from two plant vascular pathogenic bacteria

Gram-negative bacterial pathogens inject variable repertoires of effectors into host cells to interfere with defense responses. Among those, the Yersinia outer protein J (YopJ) effector family of acetyltransferases, produced by diverse animal and plant bacterial pathogens, promote pathogen virulence by acetylating specific host components. However, the range of host processes that YopJ effectors can interfere with remains elusive. Thus, we developed a proximity-dependent protein labeling involving the Turbo biotin ligase for identifying the interactomes of two well-characterized YopJ members: the Ralstonia pseudosolanacearum PopP2 and its close homolog, XopJ6 from Xanthomonas campestris. Both of them are recognized by the Arabidopsis RRS1/RPS4 pair through manipulation of an integrated WRKY domain that mimics effector primary targets, the WRKY transcription factors. Interestingly, a single residue substitution in a XopJ6 natural variant disrupts physical interaction with WRKY proteins, enabling XopJ6 to avoid host recognition while retaining XopJ6 virulence functions, likely through interference with components other than WRKYs. The different PopP2 and XopJ6 variants fused with Turbo will be expressed in both N. benthamina and Arabidopsis. Alternatively, we developed an approach consisting in Pseudomomas fluorescens-mediated delivery of the Turbo fusion proteins to remain as close as possible to the level of effectors injected in plant cells by pathogenic bacteria.