An NLR Integrated Domain toolkit to identify plant pathogen effector targets.

Plant immune receptors, known as Nod-like Receptors (NLRs), possess unique integrated decoy domains that enable plants to attract pathogen effectors and initiate a specific immune response. The aim of this study was to create a library of these integrated domains (IDs) and screen them with pathogen effectors to identify new targets for effector virulence and NLR-effector interactions. This works compiles IDs found in NLRs from seven different plant species and produced a library of 78 plasmid clones containing a total of 104 IDs, representing 43 distinct Interpro domains. They conducted a yeast-two-hybrid assay, followed by an in planta interaction test, using 32 conserved effectors from Ralstonia pseudosolanacearum type III. Through these screenings, they discovered three interactions involving different IDs (kinase, DUF3542, WRKY) interacting with two unrelated type III effectors (RipAE and PopP2). Of particular interest was the interaction between PopP2 and ID#85, an atypical WRKY domain integrated into a soybean NLR gene (GmNLR-ID#85). Using a FRET-FLIM technique to detect protein-protein interactions in living plant cells, we demonstrated that PopP2 physically associates with ID#85 in the nucleus. However, unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, GmNLR-ID#85 could not be acetylated by PopP2 and failed to activate RPS4-dependent immunity when introduced into the RRS1-R immune receptor. The generated library of 78 plasmid clones, encompassing these screenable IDs, is publicly available through Addgene. This resource is expected to be valuable for the scientific community in discovering new targets for effectors and potentially engineering novel plant immune receptors.