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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 EFR LRR domain disrupt EFR glycosylation and interaction with BAK1 in the presence of elf18.(A) Co-immunoprecipitation experiments performed using PEFR-EFR-HA with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc, in Nicotiana benthamiana. (B, C) Protein extracts from plants expressing PEFR-EFR-HA with mutations as indicated, or WT (no mutations), not digested or digested with endoglycosidase H (EndoH). Samples in (B) are from Nicotiana benthamiana, samples in (C) are from stably transformed efr −Arabidopsis leaves. EndoH-resistant (mature) EFR is present in the EndoH-treated EFR wild type (WT) samples but is not detected for EFRs carrying the indicated mutations. Degly.: EFR pool deglycosylated by EndoH. Labeling as in Figure 1. Ponceau: blots treated with Ponceau stain to confirm even loading of total protein.
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pone-0111185-g004: Mutations in the conserved C-terminal region of the EFR LRR domain disrupt EFR glycosylation and interaction with BAK1 in the presence of elf18.(A) Co-immunoprecipitation experiments performed using PEFR-EFR-HA with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc, in Nicotiana benthamiana. (B, C) Protein extracts from plants expressing PEFR-EFR-HA with mutations as indicated, or WT (no mutations), not digested or digested with endoglycosidase H (EndoH). Samples in (B) are from Nicotiana benthamiana, samples in (C) are from stably transformed efr −Arabidopsis leaves. EndoH-resistant (mature) EFR is present in the EndoH-treated EFR wild type (WT) samples but is not detected for EFRs carrying the indicated mutations. Degly.: EFR pool deglycosylated by EndoH. Labeling as in Figure 1. Ponceau: blots treated with Ponceau stain to confirm even loading of total protein.

Mentions: Mutations analogous to those of the preceding section were also engineered into EFR. These LRR domain C-terminal region mutations (Figure 2; Figure S1C, D) did cause disruption of interaction with BAK1 in the presence of elf18 (Figure 4A). This in vivo result could be attributable to direct impacts of the mutations on EFR-BAK1 interaction, or to defects in maturation and delivery of newly synthesized EFR out of the endoplasmic reticulum (ER) and golgi. Endoglycosidase H (EndoH) analyses were therefore conducted. EndoH cleaves incomplete glycosylation modifications present on proteins that have not successfully passed through the ER and related endomembrane systems [41], [42]. On the other hand, mature glycosylated proteins that are delivered to their functional location typically carry EndoH-resistant glycosylation [41], [42]. Treatment of the EFR protein extracts with EndoH revealed defects in the mutated EFR proteins, both in N. benthamiana and in stable transgenic Arabidopsis efr- plants expressing transgene EFR constructs driven by native EFR promoter sequences (Figure 4B, C). The mutations we generated in FLS2 full-length and FLS2-NoKinase did not result in glycosylation defects (Figure S2A, B). Häweker et al. 2010 [42] and Sun at al. 2012 [36] showed that single amino acid changes in glycosylation sites in the EFR ectodomain result in protein degradation and several studies reported the importance of intact glycosylation enzymes for successful processing and function of EFR [43]–[47]. FLS2 is less sensitive to mutations in glycosylation sites [36], [42]. The N590Q+S592T mutations that we placed in EFR are indeed in a Nx(S/T) predicted glycosylation site [48]. However, the EFR mutations D566E+S568T and D566F are not, yet they still disrupted correct EFR processing.


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 EFR LRR domain disrupt EFR glycosylation and interaction with BAK1 in the presence of elf18.(A) Co-immunoprecipitation experiments performed using PEFR-EFR-HA with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc, in Nicotiana benthamiana. (B, C) Protein extracts from plants expressing PEFR-EFR-HA with mutations as indicated, or WT (no mutations), not digested or digested with endoglycosidase H (EndoH). Samples in (B) are from Nicotiana benthamiana, samples in (C) are from stably transformed efr −Arabidopsis leaves. EndoH-resistant (mature) EFR is present in the EndoH-treated EFR wild type (WT) samples but is not detected for EFRs carrying the indicated mutations. Degly.: EFR pool deglycosylated by EndoH. Labeling as in Figure 1. Ponceau: blots treated with Ponceau stain to confirm even loading of total protein.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214723&req=5

pone-0111185-g004: Mutations in the conserved C-terminal region of the EFR LRR domain disrupt EFR glycosylation and interaction with BAK1 in the presence of elf18.(A) Co-immunoprecipitation experiments performed using PEFR-EFR-HA with mutations as indicated or WT (no mutations), and 35S–BAK1-Myc, in Nicotiana benthamiana. (B, C) Protein extracts from plants expressing PEFR-EFR-HA with mutations as indicated, or WT (no mutations), not digested or digested with endoglycosidase H (EndoH). Samples in (B) are from Nicotiana benthamiana, samples in (C) are from stably transformed efr −Arabidopsis leaves. EndoH-resistant (mature) EFR is present in the EndoH-treated EFR wild type (WT) samples but is not detected for EFRs carrying the indicated mutations. Degly.: EFR pool deglycosylated by EndoH. Labeling as in Figure 1. Ponceau: blots treated with Ponceau stain to confirm even loading of total protein.
Mentions: Mutations analogous to those of the preceding section were also engineered into EFR. These LRR domain C-terminal region mutations (Figure 2; Figure S1C, D) did cause disruption of interaction with BAK1 in the presence of elf18 (Figure 4A). This in vivo result could be attributable to direct impacts of the mutations on EFR-BAK1 interaction, or to defects in maturation and delivery of newly synthesized EFR out of the endoplasmic reticulum (ER) and golgi. Endoglycosidase H (EndoH) analyses were therefore conducted. EndoH cleaves incomplete glycosylation modifications present on proteins that have not successfully passed through the ER and related endomembrane systems [41], [42]. On the other hand, mature glycosylated proteins that are delivered to their functional location typically carry EndoH-resistant glycosylation [41], [42]. Treatment of the EFR protein extracts with EndoH revealed defects in the mutated EFR proteins, both in N. benthamiana and in stable transgenic Arabidopsis efr- plants expressing transgene EFR constructs driven by native EFR promoter sequences (Figure 4B, C). The mutations we generated in FLS2 full-length and FLS2-NoKinase did not result in glycosylation defects (Figure S2A, B). Häweker et al. 2010 [42] and Sun at al. 2012 [36] showed that single amino acid changes in glycosylation sites in the EFR ectodomain result in protein degradation and several studies reported the importance of intact glycosylation enzymes for successful processing and function of EFR [43]–[47]. FLS2 is less sensitive to mutations in glycosylation sites [36], [42]. The N590Q+S592T mutations that we placed in EFR are indeed in a Nx(S/T) predicted glycosylation site [48]. However, the EFR mutations D566E+S568T and D566F are not, yet they still disrupted correct EFR processing.

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