LYR3, a high‐affinity LCO‐binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor

Abstract

Edited by Julian Schroeder LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a highaffinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/F€ orster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.Keywords: FLIM/FRET; lipo-chitooligosaccharides; LysM-RLK; Medicago truncatula; membrane receptor complex; phosphoproteomics The extracellular domains of plant RLKs are built up of different protein domains which serve as sensors for endogenous and outer stimuli, as diverse as peptides, hormones, (peptido-) glycans, and (lipo-) chitooligosaccharides. Two prevalent extracellular protein domains consist of either leucine-rich repeats (LRRs) or lysin motifs (LysMs), which might interact with peptides or with N-acetyl-D-glucosamine (GlcNAc)-containing compounds, respectively [1,2].Recent advances in functional and structural analyses of plant RLKs, particularly in Arabidopsis, suggest that plant RLKs act in multimeric complexes [3]. For example, the LysM-RLK chitin elicitor receptor kinase 1 of A. thaliana (AtCERK1) interacts with other receptor proteins to mediate responses to different signals. This protein can bind chitooligosaccharides (COs) [4], but it also interacts with two CO-binding LysM-RLKs with dead kinase domains (AtLYK5/ AtLYK4) [5] and with two peptidoglycan-binding LysM receptor-like proteins (AtLYM1/AtLYM3) [6] to mediate defense responses to these GlcNAc-containing signals. In contrast, OsCERK1 from rice

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“…PCR products were purified on agarose gel with Wizard SV Gel and PCR Clean-Up System (Promega) and were cloned in a pBIN19 Gateway expression vector pBin19-35S-GW-YFP (Froidure et al, 2010), (kindly provided by Susana Rivas, LIPM Toulouse) in order to create fusion proteins with YFP in N-terminal part of the protein; the effector domain of the protein is free to interact with other molecules and potentially fully functional. CRN37 coding sequence deleted from its first 19 amino acids was fused to a peptide tag GFP under the control of 35S promoter and cloned in one step in the expression vector pCAMBIA-CR1 (Fliegmann et al, 2016) using the cloning method “Golden gate” (Engler et al, 2008, 2009). pCAMBIA-CR1 vector containing GFP under the control of 35S promoter was used as positive control for PhCRN37 transient expression experiments.…”

Know your enemy, embrace your friend: using omics to understand how plants respond differently to pathogenic and mutualistic microorganisms

“…LysM receptors are a subclass of membrane-bound plant pattern recognition proteins that have multiple repeats of the LysM domain in the extracellular portion of the protein attached to a cytoplasmic kinase signaling domain. Early characterization of these proteins found that certain LysM receptor kinases, namely NFR1 and NFR5, have a role in perceiving secreted lipochitooligosaccharides (LCOs; called 'Nod factors') from rhizobial bacteria (Amor et al, 2003;Limpens et al, 2003;Madsen et al, 2003;Malkov et al, 2016), and in inducing the initial signaling response by the plant necessary to establish the formation of these mutualistic root nodules Fliegmann et al, 2016). AM fungi also produce LCOs that are perceived by plant LysM receptors and induce a signaling cascade within the plant that fosters symbiosis (Maillet et al, 2011;Malkov et al, 2016;Carotenuto et al, 2017).…”

Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development

“…The transcriptional reporter pMtBG2:GUS, pNIN:GUS, pERN1: GUS, the pEXPA:PV cd (PV cd , parvalbumin mutated in EF hand motif, Addgene, reference Plasmid #16345):NLS:CFP [54], pEXPA:YFP, pEXPA:mCherry-ER and the pERN1:MtBG2 RNAi constructs were obtained by Golden-Gate cloning in pCambiaCR1 derived vectors [55]. DNA fragments corresponding to 1400 bp or 2100 bp upstream the ATG of NIN, 1705 bp upstream the ATG of MtBG2 (Medtr3g083580), 2200 bp upstream the ATG of ERN1, 401 bp of upstream EXPA sequences [30] were amplified by PCR, cloned in pbluescript vectors and sequenced before concatenated using Golden-Gate assembly reactions [55]. The pERN1: cals3m, pMtBG2:cals3m and pANN1:cals3m constructs were generated by combining restriction enzyme and gateway cloning.…”