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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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Mutations in the conserved C-terminal region of the FLS2 LRR domain did not have an impact on BAK1-FLS2 or BAK1-FLS2-NoKinase interaction.(A) Co-immunoprecipitation experiments performed using full-length PFLS2-FLS2-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. (B) Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. All samples in (A) and (B) are from Nicotiana benthamiana. Labeling as in Figure 1.
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pone-0111185-g003: Mutations in the conserved C-terminal region of the FLS2 LRR domain did not have an impact on BAK1-FLS2 or BAK1-FLS2-NoKinase interaction.(A) Co-immunoprecipitation experiments performed using full-length PFLS2-FLS2-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. (B) Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. All samples in (A) and (B) are from Nicotiana benthamiana. Labeling as in Figure 1.

Mentions: Site-directed mutagenesis was carried out to alter residues in the identified conserved LRR C-terminal region of FLS2 and EFR (Figure 2; Figure S1A–E). D557 and S559 mutations in FLS2 were included as control mutations located in LRR sites analogous to N704/S706 and D728/S730, but outside of the conserved LRR C-terminus. The amino acids were replaced with similar yet bulkier residues in order to impair interactions. The resulting full-length receptors were expressed in N. benthamiana and co-immunoprecipitation experiments were then carried out, using BAK1-Myc for pull-down in the presence and absence of the corresponding ligands flg22 and elf18 in the case of FLS2 or EFR, respectively. The mutations in FLS2 did not abolish the interaction with BAK1 in the presence of flg22 (Figure 3A). As is common for agroinfiltration experiments, variable levels of expression were observed for any single transgene-encoded FLS2 protein across replicates within or between experiments, but none of the mutant proteins was reproducibly present at levels different from transgene-encoded wild-type FLS2. To ensure that interaction of the kinase domains of FLS2 and BAK1 was not masking non-interaction of mutated FLS2 and BAK1 ectodomains, the same mutations were also placed into FLS2-NoKinase constructs. In these FLS2-NoKinase variants the mutations again did not prevent interaction with BAK1 (Figure 3B).


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

Koller T, Bent AF - PLoS ONE (2014)

Mutations in the conserved C-terminal region of the FLS2 LRR domain did not have an impact on BAK1-FLS2 or BAK1-FLS2-NoKinase interaction.(A) Co-immunoprecipitation experiments performed using full-length PFLS2-FLS2-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. (B) Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. All samples in (A) and (B) are from Nicotiana benthamiana. 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-g003: Mutations in the conserved C-terminal region of the FLS2 LRR domain did not have an impact on BAK1-FLS2 or BAK1-FLS2-NoKinase interaction.(A) Co-immunoprecipitation experiments performed using full-length PFLS2-FLS2-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. (B) Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA, with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc. All samples in (A) and (B) are from Nicotiana benthamiana. Labeling as in Figure 1.
Mentions: Site-directed mutagenesis was carried out to alter residues in the identified conserved LRR C-terminal region of FLS2 and EFR (Figure 2; Figure S1A–E). D557 and S559 mutations in FLS2 were included as control mutations located in LRR sites analogous to N704/S706 and D728/S730, but outside of the conserved LRR C-terminus. The amino acids were replaced with similar yet bulkier residues in order to impair interactions. The resulting full-length receptors were expressed in N. benthamiana and co-immunoprecipitation experiments were then carried out, using BAK1-Myc for pull-down in the presence and absence of the corresponding ligands flg22 and elf18 in the case of FLS2 or EFR, respectively. The mutations in FLS2 did not abolish the interaction with BAK1 in the presence of flg22 (Figure 3A). As is common for agroinfiltration experiments, variable levels of expression were observed for any single transgene-encoded FLS2 protein across replicates within or between experiments, but none of the mutant proteins was reproducibly present at levels different from transgene-encoded wild-type FLS2. To ensure that interaction of the kinase domains of FLS2 and BAK1 was not masking non-interaction of mutated FLS2 and BAK1 ectodomains, the same mutations were also placed into FLS2-NoKinase constructs. In these FLS2-NoKinase variants the mutations again did not prevent interaction with BAK1 (Figure 3B).

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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Related in: MedlinePlus