Receptor-mediated chitin perception in legume roots is functionally separable from Nod factor perception Zoltan Bozsokia,1, Jeryl Chenga,1, Feng Fengb,1, Kira Gysela, Maria Vinthera, Kasper R. Andersena, Giles Oldroydb,2, Mickael Blaisea,3, Simona Radutoiua, and Jens Stougaarda,2 aCentre for Carbohydrate Recognition and Signalling, Department of Molecular Biology and Genetics, University of Aarhus, DK–8000 Aarhus, Denmark; and bJohn Innes Centre, Norwich NR4 7UH, United Kingdom Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved August 17, 2017 (received for review April 26, 2017) The ability of root cells to distinguish mutualistic microbes from germination and prepenetration hyphal branching in AM fungi pathogens is crucial for plants that allow symbiotic microorganisms to and hyphal growth toward the root surface (28–30). AM fungi infect and colonize their internal root tissues. Here we show that appear to signal to their host plant via short-chain chitin oligo- Lotus japonicus and Medicago truncatula possess very similar LysM mers (CO4 and CO5), and a mixture of lipochitooligosaccharides pattern-recognition receptors, LjLYS6/MtLYK9 and MtLYR4, enabling with structural similarities to rhizobial Nod factors has also been root cells to separate the perception of chitin oligomeric microbe- reported to be involved (31, 32). Receptors for these different associated molecular patterns from the perception of lipochitin signals have not yet been identified in legumes, but results from oligosaccharide by the LjNFR1/MtLYK3 and LjNFR5/MtNFP receptors Parasponia and tomato suggest that LysM receptor proteins may triggering symbiosis. Inactivation of chitin-receptor genes in Ljlys6, be involved, if not in the presymbiotic phase, then later during Mtlyk9,andMtlyr4 mutants eliminates early reactive oxygen species root colonization (33, 34). responses and induction of defense-response genes in roots. Ljlys6, Proteins carrying LysM domains were also identified as recep- Mtlyk9,andMtlyr4 mutants were also more susceptible to fungal and tors of pathogen-associated molecular patterns (PAMPs) that trigger plant defense reactions to prevent or limit microbial in- bacterial pathogens, while infection and colonization by rhizobia and vasion, a process called “PAMP-triggered immunity” (PTI) (35). In arbuscular mycorrhizal fungi was maintained. Biochemical binding rice and Arabidopsis chitin is perceived by LysM proteins such as studies with purified LjLYS6 ectodomains further showed that at least OsCEBiP and/or LysM receptor kinases OsCERK1, AtCERK1, six GlcNAc moieties (CO6) are required for optimal binding efficiency. and AtLYK5 (36–41). Similarly, two Arabidopsis LysM proteins, The 2.3-Å crystal structure of the LjLYS6 ectodomain reveals three βααβ LYM1 and LYM3, together with AtCERK1, serve in a receptor LysM motifs similar to other LysM proteins and a conserved complex perceiving peptidoglycan (42). However, this distinction chitin-binding site. Theseresultsshowthatdistinctreceptorsetsin between LysM receptors recognizing PAMPs and inducing PTI legume roots respond to chitin and lipochitin oligosaccharides found and LysM receptors recognizing symbiotic signals appears too in the heterogeneous mixture of chitinaceous compounds originating simplistic. In rice OsCERK1 mediates mycorrhizal infection as well from soil microbes. This establishes a foundation for genetic and bio- as chitin-triggered defense responses toward pathogens, indicating chemical dissection of the perception and the downstream responses – separating defense from symbiosis in the roots of the 80 90% of land Significance plants able to develop rhizobial and/or mycorrhizal endosymbiosis. Like 80–90% of land plants, legumes form endosymbioses with chitin perception | plant defense | symbiosis | Lotus | Medicago arbuscular mycorrhizal fungi, host endophytes, support a rhi- zosphere community, and are attacked by pathogens. The egumes have an exceptionally wide range of interactions with ability of root cells to distinguish between these soil microbes Lbacteria and fungi. They form endosymbioses with rhizobia and the mixture of chitinaceous compounds they display as and arbuscular mycorrhizal (AM) fungi, host endophytes, support signal molecules is important for an appropriate plant re- a rhizosphere community, and, like other plants, are attacked by a sponse. We show that legumes possess very similar receptors – plethora of pathogens (1 7). Detailed studies in the two model enabling root cells to separate perception of chitin, which species Lotus japonicus (hereafter, Lotus)andMedicago truncatula triggers responses to pathogens, from perception of lipochitin (hereafter, Medicago) have identified central components of the oligosaccharides (Nod factors), which trigger endosymbiosis signal exchange initiating the mutual recognition processes that with rhizobial bacteria. The chitin receptors bind chitin in bio- establish symbiosis with rhizobia and mycorrhiza. Legume roots chemical assays, and inactivation of the corresponding genes secrete (iso)flavonoids that activate the NodD transcriptional impairs defense responses toward pathogens. Together this regulator of rhizobial Nod genes (8, 9). This activation leads to establishes a long-sought foundation for dissecting plants’ re- synthesis of lipochitooligosaccharide signal molecules (Nod fac- sponse mechanisms toward different soil microbes. tors) that are perceived by a Nod factor receptor complex con- sisting of the transmembrane LysM receptor kinase proteins Author contributions: Z.B., J.C., F.F., K.G., M.V., K.R.A., G.O., M.B., S.R., and J.S. designed LjNFR1/MtLYK3 and LjNFR5/MtNFP in Lotus and Medicago, research; Z.B., J.C., F.F., K.G., M.V., M.B., and S.R. performed research; Z.B., J.C., F.F., K.G., – K.R.A., G.O., M.B., S.R., and J.S. analyzed data; S.R. and J.S. wrote the paper; and K.R.A., respectively (10 19). Genetic and biochemical analyses have shown G.O., M.B., S.R., and J.S. conceived and interpreted the work. that LjNFR1 and LjNFR5 bind Nod factors at physiologically The authors declare no conflict of interest. relevant concentrations (20) and initiate signal transduction; one of This article is a PNAS Direct Submission. the first consequences of this is nuclear-associated calcium oscil- Data deposition: The atomic coordinates and structure factors have been deposited in the lations (spiking) in epidermal root cells, which can be detected Protein Data Bank, www.wwpdb.org (PDB ID code 5LS2). – within minutes (21 23). In Lotus the compatibility of exopoly- 1Z.B., J.C., and F.F. contributed equally to this work. saccharides is subsequently assessed by LjEPR3, another LysM 2To whom correspondence may be addressed. Email: [email protected], or stougaard@ receptor kinase that is required for the formation of infection mbg.au.dk. – threads (24 26). Interestingly, exopolysaccharides also appear to 3Present address: Institut de Recherche en Infectiologie de Montpellier, CNRS, UMR 9004, play a role in endophyte invasion (6, 27). Université de Montpellier, 34090 Montpellier, France. Mycorrhiza formation is similarly guided by mutual signal This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. exchange. Strigolactone secreted by plant roots induces spore 1073/pnas.1706795114/-/DCSupplemental. E8118–E8127 | PNAS | Published online September 5, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1706795114 Downloaded by guest on September 29, 2021 PNAS PLUS a functional overlap (43, 44). OsCERK1 may thus serve as a ligands. Kd values were subsequently calculated from the binding component in different receptor complexes triggering different curves (SI Appendix,Fig.S1) and are listed in Table 1. The Kd signal pathways. Adding further complexity, in Arabidopsis values show LjLYS6 ectodomain affinity for chitohexaose (CO6), AtLYK3 was reported to mediate suppression of defense reac- chitoheptaose (CO7), and chitooctaose (CO8) in the lower mi- tions by lipochitin oligosaccharides (LCOs) that have no endo- cromolar range (∼50 μM). Binding to chitotetraose (CO4) was symbiosis with mycorrhizal fungi or rhizobia (45). Taken together, undetectable, whereas Kd values for chitopentaose (CO5) the results from Arabidopsis, rice, and legumes highlight our limited approached the detection limit. These direct binding measure- insight into the composition and mechanisms of plant receptor ments suggest that at least six GlcNAc residues (CO6) are re- complexes that distinguish chitinaceous signal molecules. Since quired for optimal binding. Stronger binding was not apparent PAMP receptors and defense reactions were primarily studied in with CO7 and CO8. Wheat germ agglutinin binds to all chitin leaves, our insight into roots that must accommodate rhizobial ligands with comparable Kd values in the low-micromolar range. and/or mycorrhizal symbioses is even less. In legume roots both LCOs and COs induce calcium spiking LjLYS6 Crystal Structure. The purified LjLYS6 ectodomain from throughthecommonsymbiosispathwayinadose-andLCO residues Lys27 to Ser223 was crystallized. The structure was structure-dependent manner. MtNFP was not required for CO- solved by molecular replacement using the structure of the induced calcium spiking, and the corresponding LjNFR5 receptor AtCERK1 ectodomain [Protein Data Bank (PDB) ID code: from Lotus has a higher affinity for LCO than for CO, implying a 4EBZ] as a search model and refined to 2.3-Å resolution. The difference in LCO and CO perception. This raises the