A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence

Abstract

Plants sense potential microbial invaders by using patternrecognition receptors to recognize pathogen-associated molecular patterns (PAMPs) 1 . In Arabidopsis thaliana, the leucine-rich repeat receptor kinases flagellin-sensitive 2 (FLS2) (ref.2) and elongation factor Tu receptor (EFR) (ref.3) act as pattern-recognition receptors for the bacterial PAMPs flagellin 4 and elongation factor Tu (EF-Tu) (ref. 5) and contribute to resistance against bacterial pathogens. Little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. Here we show that BAK1 (BRI1-associated receptor kinase 1), a leucinerich repeat receptor-like kinase that has been reported to regulate the brassinosteroid receptor BRI1 (refs 6,7), is involved in signalling by FLS2 and EFR. Plants carrying bak1 mutations show normal flagellin binding but abnormal early and late flagellintriggered responses, indicating that BAK1 acts as a positive regulator in signalling. The bak1-mutant plants also show a reduction in early, but not late, EF-Tu-triggered responses. The decrease in responses to PAMPs is not due to reduced sensitivity to brassinosteroids. We provide evidence that FLS2 and BAK1 form a complex in vivo, in a specific ligand-dependent manner, within the first minutes of stimulation with flagellin. Thus, BAK1 is not only associated with developmental regulation through the plant hormone receptor BRI1 (refs 6,7), but also has a functional role in PRR-dependent signalling, which initiates innate immunity.PAMPs have key roles as activators of the innate immune response in animals 8 and, analogously, as 'general elicitors' of defence responses in plants [1][2][3][4][5][9][10][11] . We have previously characterized FLS2 and EFR as the pattern-recognition receptors (PRRs) for flagellin (represented by a 22-amino-acid peptide, flg22) and for EF-Tu (represented by the peptides elf18 and elf26, which correspond to its amino terminus), respectively 2-5,10 . Flagellin and EF-Tu rapidly induce a common set of Arabidopsis genes for leucine-rich repeat receptor-like kinases (LRR-RLKs), including FLS2 and EFR themselves 3,11 . This led to the assumption that some of these PAMPinduced LRR-RLKs might encode additional components of PAMP perception or signalling. Using a reverse genetic approach, we tested a collection of insertional mutants in these LRR-RLKs (previously used for identification of the EFR gene 3 ) for responsiveness to flg22 and found that two mutants with insertions in the LRR-RLK gene At4g33430 have reduced sensitivity to flg22 in seedling growth assays (Fig. 1a,b). In more than 10 repetitions of seedling growth assays with these mutants, we always observed a clear reduction (but never a complete loss) of sensitivity to flg22 and flg22-related peptides. By contrast, the mutants seemed to be as sensitive as the wild type to treatment with elf18 in more than five seedling growth assays (Fig. 1b and data not shown).

Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

“…Taken together, these results suggest that Pto -elicited ROS was only in part dependent on BAK1 . Our studies are consistent with previous reports showing that full signaling responses to the bacterial PAMP peptides flg22 require other proteins in addition to BAK1 [18,20,42]. …”

Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

“…Taken together, these results suggest that Pto -elicited ROS was only in part dependent on BAK1 . Our studies are consistent with previous reports showing that full signaling responses to the bacterial PAMP peptides flg22 require other proteins in addition to BAK1 [18,20,42]. …”

Interplay between calcium signalling and early signalling elements during defence responses to microbe‐ or damage‐associated molecular patterns

“…Similarly, MAPK activation, a downstream response to [Ca 2+ ] cyt elevations, was clearly reduced and delayed (for flg22 and elf18) or slightly reduced (for Pep1) in bak1‐4 upon elicitation but not upon ch8 elicitation (Figure 3f). Hence, these findings are in agreement with the published role of BAK1 in early signalling for FLS2, EFR (Chinchilla et al. , 2007) and PEPR1/2 (Krol et al.…”

“…To assess if the other SERKs are involved in early MAMP and DAMP signalling, the aequorin transgene was introduced into serk1‐1 , serk2‐1 , serk4‐1 and serk5‐1 . All four mutants showed normal [Ca 2+ ] cyt responses and ROS accumulation upon flg22 and elf18 elicitation (Figure 4a–d), thereby confirming earlier reports (Chinchilla et al. , 2007; Heese et al.…”

Dual Regulation of Gene Expression Mediated by Extended MAPK Activation and Salicylic Acid Contributes to Robust Innate Immunity in Arabidopsis thaliana

“…We compared the duration of MAPK activation in ETI and PTI. When wild-type seedlings in a liquid medium were treated with the PTI inducer flg22, activation of the MAPKs was observed after 10 min and returned to the basal level within one hour (Figure 3A), confirming previous observations [28]. The possibility that flg22 was rapidly degraded in the liquid culture was excluded since the MAPKs were activated similarly when fresh seedlings were placed in the liquid medium containing flg22 that had been incubated with other seedlings for 3 hours (Figure 3A).…”

Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

“…Taken together, these results suggest that Pto -elicited ROS was only in part dependent on BAK1 . Our studies are consistent with previous reports showing that full signaling responses to the bacterial PAMP peptides flg22 require other proteins in addition to BAK1 [18,20,42]. …”

Interplay between calcium signalling and early signalling elements during defence responses to microbe‐ or damage‐associated molecular patterns

“…Similarly, MAPK activation, a downstream response to [Ca 2+ ] cyt elevations, was clearly reduced and delayed (for flg22 and elf18) or slightly reduced (for Pep1) in bak1‐4 upon elicitation but not upon ch8 elicitation (Figure 3f). Hence, these findings are in agreement with the published role of BAK1 in early signalling for FLS2, EFR (Chinchilla et al. , 2007) and PEPR1/2 (Krol et al.…”

“…To assess if the other SERKs are involved in early MAMP and DAMP signalling, the aequorin transgene was introduced into serk1‐1 , serk2‐1 , serk4‐1 and serk5‐1 . All four mutants showed normal [Ca 2+ ] cyt responses and ROS accumulation upon flg22 and elf18 elicitation (Figure 4a–d), thereby confirming earlier reports (Chinchilla et al. , 2007; Heese et al.…”

Dual Regulation of Gene Expression Mediated by Extended MAPK Activation and Salicylic Acid Contributes to Robust Innate Immunity in Arabidopsis thaliana

“…We compared the duration of MAPK activation in ETI and PTI. When wild-type seedlings in a liquid medium were treated with the PTI inducer flg22, activation of the MAPKs was observed after 10 min and returned to the basal level within one hour (Figure 3A), confirming previous observations [28]. The possibility that flg22 was rapidly degraded in the liquid culture was excluded since the MAPKs were activated similarly when fresh seedlings were placed in the liquid medium containing flg22 that had been incubated with other seedlings for 3 hours (Figure 3A).…”

Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae

“…Taken together, these results suggest that Pto -elicited ROS was only in part dependent on BAK1 . Our studies are consistent with previous reports showing that full signaling responses to the bacterial PAMP peptides flg22 require other proteins in addition to BAK1 [18,20,42]. …”

Interplay between calcium signalling and early signalling elements during defence responses to microbe‐ or damage‐associated molecular patterns

“…Similarly, MAPK activation, a downstream response to [Ca 2+ ] cyt elevations, was clearly reduced and delayed (for flg22 and elf18) or slightly reduced (for Pep1) in bak1‐4 upon elicitation but not upon ch8 elicitation (Figure 3f). Hence, these findings are in agreement with the published role of BAK1 in early signalling for FLS2, EFR (Chinchilla et al. , 2007) and PEPR1/2 (Krol et al.…”

“…To assess if the other SERKs are involved in early MAMP and DAMP signalling, the aequorin transgene was introduced into serk1‐1 , serk2‐1 , serk4‐1 and serk5‐1 . All four mutants showed normal [Ca 2+ ] cyt responses and ROS accumulation upon flg22 and elf18 elicitation (Figure 4a–d), thereby confirming earlier reports (Chinchilla et al. , 2007; Heese et al.…”

Dual Regulation of Gene Expression Mediated by Extended MAPK Activation and Salicylic Acid Contributes to Robust Innate Immunity in Arabidopsis thaliana

“…We compared the duration of MAPK activation in ETI and PTI. When wild-type seedlings in a liquid medium were treated with the PTI inducer flg22, activation of the MAPKs was observed after 10 min and returned to the basal level within one hour (Figure 3A), confirming previous observations [28]. The possibility that flg22 was rapidly degraded in the liquid culture was excluded since the MAPKs were activated similarly when fresh seedlings were placed in the liquid medium containing flg22 that had been incubated with other seedlings for 3 hours (Figure 3A).…”