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The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1.

Cao Y, Liang Y, Tanaka K, Nguyen CT, Jedrzejczak RP, Joachimiak A, Stacey G - Elife (2014)

Bottom Line: Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1.Mutations in AtLYK5 resulted in a significant reduction in chitin response.The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, National Center for Soybean Biotechnology, University of Missouri, Columbia, United States.

ABSTRACT
Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.

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Computational model of the extracellular domain of AtLYK5.(A–C) The docking model of the ectodomain with chitooctaose shown in surface (A) and ribbon form (B) and a close-up surface (C). The binding affinity was calculated at −8.9 kcal mol−1. The model shows the three AtLYK5 LysM domains, that is, LysM1-3. Each LysM domain contains two beta strands and two helixes interconnected via loops. (D–E) Docking of chitooctaose to the ECP6. (D) A ribbon structure represents the docking model of ECP6 (gray color) and chitooctaose (blue, red and yellow sticks). The binding affinity was calculated at −9.0 kcal mol−1. (E) A molecular surface of ECP6 with chitooctaose binding site formed by 3 LysM motifs.DOI:http://dx.doi.org/10.7554/eLife.03766.015
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fig4s1: Computational model of the extracellular domain of AtLYK5.(A–C) The docking model of the ectodomain with chitooctaose shown in surface (A) and ribbon form (B) and a close-up surface (C). The binding affinity was calculated at −8.9 kcal mol−1. The model shows the three AtLYK5 LysM domains, that is, LysM1-3. Each LysM domain contains two beta strands and two helixes interconnected via loops. (D–E) Docking of chitooctaose to the ECP6. (D) A ribbon structure represents the docking model of ECP6 (gray color) and chitooctaose (blue, red and yellow sticks). The binding affinity was calculated at −9.0 kcal mol−1. (E) A molecular surface of ECP6 with chitooctaose binding site formed by 3 LysM motifs.DOI:http://dx.doi.org/10.7554/eLife.03766.015

Mentions: The AtCERK1 crystal structure predicted chitooctaose binding to the second LysM motif of the extracellular domain leading to homodimerization and kinase activation (Liu et al., 2012b). However, AtCERK1 appears to be a very weak chitin binding protein raising questions as to the biological relevance of the AtCERK1 homodimer model. Therefore, in order to predict the chitin binding site(s) within the AtLYK5 extracellular domain, a computational model of the AtLYK5 ectodomain was built by homology modeling against the known crystal structure of the fungal ECP6 (Sanchez-Vallet et al., 2013), a LysM effector protein, which binds chitin with very high affinity (binding at pM levels; Figure 4A,B and Figure 4—figure supplement 1). Based on the docking model of AtLYK5 with chitooctaose, the binding affinity was calculated at −8.9 kcal mol−1 (Figure 4A,B), a value comparable to the computational binding affinity of ECP6 (−9.0 kcal mol−1) (Figure 4—figure supplement 1). Four residues, that is, Thr-72, Tyr-128, Ser-206, and Ser-216, were predicted to form hydrogen bonds and hydrophobic interactions with chitooctaose based on docking model (Figure 4—figure supplement 1). Point mutations were introduced at each of these residues and transgenically expressed in Atlyk5 mutant plants from the native promoter. As shown in Figure 4C, AtLYK5S206P and AtLYK5Y128G transgenic plants could not rescue the Atlyk5-2 mutant phenotype as measured by chitin-triggered ROS production. In contrast, expression of AtLYK5T72G and AtLYK5S216P mutant proteins in the Atlyk5-2 mutant plants did restore the chitin response. Consistent with these results, AtLYK5S206P mutant proteins did not bind to chitin beads, while AtLYK5Y128G mutant proteins showed a strong reduction in chitin binding using this same assay (Figure 4D). Binding of the AtLYK5T72G and AtLYK5S216P mutant proteins to the chitin beads was similar to wild-type AtLYK5 (Figure 4D). These data indicate that residues Tyr-128 and Ser-206 of AtLYK5 are important for chitin binding and that chitin binding is essential for biological activity.10.7554/eLife.03766.014Figure 4.Tyr-128 and Ser-206 are important for AtLYK5-mediated chitin response.


The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1.

Cao Y, Liang Y, Tanaka K, Nguyen CT, Jedrzejczak RP, Joachimiak A, Stacey G - Elife (2014)

Computational model of the extracellular domain of AtLYK5.(A–C) The docking model of the ectodomain with chitooctaose shown in surface (A) and ribbon form (B) and a close-up surface (C). The binding affinity was calculated at −8.9 kcal mol−1. The model shows the three AtLYK5 LysM domains, that is, LysM1-3. Each LysM domain contains two beta strands and two helixes interconnected via loops. (D–E) Docking of chitooctaose to the ECP6. (D) A ribbon structure represents the docking model of ECP6 (gray color) and chitooctaose (blue, red and yellow sticks). The binding affinity was calculated at −9.0 kcal mol−1. (E) A molecular surface of ECP6 with chitooctaose binding site formed by 3 LysM motifs.DOI:http://dx.doi.org/10.7554/eLife.03766.015
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4356144&req=5

fig4s1: Computational model of the extracellular domain of AtLYK5.(A–C) The docking model of the ectodomain with chitooctaose shown in surface (A) and ribbon form (B) and a close-up surface (C). The binding affinity was calculated at −8.9 kcal mol−1. The model shows the three AtLYK5 LysM domains, that is, LysM1-3. Each LysM domain contains two beta strands and two helixes interconnected via loops. (D–E) Docking of chitooctaose to the ECP6. (D) A ribbon structure represents the docking model of ECP6 (gray color) and chitooctaose (blue, red and yellow sticks). The binding affinity was calculated at −9.0 kcal mol−1. (E) A molecular surface of ECP6 with chitooctaose binding site formed by 3 LysM motifs.DOI:http://dx.doi.org/10.7554/eLife.03766.015
Mentions: The AtCERK1 crystal structure predicted chitooctaose binding to the second LysM motif of the extracellular domain leading to homodimerization and kinase activation (Liu et al., 2012b). However, AtCERK1 appears to be a very weak chitin binding protein raising questions as to the biological relevance of the AtCERK1 homodimer model. Therefore, in order to predict the chitin binding site(s) within the AtLYK5 extracellular domain, a computational model of the AtLYK5 ectodomain was built by homology modeling against the known crystal structure of the fungal ECP6 (Sanchez-Vallet et al., 2013), a LysM effector protein, which binds chitin with very high affinity (binding at pM levels; Figure 4A,B and Figure 4—figure supplement 1). Based on the docking model of AtLYK5 with chitooctaose, the binding affinity was calculated at −8.9 kcal mol−1 (Figure 4A,B), a value comparable to the computational binding affinity of ECP6 (−9.0 kcal mol−1) (Figure 4—figure supplement 1). Four residues, that is, Thr-72, Tyr-128, Ser-206, and Ser-216, were predicted to form hydrogen bonds and hydrophobic interactions with chitooctaose based on docking model (Figure 4—figure supplement 1). Point mutations were introduced at each of these residues and transgenically expressed in Atlyk5 mutant plants from the native promoter. As shown in Figure 4C, AtLYK5S206P and AtLYK5Y128G transgenic plants could not rescue the Atlyk5-2 mutant phenotype as measured by chitin-triggered ROS production. In contrast, expression of AtLYK5T72G and AtLYK5S216P mutant proteins in the Atlyk5-2 mutant plants did restore the chitin response. Consistent with these results, AtLYK5S206P mutant proteins did not bind to chitin beads, while AtLYK5Y128G mutant proteins showed a strong reduction in chitin binding using this same assay (Figure 4D). Binding of the AtLYK5T72G and AtLYK5S216P mutant proteins to the chitin beads was similar to wild-type AtLYK5 (Figure 4D). These data indicate that residues Tyr-128 and Ser-206 of AtLYK5 are important for chitin binding and that chitin binding is essential for biological activity.10.7554/eLife.03766.014Figure 4.Tyr-128 and Ser-206 are important for AtLYK5-mediated chitin response.

Bottom Line: Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1.Mutations in AtLYK5 resulted in a significant reduction in chitin response.The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, National Center for Soybean Biotechnology, University of Missouri, Columbia, United States.

ABSTRACT
Chitin is a fungal microbe-associated molecular pattern recognized in Arabidopsis by a lysin motif receptor kinase (LYK), AtCERK1. Previous research suggested that AtCERK1 is the major chitin receptor and mediates chitin-induced signaling through homodimerization and phosphorylation. However, the reported chitin binding affinity of AtCERK1 is quite low, suggesting another receptor with high chitin binding affinity might be present. Here, we propose that AtLYK5 is the primary chitin receptor in Arabidopsis. Mutations in AtLYK5 resulted in a significant reduction in chitin response. However, AtLYK5 shares overlapping function with AtLYK4 and, therefore, Atlyk4/Atlyk5-2 double mutants show a complete loss of chitin response. AtLYK5 interacts with AtCERK1 in a chitin-dependent manner. Chitin binding to AtLYK5 is indispensable for chitin-induced AtCERK1 phosphorylation. AtLYK5 binds chitin at a much higher affinity than AtCERK1. The data suggest that AtLYK5 is the primary receptor for chitin, forming a chitin inducible complex with AtCERK1 to induce plant immunity.

Show MeSH