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FLS2-BAK1 extracellular domain interaction sites required for defense signaling activation.

Koller T, Bent AF - PLoS ONE (2014)

Bottom Line: However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects.In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs.However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.

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Repeat Conservation Mapping reveals conserved region near C-terminus of LRR domains of FLS2, EFR, BRI1 and PEPR1.Each row represents one leucine-rich repeat (LRR) and each square represents one solvent-exposed “x” amino acid position (as per LRR consensus sequence shown at the top). Conservation score at each amino acid position is center-weighted score for the cluster of 15, 20 or 25 predicted solvent-exposed LRR amino acids surrounding that site; blue: least conserved, red: most conserved. For FLS2, the seven rows of (A) are the same repeats (same residues) as rows 21–27 of (B). (A) Conservation map generated by comparing the most C-terminal seven repeats of the LRR sequences of the BAK1 interacting proteins FLS2, EFR, BRI1 and PEPR1. (B) Conservation map generated by comparing the entire FLS2 LRR domain sequences from eleven non-Brassicaceae plant species.
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pone-0111185-g002: Repeat Conservation Mapping reveals conserved region near C-terminus of LRR domains of FLS2, EFR, BRI1 and PEPR1.Each row represents one leucine-rich repeat (LRR) and each square represents one solvent-exposed “x” amino acid position (as per LRR consensus sequence shown at the top). Conservation score at each amino acid position is center-weighted score for the cluster of 15, 20 or 25 predicted solvent-exposed LRR amino acids surrounding that site; blue: least conserved, red: most conserved. For FLS2, the seven rows of (A) are the same repeats (same residues) as rows 21–27 of (B). (A) Conservation map generated by comparing the most C-terminal seven repeats of the LRR sequences of the BAK1 interacting proteins FLS2, EFR, BRI1 and PEPR1. (B) Conservation map generated by comparing the entire FLS2 LRR domain sequences from eleven non-Brassicaceae plant species.

Mentions: The SERK family members have been shown to interact with several different transmembrane LRR-RLKs involved in plant immunity and development. It is not known if the SERK interaction sites of these receptors evolved independently or originate from a common and potentially conserved SERK interaction site. We hypothesized the latter and also hypothesized that, to facilitate spatial proximity of potentially interacting extracellular domains, the relatively small ectodomains of SERK proteins would interact near the C-terminal end of the large LRR ectodomains of those partner receptors. Using Repeat Conservation Mapping [35] we searched the last seven repeats of the LRRs of the known Arabidopsis BAK1-interacting proteins FLS2 (28 total repeats in the LRR domain), EFR (21 LRRs), BRI1 (25 LRRs) and PEPR1 (26 LRRs), looking for the patch of solvent-exposed amino acids in this region that is most conserved across the four proteins. A conserved region of interest was identified (Figure 2A). Separately, we compared the solvent exposed amino acids of the whole LRR domains of eleven non-Brassicaceae FLS2s (Figure 2B). Both conservation maps revealed a conserved region at a similar location in the C-terminal LRRs. We hypothesized that this may be a somewhat universal site for interaction with SERK proteins.


FLS2-BAK1 extracellular domain interaction sites required for defense signaling activation.

Koller T, Bent AF - PLoS ONE (2014)

Repeat Conservation Mapping reveals conserved region near C-terminus of LRR domains of FLS2, EFR, BRI1 and PEPR1.Each row represents one leucine-rich repeat (LRR) and each square represents one solvent-exposed “x” amino acid position (as per LRR consensus sequence shown at the top). Conservation score at each amino acid position is center-weighted score for the cluster of 15, 20 or 25 predicted solvent-exposed LRR amino acids surrounding that site; blue: least conserved, red: most conserved. For FLS2, the seven rows of (A) are the same repeats (same residues) as rows 21–27 of (B). (A) Conservation map generated by comparing the most C-terminal seven repeats of the LRR sequences of the BAK1 interacting proteins FLS2, EFR, BRI1 and PEPR1. (B) Conservation map generated by comparing the entire FLS2 LRR domain sequences from eleven non-Brassicaceae plant species.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214723&req=5

pone-0111185-g002: Repeat Conservation Mapping reveals conserved region near C-terminus of LRR domains of FLS2, EFR, BRI1 and PEPR1.Each row represents one leucine-rich repeat (LRR) and each square represents one solvent-exposed “x” amino acid position (as per LRR consensus sequence shown at the top). Conservation score at each amino acid position is center-weighted score for the cluster of 15, 20 or 25 predicted solvent-exposed LRR amino acids surrounding that site; blue: least conserved, red: most conserved. For FLS2, the seven rows of (A) are the same repeats (same residues) as rows 21–27 of (B). (A) Conservation map generated by comparing the most C-terminal seven repeats of the LRR sequences of the BAK1 interacting proteins FLS2, EFR, BRI1 and PEPR1. (B) Conservation map generated by comparing the entire FLS2 LRR domain sequences from eleven non-Brassicaceae plant species.
Mentions: The SERK family members have been shown to interact with several different transmembrane LRR-RLKs involved in plant immunity and development. It is not known if the SERK interaction sites of these receptors evolved independently or originate from a common and potentially conserved SERK interaction site. We hypothesized the latter and also hypothesized that, to facilitate spatial proximity of potentially interacting extracellular domains, the relatively small ectodomains of SERK proteins would interact near the C-terminal end of the large LRR ectodomains of those partner receptors. Using Repeat Conservation Mapping [35] we searched the last seven repeats of the LRRs of the known Arabidopsis BAK1-interacting proteins FLS2 (28 total repeats in the LRR domain), EFR (21 LRRs), BRI1 (25 LRRs) and PEPR1 (26 LRRs), looking for the patch of solvent-exposed amino acids in this region that is most conserved across the four proteins. A conserved region of interest was identified (Figure 2A). Separately, we compared the solvent exposed amino acids of the whole LRR domains of eleven non-Brassicaceae FLS2s (Figure 2B). Both conservation maps revealed a conserved region at a similar location in the C-terminal LRRs. We hypothesized that this may be a somewhat universal site for interaction with SERK proteins.

Bottom Line: However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects.In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs.However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology, University of Wisconsin - Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Signaling initiation by receptor-like kinases (RLKs) at the plasma membrane of plant cells often requires regulatory leucine-rich repeat (LRR) RLK proteins such as SERK or BIR proteins. The present work examined how the microbe-associated molecular pattern (MAMP) receptor FLS2 builds signaling complexes with BAK1 (SERK3). We first, using in vivo methods that validate separate findings by others, demonstrated that flg22 (flagellin epitope) ligand-initiated FLS2-BAK1 extracellular domain interactions can proceed independent of intracellular domain interactions. We then explored a candidate SERK protein interaction site in the extracellular domains (ectodomains; ECDs) of the significantly different receptors FLS2, EFR (MAMP receptors), PEPR1 (damage-associated molecular pattern (DAMP) receptor), and BRI1 (hormone receptor). Repeat conservation mapping revealed a cluster of conserved solvent-exposed residues near the C-terminus of models of the folded LRR domains. However, site-directed mutagenesis of this conserved site in FLS2 did not impair FLS2-BAK1 ECD interactions, and mutations in the analogous site of EFR caused receptor maturation defects. Hence this conserved LRR C-terminal region apparently has functions other than mediating interactions with BAK1. In vivo tests of the subsequently published FLS2-flg22-BAK1 ECD co-crystal structure were then performed to functionally evaluate some of the unexpected configurations predicted by that crystal structure. In support of the crystal structure data, FLS2-BAK1 ECD interactions were no longer detected in in vivo co-immunoprecipitation experiments after site-directed mutagenesis of the FLS2 BAK1-interaction residues S554, Q530, Q627 or N674. In contrast, in vivo FLS2-mediated signaling persisted and was only minimally reduced, suggesting residual FLS2-BAK1 interaction and the limited sensitivity of co-immunoprecipitation data relative to in vivo assays for signaling outputs. However, Arabidopsis plants expressing FLS2 with the Q530A+Q627A double mutation were impaired both in detectable interaction with BAK1 and in FLS2-mediated responses, lending overall support to current models of FLS2 structure and function.

Show MeSH
Related in: MedlinePlus