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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 signaling output impaired to various degrees in Arabidopsis fls2− plants expressing FLS2 mutations that impact FLS2-BAK1 interaction.(A) Reactive oxygen species (ROS) production in response to flg22 in Arabidopsis Col-0 fls2− plants stably transformed to express full-length FLS2 proteins carrying single mutations as noted, under control of FLS2 promoter sequences. For each mutation, ROS production was recorded for 30 min. and the average for seven separately monitored leaf discs is shown for each of four independent transgenic lines (or three lines for S554W). WT: Average ROS response for six independent fls2− transformants expressing wild-type FLS2 (42 total leaf discs for WT), from same experiment. (B) FLS2-mediated seedling growth inhibition (SGI) in response to flg22, for plant lines as in (A). Mean and std. error of mean shown for six to eight independent transformants for each FLS2 construct. (C) ROS experiment as in (A), except with five independent lines expressing FLS2 Q530A+Q627A double mutations. (D) Seedling growth experiment as in (B), except with twelve independent lines expressing Q530A+Q627A double mutations.
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pone-0111185-g006: FLS2 signaling output impaired to various degrees in Arabidopsis fls2− plants expressing FLS2 mutations that impact FLS2-BAK1 interaction.(A) Reactive oxygen species (ROS) production in response to flg22 in Arabidopsis Col-0 fls2− plants stably transformed to express full-length FLS2 proteins carrying single mutations as noted, under control of FLS2 promoter sequences. For each mutation, ROS production was recorded for 30 min. and the average for seven separately monitored leaf discs is shown for each of four independent transgenic lines (or three lines for S554W). WT: Average ROS response for six independent fls2− transformants expressing wild-type FLS2 (42 total leaf discs for WT), from same experiment. (B) FLS2-mediated seedling growth inhibition (SGI) in response to flg22, for plant lines as in (A). Mean and std. error of mean shown for six to eight independent transformants for each FLS2 construct. (C) ROS experiment as in (A), except with five independent lines expressing FLS2 Q530A+Q627A double mutations. (D) Seedling growth experiment as in (B), except with twelve independent lines expressing Q530A+Q627A double mutations.

Mentions: To investigate if mutations in predicted FLS2 BAK1-interaction residues not only disrupt FLS2-BAK1 interactions in co-immunoprecipitation experiments but also have an impact on FLS2 signaling, we made the analogous single mutations and one of the double mutations in full-length FLS2s. We then tested FLS2 signaling in stably transformed fls2− Arabidopsis plants containing the mutated and HA-tagged full-length FLS2s under control of native FLS2 promoter sequences. The two most widely used assays for FLS2 signaling were utilized: ROS burst assays and seedling growth inhibition assays [1]. Surprisingly, in vivo FLS2-mediated signaling persisted and was only minimally reduced in plants expressing most single-mutant forms of FLS2 (Figure 6A, B), including mutants that exhibited no detectable flg22-induced co-immunoprecipitation with BAK1 (Figure 5A, B). As a general trend across the multiple independent transgenic lines tested for each FLS2 construct, mutations to alanine allowed stronger FLS2 signaling than mutations to tryptophan (Figure 6A, B). The results suggest that reduced-affinity or more transient interactions of FLS2 and BAK1 occur with many of the FLS2 mutants described in Figures 5 and 6, and that those interactions are sufficient for flg22-stimulated FLS2 signaling even if the stability of FLS2-BAK1 interactions is reduced below levels detectable in standard co-immunoprecipitation experiments. Although some FLS2 signaling capacity was still conferred by FLS2 constructs mutated at single predicted FLS2 BAK1-interaction sites, with the double mutation Q530A+Q627A FLS2-mediated signaling was significantly impaired (Figure 6C, D), supporting current models of FLS2 structure and function.


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

Koller T, Bent AF - PLoS ONE (2014)

FLS2 signaling output impaired to various degrees in Arabidopsis fls2− plants expressing FLS2 mutations that impact FLS2-BAK1 interaction.(A) Reactive oxygen species (ROS) production in response to flg22 in Arabidopsis Col-0 fls2− plants stably transformed to express full-length FLS2 proteins carrying single mutations as noted, under control of FLS2 promoter sequences. For each mutation, ROS production was recorded for 30 min. and the average for seven separately monitored leaf discs is shown for each of four independent transgenic lines (or three lines for S554W). WT: Average ROS response for six independent fls2− transformants expressing wild-type FLS2 (42 total leaf discs for WT), from same experiment. (B) FLS2-mediated seedling growth inhibition (SGI) in response to flg22, for plant lines as in (A). Mean and std. error of mean shown for six to eight independent transformants for each FLS2 construct. (C) ROS experiment as in (A), except with five independent lines expressing FLS2 Q530A+Q627A double mutations. (D) Seedling growth experiment as in (B), except with twelve independent lines expressing Q530A+Q627A double mutations.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111185-g006: FLS2 signaling output impaired to various degrees in Arabidopsis fls2− plants expressing FLS2 mutations that impact FLS2-BAK1 interaction.(A) Reactive oxygen species (ROS) production in response to flg22 in Arabidopsis Col-0 fls2− plants stably transformed to express full-length FLS2 proteins carrying single mutations as noted, under control of FLS2 promoter sequences. For each mutation, ROS production was recorded for 30 min. and the average for seven separately monitored leaf discs is shown for each of four independent transgenic lines (or three lines for S554W). WT: Average ROS response for six independent fls2− transformants expressing wild-type FLS2 (42 total leaf discs for WT), from same experiment. (B) FLS2-mediated seedling growth inhibition (SGI) in response to flg22, for plant lines as in (A). Mean and std. error of mean shown for six to eight independent transformants for each FLS2 construct. (C) ROS experiment as in (A), except with five independent lines expressing FLS2 Q530A+Q627A double mutations. (D) Seedling growth experiment as in (B), except with twelve independent lines expressing Q530A+Q627A double mutations.
Mentions: To investigate if mutations in predicted FLS2 BAK1-interaction residues not only disrupt FLS2-BAK1 interactions in co-immunoprecipitation experiments but also have an impact on FLS2 signaling, we made the analogous single mutations and one of the double mutations in full-length FLS2s. We then tested FLS2 signaling in stably transformed fls2− Arabidopsis plants containing the mutated and HA-tagged full-length FLS2s under control of native FLS2 promoter sequences. The two most widely used assays for FLS2 signaling were utilized: ROS burst assays and seedling growth inhibition assays [1]. Surprisingly, in vivo FLS2-mediated signaling persisted and was only minimally reduced in plants expressing most single-mutant forms of FLS2 (Figure 6A, B), including mutants that exhibited no detectable flg22-induced co-immunoprecipitation with BAK1 (Figure 5A, B). As a general trend across the multiple independent transgenic lines tested for each FLS2 construct, mutations to alanine allowed stronger FLS2 signaling than mutations to tryptophan (Figure 6A, B). The results suggest that reduced-affinity or more transient interactions of FLS2 and BAK1 occur with many of the FLS2 mutants described in Figures 5 and 6, and that those interactions are sufficient for flg22-stimulated FLS2 signaling even if the stability of FLS2-BAK1 interactions is reduced below levels detectable in standard co-immunoprecipitation experiments. Although some FLS2 signaling capacity was still conferred by FLS2 constructs mutated at single predicted FLS2 BAK1-interaction sites, with the double mutation Q530A+Q627A FLS2-mediated signaling was significantly impaired (Figure 6C, D), supporting current models of FLS2 structure and function.

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