An NLR Integrated Domain toolkit to identify plant pathogen effector targets

Abstract

Plant resistance genes (or NLR, Nod-like Receptors) are known to contain atypical domains procuring them with a decoy capacity. Some of these integrated domains (or ID) allow the plant to lure the virulence determinants (effectors) of pathogens and triggering a specific NLR immune reaction. In this work, our goal was to generate a library of known IDs that could be screened with plant pathogen effectors in order to identify putative new effector virulence targets and NLR-effector pairs. We curated the IDs contained in NLRs from seven model and crop plant species. We cloned 52 IDs representing 31 distinct Pfam domains. This library was screened for interaction by yeast-two-hybrid with a set of 31 conserved Ralstonia solanacearum type III effectors. This screening and the further in planta interaction assay allowed us to identify three interactions, involving different IDs (kinase, DUF3542, WRKY) and two type III effectors (RipAE and PopP2). PopP2 was found to physically interact with ID#85, an atypical WRKY domain integrated in the GmNLR-ID85 NLR protein from Soybean. Using a imaging method in living plant cells, we showed that PopP2 associates with ID#85 in the nucleus. But unlike the known WRKY-containing Arabidopsis RRS1-R NLR receptor, this newly identified soybean WRKY domain could not be acetylated by PopP2 and its atypical sequence (WRKYGKR) also probably renders it inefficient in plant immunity triggering. This ID toolkit is available for screening with other plant pathogen effectors and should prove useful to discover new effectors targets and potentially engineer new plant resistance genes.

Unmasking the invaders: NLR-mal function in plant defense

“…The RRS1/RPS4 pair exists in an inhibited resting state until a lysine residue within the WRKY domain is acetylated, which switches it from an inhibited complex to an activated one to trigger an immune response (Le Roux et al, 2015). One study has generated a library of known IDs that can be utilized to screen with pathogen effectors to better identify virulence targets (Landry et al, 2021). Here, PopP2 was shown to physically interact with the WRKY domain within the GmNLR-ID85 in soybean.…”

Show me your ID: NLR immune receptors with integrated domains in plants

“…All the identified and studied IDs potentially reflect the original targets of the pathogens at the subcellular level so far. This indicates that these integrated domains can be used as a toolbox toward the identification of the original effector targets in the host cells as well as novel host susceptibility components that could be utilized further [ 70 , 71 ].…”

Engineering effector‐triggered immunity in rice: Obstacles and perspectives

“…This prompted the search for effectors that interact with the identified IDs in the NLR repertoire. An ID toolkit comprising 52 IDs representing 31 distinct Pfam domains from seven plant species, including rice, was developed to screen for interactions with 31 conserved Ralstonia solanacearum type III effectors (Landry et al, 2021). Three interactions involving different IDs (kinase, DUF3542 and an atypical WRKY sequence, WRKYGKR) and two type III effectors, RipAE and PopP2, were determined (Landry et al, 2021).…”

“…An ID toolkit comprising 52 IDs representing 31 distinct Pfam domains from seven plant species, including rice, was developed to screen for interactions with 31 conserved Ralstonia solanacearum type III effectors (Landry et al, 2021). Three interactions involving different IDs (kinase, DUF3542 and an atypical WRKY sequence, WRKYGKR) and two type III effectors, RipAE and PopP2, were determined (Landry et al, 2021). However, no interaction between effectors and IDs from rice or another monocot species, Brachypodium distachyon , was identified.…”