Role of N-Glycosylation Sites and CXC Motifs in Trafficking of Medicago truncatula Nod Factor Perception Protein to Plasma Membrane

Lefebvre, Benoît; Klaus-Heisen, Doerte; Pietraszewska-Bogiel, Anna; Hervé, Christine; Camut, Sylvie; Auriac, Marie‐Christine; Gasciolli, Virginie; Nurisso, Alessandra; Gadella, Theodorus W. J.; Cullimore, Julie V.

doi:10.1074/jbc.m111.281634

Cited by 52 publications

(49 citation statements)

References 56 publications

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“…Similar findings were also obtained for their orthologs in Arabidopsis (Willmann et al, 2011). Recently, it has been revealed that the Nglycosylation of NFP, the LysM-RLK for Nod factor perception, was not essential for its biological activity including the ligand binding (Lefebvre et al, 2012). Our work and others thus suggest a role of N-glycosylation in regulating the protein trafficking of these receptors.…”

Section: Discussionsupporting

confidence: 72%

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

et al. 2012

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Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif-containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN-or chitin-but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity.

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Section: Discussionsupporting

confidence: 72%

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

et al. 2012

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“…Glycosylation was also detected in the NFP protein from transgenic Medicago by polyacrylamide gel analysis (38). Together with our detailed MS analysis of glycan structures, this observation indicates that NFRs have highly N-glycosylated ectodomains although a functional role could not be assigned in complementation studies using NFP (34). Overall our N-glycan analysis of NFR5 indicates that the NFR5 protein is folded and processed in both expression systems.…”

Section: Discussionmentioning

confidence: 50%

“…Both NFR1 and NFR5 have six cysteines arranged in three pairs in their processed ectodomains and disulfide bridges between them probably affect the structure of the ectodomains as recently shown for CERK1 (33). Highlighting the importance of these cysteine pairs, amino acid substitutions in the corresponding cysteines of the NFP protein were shown to be functionally significant in complementation studies (34). Detailed analysis of the N-glycan structure of NFR5 by tandem mass spectrometry revealed two complex structures with identical sugar compositions (Man 3 XylFucGlcNAc 4 ) at N68 and N198.…”

Section: Discussionmentioning

confidence: 99%

Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

Broghammer

Krusell

Blaise

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

317

228

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Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man 3 XylFucGlcNAc 4 , were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor-ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The K d values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes.lysin motif proteins | plant-microbe interactions | symbiotic signalling | lysin motif receptor-like kinase | non-self recognition F ormation of nitrogen-fixing root nodules relies on an intriguing signal exchange between the legume host and the bacterial microsymbiont (1). In this two-way exchange, Nod factors synthesized by rhizobia function as central signal molecules, which induce early physiological responses, gene expression, and cell division in susceptible legumes (1-3). In root cells of the legume hosts, perception is mediated by Nod factor receptors (NFRs) containing ectodomains with three lysin motif (LysM) modules and cytoplasmic serine/threonine kinase domains (4-9). Mutant studies in the model legumes Lotus japonicus (Lotus) and Medicago truncatula (Medicago) as well as the crop legumes soybean (10, 11) and pea (5, 6) have shown that NFRs are required for nodulation. Mutant analysis in Lotus identified two NFRs, NFR1 and NFR5, and phenotypic analysis showed that both nfr1 and nfr5 mutants are equally impaired in nodule initiation (4, 5). In Medicago, only mutation of the Nfr5 ortholog, Nfp, results in a nonnodulating phenotype, whereas the Nfr1 homolog Lyk3 is required for progression of infection threads (7-9, 12).The earliest plant responses to Nod factor, such as membrane depolarization, cytoplasmic alkalinization, calcium fluxes, calcium spiking, and root hair deformation (3, 13), are either strongly at...

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“…Moreover, even in the case of the one N-glycosylation site, Asn-389 in the N389-C390-T391A motif, whose elimination did abrogate the SI response, our results indicated that it was the structural disruption caused by the T391A mutation rather than the lack of an N-glycan chain at this site that caused breakdown of SI. Thus, SRKb is more similar to the A. thaliana FLS2 (Sun et al, 2012) and the Medicago truncatula NFP (Lefebvre et al, 2012) receptor-like kinases, which are insensitive to disruption of its N-glycosylation sites, than to the A. thaliana EFR and tomato (Solanum lycopersicum) Cf-9 receptors, which are inactivated by disruption of even single N-glycosylation sites (van der Hoorn et al, 2005;Häweker et al, 2010;Saijo, 2010).…”

Section: Discussionmentioning

confidence: 99%

Site-SpecificN-Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae

et al. 2014

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The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allelespecific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single-and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.

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Role of N-Glycosylation Sites and CXC Motifs in Trafficking of Medicago truncatula Nod Factor Perception Protein to Plasma Membrane

Cited by 52 publications

References 56 publications

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity

Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

Site-SpecificN-Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae

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