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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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FLS2 residues Q530, S554, Q627 and N674 are important for FLS2-BAK1 ectodomain interaction in the presence of flg22.Co-immunoprecipitation experiments performed in N. benthamiana with 35S–FLS2-NoKinase-HA with mutations as indicated or WT (no mutations), and with 35S–BAK1-Myc. Flg22-dependent interaction between FLS2-NoKinase and BAK1 not detected for (A) FLS2 carrying Q530A, Q530W or S554W mutations, (B) FLS2 carrying N674A, N674W or Q627W mutations, or (C) FLS2 carrying Q530A+Q627A or S554W+N674W double mutations. Labeling as in Figure 1.
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pone-0111185-g005: FLS2 residues Q530, S554, Q627 and N674 are important for FLS2-BAK1 ectodomain interaction in the presence of flg22.Co-immunoprecipitation experiments performed in N. benthamiana with 35S–FLS2-NoKinase-HA with mutations as indicated or WT (no mutations), and with 35S–BAK1-Myc. Flg22-dependent interaction between FLS2-NoKinase and BAK1 not detected for (A) FLS2 carrying Q530A, Q530W or S554W mutations, (B) FLS2 carrying N674A, N674W or Q627W mutations, or (C) FLS2 carrying Q530A+Q627A or S554W+N674W double mutations. Labeling as in Figure 1.

Mentions: For FLS2 amino acids predicted in the crystal structure to form FLS2-BAK1 interaction sites [27], we changed single residues to alanine (small and relatively inactive) or to tryptophan (bulky). In addition to the single mutations we made two FLS2-NoKinase constructs with double mutations and one full-length FLS2 construct with a double mutation. We had previously shown that the FLS2-NoKinase used in this work performed similarly to FLS2-full-length in flg22-dependent BAK1 co-immunoprecipitation experiments (Figure 3A, B). In in vivo tests of the newly predicted FLS2-BAK1 interaction sites, mutation of FLS2 residues Q530, S554, Q627 or N674 to tryptophan disrupted the flg22-stimulated interaction of FLS2-NoKinase with BAK1 (Figure 5A, B). The interaction was disrupted as well when FLS2 Q530 and N674 were changed to alanine (Figure 5A, B). However, the FLS2 S554 and Q627 single mutations to alanine had much less impact on flg22-dependent interaction with BAK1 (Figure 5A, B), suggesting a stronger role for Q530 and N674 than S554 or Q627 in mediating FLS2-BAK1 interaction. The double alanine mutation Q530A+Q627A and the double tryptophan mutation S554W+Q627W disrupted BAK1 interaction as well (Figure 5C). The presence of abundant EndoH-insensitive bands suggested that FLS2 maturation had proceeded successfully for each of the representative FLS2 mutants S554A, Q627W and Q530A+Q627A (Figure S2C).


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

Koller T, Bent AF - PLoS ONE (2014)

FLS2 residues Q530, S554, Q627 and N674 are important for FLS2-BAK1 ectodomain interaction in the presence of flg22.Co-immunoprecipitation experiments performed in N. benthamiana with 35S–FLS2-NoKinase-HA with mutations as indicated or WT (no mutations), and with 35S–BAK1-Myc. Flg22-dependent interaction between FLS2-NoKinase and BAK1 not detected for (A) FLS2 carrying Q530A, Q530W or S554W mutations, (B) FLS2 carrying N674A, N674W or Q627W mutations, or (C) FLS2 carrying Q530A+Q627A or S554W+N674W double mutations. Labeling as in Figure 1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111185-g005: FLS2 residues Q530, S554, Q627 and N674 are important for FLS2-BAK1 ectodomain interaction in the presence of flg22.Co-immunoprecipitation experiments performed in N. benthamiana with 35S–FLS2-NoKinase-HA with mutations as indicated or WT (no mutations), and with 35S–BAK1-Myc. Flg22-dependent interaction between FLS2-NoKinase and BAK1 not detected for (A) FLS2 carrying Q530A, Q530W or S554W mutations, (B) FLS2 carrying N674A, N674W or Q627W mutations, or (C) FLS2 carrying Q530A+Q627A or S554W+N674W double mutations. Labeling as in Figure 1.
Mentions: For FLS2 amino acids predicted in the crystal structure to form FLS2-BAK1 interaction sites [27], we changed single residues to alanine (small and relatively inactive) or to tryptophan (bulky). In addition to the single mutations we made two FLS2-NoKinase constructs with double mutations and one full-length FLS2 construct with a double mutation. We had previously shown that the FLS2-NoKinase used in this work performed similarly to FLS2-full-length in flg22-dependent BAK1 co-immunoprecipitation experiments (Figure 3A, B). In in vivo tests of the newly predicted FLS2-BAK1 interaction sites, mutation of FLS2 residues Q530, S554, Q627 or N674 to tryptophan disrupted the flg22-stimulated interaction of FLS2-NoKinase with BAK1 (Figure 5A, B). The interaction was disrupted as well when FLS2 Q530 and N674 were changed to alanine (Figure 5A, B). However, the FLS2 S554 and Q627 single mutations to alanine had much less impact on flg22-dependent interaction with BAK1 (Figure 5A, B), suggesting a stronger role for Q530 and N674 than S554 or Q627 in mediating FLS2-BAK1 interaction. The double alanine mutation Q530A+Q627A and the double tryptophan mutation S554W+Q627W disrupted BAK1 interaction as well (Figure 5C). The presence of abundant EndoH-insensitive bands suggested that FLS2 maturation had proceeded successfully for each of the representative FLS2 mutants S554A, Q627W and Q530A+Q627A (Figure S2C).

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