Cautionary Notes on the Use of C-Terminal BAK1 Fusion Proteins for Functional Studies

Ntoukakis, Vardis; Schwessinger, Benjamin; Segonzac, Cécile; Zipfel, Cyril

doi:10.1105/tpc.111.090779

Cited by 60 publications

(53 citation statements)

References 43 publications

(79 reference statements)

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“…Flg22 perception initiates downstream immune responses including production of reactive oxygen species (ROS), phosphorylation and activation of mitogen-activated protein kinases (MAPKs), and transcriptional changes (Nicaise et al, 2009;Tena et al, 2011;Monaghan and Zipfel, 2012), and increasing evidence suggests that these signaling events constitute a signaling network rather than a single linear pathway (Lu et al, 2009;Korasick et al, 2010;Tena et al, 2011). The regulatory RLK BRI1-ASSOCIATED KINASE1 (BAK1) forms a flg22-induced complex with FLS2 within seconds and functions very early in flg22 response pathways because BAK1 is required for all known downstream flg22-signaling responses (Chinchilla et al, 2007(Chinchilla et al, , 2009Heese et al, 2007;Schulze et al, 2010;Ntoukakis et al, 2011). The importance of FLS2-dependent responses in plant immunity is underlined by the fact that in the absence of functional FLS2, cells exhibit enhanced susceptibility to bacterial infection Li et al, 2005;Hann and Rathjen, 2007;Zeng and He, 2010).…”

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confidence: 99%

Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2

et al. 2013

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FLAGELLIN-SENSING2 (FLS2) is the plant cell surface receptor that perceives bacterial flagellin or flg22 peptide, initiates flg22-signaling responses, and contributes to bacterial growth restriction. Flg22 elicitation also leads to ligand-induced endocytosis and degradation of FLS2 within 1 h. Why plant cells remove this receptor precisely at the time during which its function is required remains mainly unknown. Here, we assessed in planta flg22-signaling competency in the context of ligand-induced degradation of endogenous FLS2 and chemical interference known to impede flg22-dependent internalization of FLS2 into endocytic vesicles. Within 1 h after an initial flg22 treatment, Arabidopsis (Arabidopsis thaliana) leaf tissue was unable to reelicit flg22 signaling in a ligand-, time-, and dose-dependent manner. These results indicate that flg22-induced degradation of endogenous FLS2 may serve to desensitize cells to the same stimulus (homologous desensitization), likely to prevent continuous signal output upon repetitive flg22 stimulation. In addition to impeding ligand-induced FLS2 degradation, pretreatment with the vesicular trafficking inhibitors Wortmannin or Tyrphostin A23 impaired flg22-elicited reactive oxygen species production that was partially independent of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1. Interestingly, these inhibitors did not affect flg22-induced mitogenactivated protein kinase phosphorylation, indicating the ability to utilize vesicular trafficking inhibitors to target different flg22-signaling responses. For Tyrphostin A23, reduced flg22-induced reactive oxygen species could be separated from the defect in FLS2 degradation. At later times (.2 h) after the initial flg22 elicitation, recovery of FLS2 protein levels positively correlated with resensitization to flg22, indicating that flg22-induced new synthesis of FLS2 may prepare cells for a new round of monitoring the environment for flg22.

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confidence: 99%

Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2

et al. 2013

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“…To explain this, we propose that a negative regulator is out-titrated, rendering the roots almost constitutively sensitive to BRs. Alternatively, the GFP tag on the C-terminal end of BRI1 may interfere with the interaction with downstream targets, similar to what has been demonstrated for BAK1 (SERK3), one of the BRI1 coreceptors (Ntoukakis et al, 2011). In modeling terms, both mechanisms would result in a change in half-maximum response values.…”

Section: Discussionmentioning

confidence: 78%

A Mathematical Model for BRASSINOSTEROID INSENSITIVE1-Mediated Signaling in Root Growth and Hypocotyl Elongation

Esse¹,

Mourik

Stigter

et al. 2012

Plant Physiology

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(S.v.M., J.M.) Brassinosteroid (BR) signaling is essential for plant growth and development. In Arabidopsis (Arabidopsis thaliana), BRs are perceived by the BRASSINOSTEROID INSENSITIVE1 (BRI1) receptor. Root growth and hypocotyl elongation are convenient downstream physiological outputs of BR signaling. A computational approach was employed to predict root growth solely on the basis of BRI1 receptor activity. The developed mathematical model predicts that during normal root growth, few receptors are occupied with ligand. The model faithfully predicts root growth, as observed in bri1 loss-of-function mutants. For roots, it incorporates one stimulatory and two inhibitory modules, while for hypocotyls, a single inhibitory module is sufficient. Root growth as observed when BRI1 is overexpressed can only be predicted assuming that a decrease occurred in the BRI1 half-maximum response values. Root growth appears highly sensitive to variation in BR concentration and much less to reduction in BRI1 receptor level, suggesting that regulation occurs primarily by ligand availability and biochemical activity.

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“…We performed FRET-APB measurements, which only detect hetero-FRET between GFP and mCherry, with GFP-and mCherry-tagged versions of the proteins. Although C-terminal fusions to BAK1 impair the protein's signaling capacity, ligand-dependent complex formation with FLS2 is not impaired by the tag (25). We measured donor dequenching, which we quantified as apparent FRET efficiency (ap.…”

Section: Mfis Is Used In Mammalian and Plant Cells To Study Molecularmentioning

confidence: 99%

Real-time dynamics of peptide ligand–dependent receptor complex formation in planta

Somssich

Weidtkamp‐Peters

et al. 2015

Sci. Signal.

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The CLAVATA (CLV) and flagellin (flg) signaling pathways act through peptide ligands and closely related plasma membrane-localized receptor-like kinases (RLKs). The plant peptide CLV3 regulates stem cell homeostasis, whereas the bacterial flg22 peptide elicits defense responses. We applied multiparameter fluorescence imaging spectroscopy (MFIS) to characterize the dynamics of RLK complexes in the presence of ligand in living plant cells expressing receptor proteins fused to fluorescent proteins. We found that the CLV and flg pathways represent two different principles of signal transduction: flg22 first triggered RLK heterodimerization and later assembly into larger complexes through homomerization. In contrast, CLV receptor complexes were preformed, and ligand binding stimulated their clustering. This different behavior likely reflects the nature of these signaling pathways. Pathogen-triggered flg signaling impedes plant growth and development; therefore, receptor complexes are formed only in the presence of ligand. In contrast, CLV3-dependent stem cell homeostasis continuously requires active signaling, and preformation of receptor complexes may facilitate this task.

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Cautionary Notes on the Use of C-Terminal BAK1 Fusion Proteins for Functional Studies

Cited by 60 publications

References 43 publications

Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2

Sensitivity to Flg22 Is Modulated by Ligand-Induced Degradation and de Novo Synthesis of the Endogenous Flagellin-Receptor FLAGELLIN-SENSING2

A Mathematical Model for BRASSINOSTEROID INSENSITIVE1-Mediated Signaling in Root Growth and Hypocotyl Elongation

Real-time dynamics of peptide ligand–dependent receptor complex formation in planta

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