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Functional Analysis of Chimeric Lysin Motif Domain Receptors Mediating Nod Factor-Induced Defense Signaling in Arabidopsis Thali

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Functional Analysis of Chimeric Lysin Motif Domain Receptors Mediating Nod Factor-Induced Defense Signaling in Arabidopsis Thali The Plant Journal (2014) 78, 56–69 doi: 10.1111/tpj.12450 Functional analysis of chimeric lysin motif domain receptors mediating Nod factor-induced defense signaling in Arabidopsis thaliana and chitin-induced nodulation signaling in Lotus japonicus Wei Wang1,2, Zhi-Ping Xie1,* and Christian Staehelin1,* 1State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, East Campus, Guangzhou 510006, China, and 2Anhui Key Laboratory of Plant Genetics & Breeding, School of Life Sciences, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China Received 12 October 2013; revised 11 January 2014; accepted 16 January 2014; published online 8 February 2014. *For correspondence (e-mails [email protected] or [email protected]). SUMMARY The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corre- sponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1–AtCERK1 and LjNFR5–AtCERK1, were expressed in cerk1-2, an A. thaliana CERK1 mutant lacking chitin-induced defense signaling. When treated with NFs from Rhizobi- um sp. NGR234, cerk1-2 expressing both chimeras accumulated reactive oxygen species, expressed chitin- responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor-bind- ing protein), respectively. The chimeras, named OsCERK1–LjNFR1 and OsCEBiP–LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1-1; nfr5-2) carrying a GUS (b-glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF-induced defense signaling in non-legumes and chitin-induced nodulation signaling in legumes. Keywords: defense signaling, chitin, nodulation signaling, Nod factors, LysM domain receptor, chimeric receptor, Arabidopsis thaliana, Lotus japonicus. INTRODUCTION The activation of plant defense responses against the important structural component in fungi. Chitin fragments unwanted invasion of bacteria or fungi (innate immunity) (chitooligosaccharides) released from fungal cell walls by includes recognition of microbe-associated molecular plant chitinase function as MAMPs in a similar way (Felix patterns (MAMPs) by pattern recognition receptors (PRRs). et al., 1993; Boller and Felix, 2009). An example of a bacte- Microbe-associated molecular patterns are conserved rial MAMP is peptidoglycan, which is a conserved structural molecular components from a specific class of microbes part of the bacterial cell wall and contains alternating that function as elicitors of defense signaling, resulting in residues of b-1,4-linked N-acetylglucosamine and N-acetyl- immunity to fungi and bacteria (Boller and Felix, 2009). A muramic acid residues (Gust et al., 2007). Chitin, chitooligo- typical MAMP is chitin (a polymer with b-1,4-linked N-ace- saccharides and peptidoglycan are recognized by PRRs tyl-D-glucosamine residues; poly-GlcNAc), which is an containing extracellular lysin motif (LysM) domains, which 56 © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd Chimeric LysM domain receptors 57 are known to bind to GlcNAc-containing molecules. Ligand- molecular flux through plasmodesmata, a chitin response mediated receptor activation triggers defense signaling, that is independent of AtCERK1-regulated defense gene which culminates in accumulation of reactive oxygen spe- activation (Faulkner et al., 2013). The related AtLYM1 and cies (ROS), mitogen-activated protein (MAP) kinase signal- AtLYM3 proteins are involved in peptidoglycan perception ing, activation of defense genes and ultimately in immunity and presumably form a receptor complex with AtCERK1 against invading microbes (Hamel and Beaudoin, 2010; (Willmann et al., 2011). Similar to rice, activated AtCERK1 Gust et al., 2012). initiates MAP kinase signaling (via AtMPK3/AtMPK6), In rice (Oryza sativa L.), OsCEBiP (chitin elicitor-binding which triggers the expression of chitin-responsive defense protein) and OsCERK1 (chitin elicitor receptor kinase 1) are genes (Wan et al., 2004; Miya et al., 2007). LysM domain PRRs required for the induction of chitin- Nod factors (nodulation factors; NFs) are chitin-related induced defense signaling. The chitin-binding OsCEBiP is bacterial signals that are secreted by nitrogen-fixing rhizo- a glycosylphosphatidylinositol-anchored protein, whereas bia to initiate nodule symbiosis with host legumes. Nod OsCERK1 possesses an intracellular kinase domain. Bio- factors are lipo-chitooligosaccharides, i.e. they consist of a chemical studies suggest that the two proteins form hete- carbohydrate moiety usually containing four or five rodimers in a ligand-dependent manner to initiate defense b-1,4-linked GlcNAc oligomers and an acyl residue (fatty signaling (Kaku et al., 2006; Shimizu et al., 2010). More- acid) at the non-reducing end. Furthermore, the terminal over, rice possesses additional LysM domain PRRs, namely sugars of NFs carry strain-specific modifications, which OsLYP4 and OsLYP6 (lysin motif-containing proteins 4 and may be required for nodulation of certain host plants 6), which seem to play a dual role in perception of chitin (Perret et al., 2000). Arbuscular mycorrhizal fungi pro- and peptidoglycan (Liu et al., 2012a). The OsCERK1 and duce structurally related lipo-chitooligosaccharides, the OsCEBiP receptors appear to interact with the Rho-like so-called Myc factors, but their role in establishing symbio- small GTPase OsRac1 and the guanine nucleotide exchange sis with host roots remains elusive (Maillet et al., 2011; factor OsRacGEF1, forming a so-called ‘defensome’. Phos- Genre et al., 2013). Similar to the PRRs perceiving chitin, phorylation of OsRacGEF1 by the kinase domain of chitooligosaccharides and peptidoglycan, specific LysM OsCERK1 was found to be required for downstream domain receptors of host plants recognize NFs and Myc fac- defense signaling (Akamatsu et al., 2013). Furthermore, tors to initiate symbiotic signaling (Ferguson et al., 2010; OsCERK1 phosphorylates the receptor-like cytoplasmic Oldroyd, 2013). LjNFR1 and LjNFR5 (Nod factor receptors 1 kinase OsRLCK185, which is a downstream signaling com- and 5) of Lotus japonicus are prime examples of NF recep- ponent required for activation of the chitin-induced MAP tor genes, which have been also characterized in other kinase cascade OsMKK4–OsMPK3/OsMPK6 (Kishi-Kaboshi legumes such as soybean (Glycine max) and Medicago et al., 2010; Yamaguchi et al., 2013). truncatula (Limpens et al., 2003; Madsen et al., 2003; Radu- In Arabidopsis thaliana, AtCERK1 (also called LYK1; toiu et al., 2003; Arrighi et al., 2006; Indrasumunar et al., LysM-containing receptor-like kinase 1) is a similar LysM 2010, 2011). Nod factors of Mesorhizobium loti bind to domain PRR required for chitin perception and activation LjNFR5 and LjNFR1 at nanomolar concentrations and the of chitin-responsive defense genes (Miya et al., 2007; Wan two receptors seem to form a ligand-dependent heterodi- et al., 2008). AtCERK1 possesses a functional intracellular merization complex (Madsen et al., 2011; Broghammer kinase domain and its activation does not require interac- et al., 2012). LjNFR5 interacts with the Rho-like small tion with an OsCEBiP-related protein (Shinya et al., 2012; GTPase LjROP6, which positively regulates the develop- Wan et al., 2012). Biochemical data and crystal structure ment of infection threads (Ke et al., 2012). In contrast to analysis showed that chitin and chitooligosaccharides LjNFR5, which lacks protein kinase activity, the kinase activ- [degree of polymerization (DP) = 4–8] bind directly to the ity of LjNFR1 was found to be essential for autophosphory- ectodomain of AtCERK1. Long-chain chitooligosaccharides lation and downstream signaling (Madsen et al., 2011). A [namely chitooctaose, (GlcNAc)8] and chitin act as bivalent specific component of nodulation signaling is the transcrip- ligands, having the capacity to trigger dimerization of two tion factor NIN (nodule inception), which is required for AtCERK1 ectodomains and subsequent phosphorylation of expression of early nodulin genes and subsequent initiation the kinase domain (Iizasa et al., 2010; Petutschnig et al., of nodule organogenesis (Schauser et al., 1999). The NIN 2010; Liu et al., 2012b). AtLYK4, an AtCERK1-related recep- protein of L. japonicus binds to the promoters of two NF-Y tor with a kinase domain, may assist AtCERK1-dependent (nuclear factor-Y) subunit genes, which are thought to regu- chitin signaling (Wan et al., 2012). The OsCEBiP-related late cell division in nodule primordia (Soyano et al., 2013). protein AtLYM2 (lysin motif
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