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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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Extracellular domain of FLS2 can mediate interaction with BAK1.Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA (construct lacking the FLS2 intracellular domain) and 35S–BAK1-Myc transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Samples were prepared for SDS-PAGE two days after agroinfiltration, two minutes after flg22 or water (mock) was infiltrated into leaves. IP: antibody used for immunoprecipitation prior to SDS-PAGE; WB: antibody used for immunodetection on protein blot; crude: SDS-PAGE and blotting of total (crude extract) protein samples. The experiment was repeated three times with similar results.
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pone-0111185-g001: Extracellular domain of FLS2 can mediate interaction with BAK1.Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA (construct lacking the FLS2 intracellular domain) and 35S–BAK1-Myc transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Samples were prepared for SDS-PAGE two days after agroinfiltration, two minutes after flg22 or water (mock) was infiltrated into leaves. IP: antibody used for immunoprecipitation prior to SDS-PAGE; WB: antibody used for immunodetection on protein blot; crude: SDS-PAGE and blotting of total (crude extract) protein samples. The experiment was repeated three times with similar results.

Mentions: Full-length FLS2 and BAK1 do not detectably interact until exposure to flg22 or similar flagellin ligands, at which time interaction is immediately observed [14], [15], [38]. Flg22-elicited immune signaling then requires phosphorylation events among the respective kinase domains [26], [38], [39]. We hypothesized that the FLS2-BAK1 interaction is mediated not only intracellularly by the respective kinase domains, but also by interaction of the ectodomains. To test this we used a truncated FLS2 carrying the N-terminal ∼70% of the protein including the LRR and transmembrane domains but not the predicted intracellular domains (FLS2-NoKinase-HA; [36]). FLS2-NoKinase-HA was expressed in Nicotiana benthamiana together with a plasmid encoding a full-length, epitope tagged BAK1-Myc. The transiently transformed leaves were treated with flg22 and co-immunoprecipitation experiments were performed. BAK1 and FLS2-NoKinase interact in the presence of flg22, indicating that the kinase domain of FLS2 is not needed for interaction with BAK1 in planta (Figure 1).


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

Koller T, Bent AF - PLoS ONE (2014)

Extracellular domain of FLS2 can mediate interaction with BAK1.Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA (construct lacking the FLS2 intracellular domain) and 35S–BAK1-Myc transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Samples were prepared for SDS-PAGE two days after agroinfiltration, two minutes after flg22 or water (mock) was infiltrated into leaves. IP: antibody used for immunoprecipitation prior to SDS-PAGE; WB: antibody used for immunodetection on protein blot; crude: SDS-PAGE and blotting of total (crude extract) protein samples. The experiment was repeated three times with similar results.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111185-g001: Extracellular domain of FLS2 can mediate interaction with BAK1.Co-immunoprecipitation experiments performed using 35S–FLS2-NoKinase-HA (construct lacking the FLS2 intracellular domain) and 35S–BAK1-Myc transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Samples were prepared for SDS-PAGE two days after agroinfiltration, two minutes after flg22 or water (mock) was infiltrated into leaves. IP: antibody used for immunoprecipitation prior to SDS-PAGE; WB: antibody used for immunodetection on protein blot; crude: SDS-PAGE and blotting of total (crude extract) protein samples. The experiment was repeated three times with similar results.
Mentions: Full-length FLS2 and BAK1 do not detectably interact until exposure to flg22 or similar flagellin ligands, at which time interaction is immediately observed [14], [15], [38]. Flg22-elicited immune signaling then requires phosphorylation events among the respective kinase domains [26], [38], [39]. We hypothesized that the FLS2-BAK1 interaction is mediated not only intracellularly by the respective kinase domains, but also by interaction of the ectodomains. To test this we used a truncated FLS2 carrying the N-terminal ∼70% of the protein including the LRR and transmembrane domains but not the predicted intracellular domains (FLS2-NoKinase-HA; [36]). FLS2-NoKinase-HA was expressed in Nicotiana benthamiana together with a plasmid encoding a full-length, epitope tagged BAK1-Myc. The transiently transformed leaves were treated with flg22 and co-immunoprecipitation experiments were performed. BAK1 and FLS2-NoKinase interact in the presence of flg22, indicating that the kinase domain of FLS2 is not needed for interaction with BAK1 in planta (Figure 1).

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