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Keywords:

  • Arabidopsisthaliana;
  • microbe-associated molecular patterns;
  • calcium;
  • reactive oxygen species;
  • BAK1;
  • SERK

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

While diverse microbe- or damage-associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP-specific differences in Ca2+ signalling, defence gene expression and MAMP-mediated growth arrest in Arabidopsis thaliana. Such MAMP-specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca2+ elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1-mediated signal amplification to attain MAMP/DAMP-specific Ca2+ amplitudes in wild-type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca2+ peak, demonstrating a feedback effect of ROS on Ca2+ signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca2+ response. Thus, fine-tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up- or downstream of Ca2+ signalling.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant defence is initiated through the recognition of conserved microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs) or plant-derived damage-associated molecular patterns (DAMPs) by specific pattern-recognition receptors (PRR). Activation of PRRs initiates a set of common defence responses eventually leading to immunity (reviewed in Boller and Felix, 2009). One of the earliest signalling events after MAMP/DAMP perception is a rapid change in the cytosolic Ca2+ concentration ([Ca2+]cyt) and concomitant membrane depolarization (Blume et al., 2000; Lecourieux et al., 2002; Ranf et al., 2008; Jeworutzki et al., 2010). Subsequently, the generation of reactive oxygen species (ROS) may directly confine pathogen growth via toxic effects and cell wall strengthening or may exert signalling functions (Torres et al., 2006). The Arabidopsis NADPH oxidases, RbohD and RbohF, contribute to the generation of ROS in response to pathogen attack (Torres et al., 2002). Superoxide dismutases rapidly convert membrane-impermeable superoxide (inline image), produced in the apoplast by NADPH oxidases, into hydrogen peroxide (H2O2), which can enter cytosol and nucleus to execute intracellular functions. Activation of mitogen-activated protein kinase (MAPK) cascades (MAP3K–MKK4/MKK5–MPK3/MPK6 and MEKK1–MKK1/MKK2–MPK4) and Ca2+-dependent kinases (CPK4/-5/-6/-11) leads to re-programming of gene expression (Boudsocq et al., 2010; Rodriguez et al., 2010).

The best-studied PAMP/PRR pairs in Arabidopsis to date are flagellin/FLS2 (Flagellin sensitive 2) and EF-Tu/EFR (EF-Tu receptor), with the peptides flg22 and elf18 functioning as the elicitor-active PAMPs, respectively (Felix et al., 1999; Gomez-Gomez et al., 1999; Gomez-Gomez and Boller, 2000; Kunze et al., 2004; Zipfel et al., 2006). Upon flg22 binding, FLS2 hetero-oligomerizes with another receptor-like kinase (RLK), BRI1-associated kinase 1 (BAK1), which was originally found as an interactor with the brassinosteroid receptor BRI1 (Brassinosteroid insensitive 1; Li et al., 2002; Nam and Li, 2002). While sometimes referred to as a ‘co-receptor’, BAK1 is not involved in flg22 binding but its association with FLS2 is necessary for full responsiveness (Chinchilla et al., 2007; Heese et al., 2007). BAK1 also contributes to signalling pathways activated by several other PAMPs, as well as AtPep1, a plant-derived DAMP (Shan et al., 2008; Krol et al., 2010). Similarly, further signalling components like MAPKs, CPKs and WRKY transcription factors are shared not only between multiple MAMPs/DAMPs but also by other stimuli, such as abiotic stresses, hormones and developmental cues (Kudla et al., 2010; Rodriguez et al., 2010; Rushton et al., 2010). Moreover, Ca2+ is a ubiquitous second messenger involved in nearly all aspects of plant life (Dodd et al., 2010; Kudla et al., 2010). This raises the question of how and to which extent signal specificity is maintained by common signalling components. Whereas MAPKs, CPKs and WRKY transcription factors comprise large gene families of several individuals with, for example, specific domain structures and expression patterns to determine specific outputs, Ca2+ is just a simple ion. A longstanding paradigm to explain stimulus-specific responses is the concept of the ‘Ca2+ signature’ (Webb et al., 1996), where duration, amplitude, frequency and spatial distribution are thought to encode stimulus-specific information that is decoded by various Ca2+-binding proteins, like calmodulins (CaM), Ca2+-dependent CaM-binding transcription factors (CAMTAs) and CPKs. Alternatively, Ca2+ may act as a ‘chemical on–off switch’, where Ca2+ levels beyond a certain threshold are necessary and sufficient to trigger responses (Scrase-Field and Knight, 2003; Dodd et al., 2010).

The relevance for these signalling components in immunity can be inferred from the strategies used by pathogens to interfere with their function. These include effector proteins delivered into host cells (Göhre and Robatzek, 2008) or secretion of extracellular polysaccharides that sequester apoplastic Ca2+ to impair MAMP/DAMP signalling (Aslam et al., 2008). Thus, changes in [Ca2+]cyt are one of the earliest and vital events for signalling. As the interaction and mutual relations of many early MAMP/DAMP signalling components in Arabidopsis are not well understood, these were analysed for a possible impact on Ca2+ signalling. We concentrated on such components that act in the same time frame as Ca2+ signalling, like BAK1 and homologous somatic embryogenesis receptor-like kinases (SERK), MAPKs and NADPH oxidases.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Different MAMPs and DAMPs induce specific [Ca2+]cyt elevations in Arabidopsis seedlings

As assessed by aequorin luminescence-based Ca2+ imaging, several MAMPs or DAMPs, such as bacterial flg22 and elf18, fungal N-acetylchitooctaose (ch8), and the plant-derived DAMP, Pep1, elicit [Ca2+]cyt increases in Arabidopsis seedlings (Figure 1a). Specificity is demonstrated by the lack of any [Ca2+]cyt increase in the controls, comprising either water or inactive forms of the MAMPs/DAMPs (Figure 1 and Figure S1 in Supporting Information; Felix et al., 1999; Kunze et al., 2004). Pre-treatment of seedlings with either LaCl3, a Ca2+ -channel blocker, or BAPTA, a membrane-impermeable Ca2+ chelator, completely abolished the MAMP/DAMP-induced [Ca2+]cyt elevations (Figure S2), showing that an initial influx of apoplastic Ca2+ across the plasma membrane is required for establishing the [Ca2+]cyt response.

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Figure 1.  Microbe- or damage-associated molecular patterns (MAMPs/DAMPs) induce specific elevations in cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis seedlings and roots. (a, b) The [Ca2+]cyt elevations upon application (marked by arrow) of the MAMPs/DAMPs flg22, elf18, ch8, Pep1 and water as a control in (a) intact Col-0 seedlings or (b) isolated roots were monitored over time. Data represent the mean ± SD of four or more independent experiments (n ≥ 30). Letters indicate statistically significant differences between the [Ca2+]cyt amplitudes induced by the distinct MAMPs/DAMPs, with the statistically significant groups categorized by different letters (Kruskal–Wallis/Dunn’s post-test; P < 0.001). (c) Comparison of the maximum [Ca2+]cyt amplitudes in intact seedlings (black bars), root-dissected seedlings [without (w/o) roots; grey bars] or isolated roots (shaded bars) induced by the indicated MAMPs/DAMPs (all 1 μm) or water as a control. Note that the different letters indicate statistically significant groups (Kruskal–Wallis/Dunn’s post-test, P < 0.001) within each MAMP/DAMP treatment (and not between MAMP/DAMP). (d) Expression of MAMP/DAMP receptor genes in Arabidopsis seedlings, rosettes and roots (upper panel; AT-93) or in distinct Arabidopsis root tissues (lower panel; AT-191). Data were obtained from a public database (Genevestigator v3).

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Dose–response curves showed that, with the exception of Pep1, the tested MAMPs/DAMPs reached apparent saturating concentrations for inducing [Ca2+]cyt elevations between 100 nm and 1 μm (Figure S3). The MAMPs or DAMPs, which were applied at 1 μm for general comparison, induced [Ca2+]cyt elevations with a typical pattern (Figure 1a): after a lag phase of about 40 sec (flg22/ch8) to 1 min (elf18/Pep1), the [Ca2+]cyt steeply increased, followed by a short plateau phase and a slow decline to resting level over 30–40 min. Maximum peak heights differed between the tested MAMPs/DAMPs, with flg22 having the highest [Ca2+]cyt amplitude (Figure 1a). Interestingly, two distinct peaks were detectable for flg22, whereas for elf18 and Pep1 the ‘twin peaks’, while regularly visible in individual plots (Figure S4), were merged to a prolonged plateau phase in the composite plot representing the average of more than 30 seedlings (Figure 1a).

Root- and shoot-specific differences in response to MAMPs/DAMPs

Since flagellin, EF-Tu and chitin are not specific for foliar microbes, their ability to induce [Ca2+]cyt responses in isolated roots was compared with that of the aerial seedling parts. No significant differences in amplitude or kinetics of MAMP/DAMP-induced [Ca2+]cyt elevations between intact and root-dissected seedlings (Figures 1c and S5) were found for flg22, elf18 and ch8. Pep1-induced [Ca2+]cyt amplitudes were slightly enhanced in root-dissected seedlings, probably due to wounding effects (Figures 1c and S5). Taken together, the detected flg22/elf18-induced [Ca2+]cyt elevations of intact seedlings represent those of the aerial tissues. Whereas ch8 and Pep1 induced rather similar responses in seedling shoots and roots, roots were insensitive to elf18 and showed only a minor response to flg22 (Figure 1b). The shape of the flg22-induced [Ca2+]cyt curve in roots resembled that in seedlings induced by lower flg22 concentrations (Figure S3a). Accordingly, the FLS2 and EFR genes are highly expressed in whole seedlings and rosette leaves but only marginally in roots, while CERK1 (Chitin elicitor receptor kinase 1), PEPR1/2 (Pep receptor 1/2) and BAK1 are similarly expressed in roots and aerial parts (Figure 1d). Thus, the observed MAMP/DAMP-induced [Ca2+]cyt elevations in roots mirror the expression levels of the corresponding receptors.

Plant growth arrest in the presence of MAMPs/DAMPs is well documented, but there appear to be organ-specific differences, e.g. for Pep1 (Krol et al., 2010). We therefore re-evaluated growth inhibition of Col-0 seedlings on flg22- or elf18-containing agar plates. Seedlings on flg22 plates had short roots with many lateral roots, while the shoots were smaller, sometimes with slightly yellowish leaves, and very variable in size. By contrast, seedlings on elf18 plates had tiny, dark brown cotyledons and long, thin primary roots without lateral roots (Figure 2a). Thus, flg22 mainly inhibits root growth whereas elf18 predominantly affects shoots (Figure 2b,c). Quantification, based on root length for flg22 and fresh weight for elf18 (Figure 2b,c), corroborates the [Ca2+]cyt elevation dose–response data (Figure S3) showing that maximum inhibition is attained between 100 nm and 1 μm. Hence, while at first sight many MAMP-induced responses appear similar, detailed comparison based on flg22 and elf18 revealed that MAMP-specific differences exist in both ‘late’ responses like MAMP-induced growth inhibition and ‘early’ events such as [Ca2+]cyt elevations.

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Figure 2.  Flg22- and elf18-induced growth inhibition reveals different phenotypes in Arabidopsis seedlings. (a) Col-0 seedlings were grown on agar plates ±1 μm flg22 or elf18 for 14 days and representative seedlings were photographed. Inserts show enlarged photographs of elf18-induced upper seedling parts. Similar results were obtained in three or more independent experiments. (b) Root length of Col-0 seedlings grown for 14 days on agar plates containing the indicated concentrations of flg22 (black bars) or elf18 (shaded bars). (c) Fresh weight of 5-day-old Col-0 seedlings grown for a further 15 days in liquid medium containing the indicated concentrations of flg22 (black bars) or elf18 (shaded bars).

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[Ca2+]cyt elevations induced by flg22, elf18 and Pep1, but not ch8 are delayed and reduced in bak1 mutants

To investigate the role of Ca2+signalling, several signalling components were tested for their contribution to MAMP-induced [Ca2+]cyt elevations. The receptor mutants, fls2, efr and cerk1, were insensitive to their respective MAMPs, but reacted normally to other tested MAMPs/DAMPs (Figure S6). By contrast, mutants of the BAK1‘co-receptor’ did not show complete insensitivity, but had delayed and reduced [Ca2+]cyt responses to flg22, elf18 and Pep1 (Figure 3a–c,e). The ch8-induced [Ca2+]cyt elevations in bak1 mutants, in contrast, were essentially identical to the wild type (Figure 3d). Similarly, MAPK activation, a downstream response to [Ca2+]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., 2010), but not CERK1 (Shan et al., 2008).

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Figure 3.  Elevations in cytosolic Ca2+ concentration ([Ca2+]cyt) and mitogen-activated protein kinase (MAPK) activation is delayed and reduced in bak1 seedlings upon flg22, elf18 and Pep1, but not ch8, elicitation. (a–d) Elevations of [Ca2+]cyt in bak1 seedlings induced by the indicated microbe- or damage-associated molecular patterns (MAMPs/DAMPs) compared to Col-0. Dashed lines/arrows indicate reduction of peak height in bak1-4 compared with Col-0. Black arrows mark the time of MAMP/DAMP application. Data represent the mean ± SD of four or more independent experiments (n ≥ 10). Letters indicate statistically significant differences between the [Ca2+]cyt amplitudes in the distinct genotypes, separately calculated for each MAMP/DAMP, with the statistically significant groups categorized by different letters (Kruskal–Wallis/Dunn’s post-test; P < 0.001). (e) Comparison of the maximum [Ca2+]cyt amplitudes in Col-0 or bak1 mutants induced by 1 μm flg22 (black bars), 1 μm elf18 (grey bars) or 1 μm Pep1 (shaded bars). Letters indicate statistically significant differences between [Ca2+]cyt amplitudes induced by the distinct MAMPs/DAMPs, separately calculated for each genotype, with the statistically significant groups categorized by different letters (Kruskal–Wallis/Dunn’s post-test; P < 0.001). (f) Activation of MAPK upon application of the indicated MAMPs/DAMPs in bak1-4 compared with Col-0 was analysed by anti-pTEpY western blot at indicated time points. Amido-black-stained membranes show equal loading. Three independent experiments revealed comparable results.

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Strikingly, the distinct flg22-, elf18- and Pep1-induced [Ca2+]cyt elevations in the wild type were reduced in bak1 mutants to virtually identical peak heights (Figure 3e) and shapes for all three MAMPs/DAMPs (Figure 3a–c). Different amplitudes for MAMP/DAMP-induced membrane depolarization have similarly been observed between bak1-4 and wild-type plants (Krol et al., 2010). Thus, BAK1-receptor interaction determines the distinct amplitudes of [Ca2+]cyt elevation (Figure 3a–c,e) and membrane depolarization induced by different MAMPs/DAMPs in wild-type plants, with BAK1 acting as signal amplifier to accelerate and boost the overall response. The residual responses observed in bak1 mutants might originate from signalling solely via the receptors or from partially redundant BAK1 homologues (see below) or other receptor complex constituents.

Other SERK family members do not show altered flg22/elf18-induced [Ca2+]cyt elevations and ROS accumulation, but enhanced flg22-mediated root growth arrest

BAK1 belongs to the five-membered family of somatic-embryogenesis receptor-like kinases (SERKs) and is accordingly also named SERK3 (Hecht et al., 2001). 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 [Ca2+]cyt responses and ROS accumulation upon flg22 and elf18 elicitation (Figure 4a–d), thereby confirming earlier reports (Chinchilla et al., 2007; Heese et al., 2007). While ch8-induced [Ca2+]cyt responses were not altered in any of the other serk mutants (Figure S7), Pep1 induced wild-type-like [Ca2+]cyt elevations in serk1-1, serk2-1 and serk5-1, but a slightly reduced [Ca2+]cyt increase in serk4-1 (Figure S8). Whereas none of the bak1 or serk mutants showed any differential growth arrest to elf18 compared with the wild type (Figure S9), root growth of the bak1 mutants was considerably less sensitive to flg22 (Figure 4e,f). Interestingly, flg22-mediated inhibition of root growth was significantly enhanced in serk2-1 and serk4-1 mutants, while it was comparable to the wild type for serk1-1 and serk5-1 (Figure 4e,f). As [Ca2+]cyt elevations (Figure 4a,b) and ROS accumulation (Figure 4c,d) were unaltered in both mutants, SERK2 and SERK4 probably do not exert their function in flg22 signalling at the early stages, but act further downstream in the pathway leading to growth arrest.

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Figure 4.  Flg22-mediated root growth inhibition, but not elevation in cytosolic Ca2+ concentration ([Ca2+]cyt), is enhanced in serk2-1 and serk4-1. (a, b) Elevations in [Ca2+]cyt in serk mutant seedlings induced by (a) 1 μm flg22 or (b) 1 μm elf18 compared with Col-0. For clarity, graphs are depicted offset. Arrows mark the time of microbe-associated molecular pattern (MAMP) application. Data represent the mean ± SD of three or more independent experiments (n ≥ 40). Letters indicate statistically significant differences between the [Ca2+]cyt amplitude of the mutant compared with the wild type, separately calculated for each mutant–wild-type pair, with the statistically significant groups categorized by different letters (Student’s t-test; P < 0.001). (c, d) Reactive oxygen species (ROS; H2O2) production induced by (c) 1 μm flg22 or (d) 1 μm elf18 was monitored using a luminol-based assay in leaf discs of bak1 and serk mutants compared with Col-0. Data are given as relative light units (RLU) and represent the mean ± SE of three or more independent experiments (n ≥ 20). Letters indicate statistically significant differences at the time of maximum ROS accumulation in the wild type, with the statistically significant groups categorized by different letters (Kruskal–Wallis/Dunn’s post-test; P < 0.05). (e) Root growth inhibition (1 μm flg22) of different serk mutants (black bars) each compared with its respective wild-type control (shaded bars). Data are given as percentage inhibition compared with the untreated control; mean ± SE of three or more independent experiments (n ≥ 60). *Statistically significant difference; n.s., not significant (two-way anova genotype × treatment; P < 0.001). (f) Photographs of representative Col-0 and serk mutant seedlings grown in the presence (+; two seedlings) or absence (−; one seedling) of 1 μm flg22. Similar results were obtained in three or more independent experiments.

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Taken together, unlike bak1, none of the four serk mutants showed a phenotype with regard to [Ca2+]cyt responses or ROS accumulation. Nevertheless, it cannot be excluded that additional phenotypes might only become visible in higher-order mutants. For instance, a role for SERK4 in control of cell death only became evident in the serk3serk4 double mutant (He et al., 2007).

BAK1 differentially contributes to responses induced by flg22 and elf18

To further elucidate the role of BAK1 in conferring MAMP- and DAMP-specific information, defence-related gene expression upon flg22 and elf18 elicitation was monitored. For better comparison, the protoplast-based FRK1-promoter-luciferase (pFRK1-LUC) expression assay, previously used to demonstrate differential roles of BAK1 between flg22, elf18 and chitin (Shan et al., 2008), was employed. LUC expression, driven by two additional defence-related promoters, NHL10 and PHI1 (Boudsocq et al., 2010), was also included. Interestingly, by incorporating a time-course profiling aspect, different kinetics could already be detected between flg22- and elf18-induced expression of pNHL10-LUC, pFRK1-LUC and pPHI1-LUC in wild-type protoplasts. Whereas expression of all three genes was generally more transient upon flg22 treatment, a long-lasting response was observed after elf18 application within the tested time frame (Figure 5).

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Figure 5.  Different patterns of defence gene promoter activity were observed in Col-0 and bak1 protoplasts upon flg22 and elf18 elicitation. Promoter activities of the defence genes (a, d) NHL10, (b, e) FRK1 and (c, f) PHI1 were monitored in Col-0 and bak1 protoplasts upon application of (a–c) 100 nm flg22 and (d–f) 100 nm elf18. Data are depicted as fold-induction compared with the untreated control; mean ± SE of four or more independent experiments (n ≥ 20). Letters indicate statistically significant differences at selected time points, with the statistically significant groups categorized by different letters (Kruskal–Wallis/Dunn’s post-test; P < 0.05).

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Moreover, BAK1 differentially contributed to flg22- and elf18-induced expression of pNHL10-LUC, pFRK1-LUC and pPHI1-LUC. In bak1 mutants, all three constructs showed a loss of flg22-induced expression (Figure 5a–c), whereas elf18 activated all three promoter-constructs, although with a slight delay for pNHL10 (Figure 5d–f). Two to three hours after elf18 elicitation, pNHL10 and pFRK1 activity in the bak1 mutants was even higher than in the wild type, whereas pPHI1 activity did not reach wild-type levels (Figure 5d–f). Hence, BAK1 is not or is only partially required for the early phase of elf18-induced NHL10 and FRK1 expression but is required to attenuate the expression of these two genes subsequently.

Like Chinchilla et al. (2007), we found that flg22-mediated growth inhibition is strongly reduced in bak1 seedlings compared with the wild type (Figure 4e,f), while no reduction was observed after elf18 treatment (Figure S9). In conclusion, besides the obvious distinct characteristics in the kinetics of defence gene expression and the growth inhibition phenotype upon flg22 versus elf18 application, striking differences were noticed regarding the involvement of BAK1 in both responses.

Feedback effect of MAMP- and DAMP-induced RbohD-mediated ROS accumulation on [Ca2+]cyt signalling

Another typical early response, occurring within minutes of MAMP and DAMP perception, is an accumulation of ROS. This is dependent on [Ca2+]cyt changes, since pre-treatment with LaCl3, to inhibit Ca2+ channels, also abrogates accumulation of ROS in Arabidopsis leaf discs (Figure S10). Nevertheless, some ROS, like H2O2, are themselves capable of inducing [Ca2+]cyt elevations (Pei et al., 2000; Rentel and Knight, 2004; Ranf et al., 2008). To test any possible feedback impact of ROS on [Ca2+]cyt elevations in response to different MAMPs and DAMPs, a pharmacological as well as a genetic approach was used. The NADPH oxidases rbohD and rbohF account for most of the pathogen-responsive oxidative burst (Torres et al., 2002). In agreement with published results (Zhang et al., 2007; Mersmann et al., 2010), RbohD appears to be the major source of MAMP-induced ROS since flg22/elf18-induced ROS accumulation was eliminated in the rbohD mutant (see Figure 7a,b) but not significantly altered in rbohF (Zhang et al., 2007). Interestingly, when the flg22- and elf18-induced [Ca2+]cyt elevations in aequorin-transgenic rbohD mutant lines were analysed the first Ca2+ peak was comparable to the wild type while the second peak was abolished (Figure 6a,b). The plateau-phase of the Pep1-induced [Ca2+]cyt elevation was likewise reduced in rbohD (Figure 6c). A minor phenotype was observed after ch8 elicitation (Figure 6d). Similar results were obtained with the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI; Figure 6e–h). Thus, MAMP/DAMP-induced ROS indeed have a feedback effect on the [Ca2+]cyt response by inducing an additive [Ca2+]cyt elevation causing a second peak or prolonged plateau. However, flg22-mediated root growth arrest in rbohD was comparable to the wild type (Mersmann et al., 2010). Thus, while ROS have a significant but overall minor feedback contribution to the ‘Ca2+ signature’, the later growth inhibition response is independent of this rapid ROS generation.

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Figure 7.  Microbe-associated molecular pattern (MAMP)-induced accumulation of reactive oxygen species (ROS), but not elevation in cytosolic Ca2+ concentration ([Ca2+]cyt), is prolonged in mpk3. (a, b) Production of ROS (H2O2) induced by (a) 1 μm flg22 or (b) 1 μm elf18 was monitored using a luminol-based assay in leaf discs of mpk3 and mpk6 mutants compared with Col-0 and rbohD as controls. While mpk3-1 and mpk6-3 are T-DNA insertion lines, mpk3-DG is a fast neutron deletion mutant (see Table S1). Data are given as relative light units (RLU) and represent the mean ± SE of five or more independent experiments (n ≥ 120). Letters indicate statistically significant differences, separately calculated for each time point, with the statistically significant groups categorized by different letters (Kruskal–Wallis/Dunn’s post-test; P < 0.05). (c, d) The [Ca2+]cyt elevations in mpk3 and mpk6 seedlings compared with Col-0 induced by (c) 1 μm flg22 or (d) 1 μm elf18. For clarity, graphs are depicted offset. Arrows mark the time of MAMP application. Data represent mean ± SD of four or more independent experiments (n ≥ 20). (e) Root growth inhibition (10 μm flg22) of mpk3 and mpk6 mutants (black bars) each compared with its respective wild-type control (shaded bars). Data are given as percentage inhibition compared with untreated controls; mean ± SE of four or more independent experiments (n ≥ 40). *A statistically significant difference (two-way anova genotype × treatment; P < 0.001).

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Figure 6.  Feedback effect of microbe- or damage-associated molecular pattern (MAMP/DAMP)-induced accumulation of reactive oxygen species (ROS) on elevation in cytosolic Ca2+ concentration ([Ca2+]cyt). (a–d) Elevations in [Ca2+]cyt in rbohD seedlings induced by the indicated MAMPs/DAMPs compared with Col-0. Arrows mark the time of MAMP/DAMP application. Data represent the mean ± SD of three or more independent experiments (n ≥ 20). (e–h) Elevations in [Ca2+]cyt induced by the indicated MAMPs/DAMPs in Col-0 seedlings pre-treated with 25 μm diphenylene iodonium chloride (DPI; in DMSO) or DMSO alone as control for 5 min. Arrows mark the time of MAMP/DAMP application. Data represent the mean ± SD of two or more independent experiments (n ≥ 15). Letters indicate statistically significant differences, separately calculated for each time point, with the statistically significant groups categorized by different letters (Student’s t-test; P < 0.001).

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Flg22 induces a wild type-like [Ca2+]cyt elevation, but a prolonged ROS generation in mpk3 mutants

Activation of MAPK is usually detectable 2–3 min after flg22 or elf18 elicitation (Figure 3f) and therefore occurs in the same time frame as the [Ca2+]cyt elevations. Although MAPK activation has been reported to be downstream of [Ca2+]cyt elevation in tobacco (Lecourieux et al., 2002), LaCl3 and BAPTA reduced but did not completely abolish MAPK activation in flg22-treated Arabidopsis suspension-cultured cells (Figure S11). Nevertheless, this demonstrates that MAPK activation is at least partially dependent on the [Ca2+]cyt elevation. Conversely, unlike the rbohD mutant shown above (Figure 6a,b), mutations in MPK3 or MPK6 did not significantly alter the flg22- or elf18-induced [Ca2+]cyt elevation (Figure 7c,d).

By contrast, the accumulation of ROS in leaf discs of mpk3, but not mpk6, mutants was significantly prolonged upon flg22 and elf18 elicitation (Figure 7a,b). This correlates to a mild but statistically significant enhancement of growth inhibition by flg22 in the mpk3 mutants, while the mpk6 mutant is slightly less sensitive to flg22 (Figure 7e).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Response specificity encoding in [Ca2+] amplitude or signature

A hallmark of MAMP- and DAMP-induced [Ca2+]cyt elevation, either in Arabidopsis shown here or in other systems such as parsley, tobacco and soybean cell suspensions (Mithöfer et al., 1999; Blume et al., 2000; Lecourieux et al., 2002), is [Ca2+]cyt elevations that are sustained compared with the ‘spike-like’ transient [Ca2+]cyt elevations induced by most abiotic stimuli (Knight et al., 1996, 1997; Rentel and Knight, 2004; Ranf et al., 2008). However, for valid comparison between different stimuli, nearly synchronous elicitation in the multicellular Arabidopsis seedlings should be attained. Thus, liquid-grown seedlings were used here, which, due to direct contact of the complete surface with the surrounding medium and presumably due to a less developed cuticle, facilitated MAMP/DAMP accessibility. Additionally, near saturating MAMP concentrations were applied to further reduce the variation in accessibility and to compare the maximum [Ca2+]cyt elevation-inducing capacities of different MAMPs and DAMPs. Another advantage, over leaf discs, is that intact seedlings are not ‘primed’ by or ‘refractory’ from wounding.

Under the above-mentioned conditions, differences in lag phases and [Ca2+]cyt amplitudes were observed (Figure 1). Aslam et al. (2009), who also observed different [Ca2+]cyt amplitudes, suggested that this may be accounted by differential MAMP diffusion rates through the cell wall matrix. However, such differences in diffusion rates for the relatively small peptide MAMPs or DAMPs being compared here are unlikely. Distinct lag phases for flg22 and elf18 were also observed for medium alkalinization in Arabidopsis suspension-cultured cells (Zipfel et al., 2006) and distinct MAMP/DAMP-induced membrane depolarization in leaves with the epidermis removed (Krol et al., 2010). Thus, the differences are most probably intrinsic characteristics of the receptor-mediated perception of the distinct MAMPs.

Despite the overall similarity of the [Ca2+]cyt kinetics, the lag phases and amplitudes differed between the tested MAMPs and DAMPs. It is conceivable that such qualitative, quantitative or kinetic differences in [Ca2+]cyt elevations encode information contributing to the MAMP-specific responses. On the contrary, bak1 mutants show similar MAPK activation (Figure 3f) as well as distinct downstream responses, like gene expression (Figure 5) and growth arrest (Figure 4e and S9) to different MAMPs, despite virtually identical [Ca2+]cyt elevations (Figure 3a,b,e). This strongly argues against the concept that the ‘Ca2+ signatures’ convey MAMP-specific information into downstream responses. The presented data therefore substantiate the notion of a ‘chemical on–off switch’ or ‘threshold’ function for Ca2+ in MAMP and DAMP signalling (Scrase-Field and Knight, 2003; Dodd et al., 2010).

Concerning the question of whether [Ca2+]cyt elevations encode and transmit MAMP-specific information, it should also be considered that aequorin-based [Ca2+] imaging provides an average response of whole seedlings or tissues consisting of different cell types whose individual responses can differ substantially (see below). Hence, while it may reveal differences in some cases, as illustrated by the rbohD analysis, the underlying single cell/tissue Ca2+ signatures may not always be deducible from aequorin-based [Ca2+] imaging of whole seedlings (Dodd et al., 2010).

Organ/tissue-specific differences in MAMP/DAMP responses

As the [Ca2+]cyt measurements show that both flg22 and elf18 are effectively sensed in aerial parts and only marginally in roots (Figure 1), the distinct shoot and root growth arrest phenotypes cannot be simply explained by specific perception in these tissues. Based on callose deposition and reporter gene expression, Millet et al. (2010) also reported that roots are insensitive to elf18 but react strongly to chitin all over the mature zone, whereas flg22-induced responses were only detectable in the root elongation zone (EZ). By contrast, flg22-induced membrane depolarization was also detected in root hairs (Jeworutzki et al., 2010). As FLS2 is thought to be expressed throughout the entire root (Robatzek et al., 2006) and no enhanced FLS2 transcript level is observed in the EZ (Figure 1d), the minor flg22-induced [Ca2+]cyt elevation measured in whole roots is probably due to a weak response in the entire root rather than a localized response in the EZ.

Thus, in contrast to chitin, the bacterial MAMPs flg22 and elf18 apparently only make minor contributions to PTI in roots compared with shoots. However, as illustrated by the pivotal roles of Ca2+ signalling in roots during legume–rhizobia arbuscular mycorrhizal symbiosis and beneficial interaction of Arabidopsis with the endophytic growth-promoting fungus Piriformospora indica (Harper and Harmon, 2005; Oldroyd and Downie, 2006; Navazio et al., 2007; Vadassery et al., 2009), perception of other MAMPs in roots is important for several other plant–microbe interactions.

The role of BAK1 in early and late signalling

The nearly identical [Ca2+]cyt kinetics (Figure 3a–c) and membrane-depolarization (Jeworutzki et al., 2010) induced by flg22, elf18 and Pep1 in bak1 mutants led to the hypothesis that a similar basal level of early signal transduction activation by these diverse MAMPs or DAMPs exists, and this is further differentially amplified and accelerated by the receptor-associated kinase BAK1. Using ROS accumulation and growth arrest as markers for early and late responses, respectively, Chinchilla et al. (2007) surmised that flg22-induced early and late responses are both altered in bak1, whereas BAK1 is only required for early signalling but not for late responses induced by elf18. Our current gene expression studies now suggest that BAK1 also has different regulatory functions in elf18-induced defence gene expression at early time points, well within the time frame of [Ca2+]cyt elevations and MAPK activation. Whereas [Ca2+]cyt elevations and MAPK activation revealed nearly identical phenotypes for both flg22 and elf18 in bak1 (Figure 3), different phenotypes were observed regarding gene expression in bak1 (Figure 5), although regulation of the analysed promoters was reported to be dependent on MAPK (pFRK1) or CPK activity (pPHI1) or both (pNHL10) (Boudsocq et al., 2010). Taken together, a differential involvement of BAK1 can be observed at early and late signalling phases. One possible explanation for the diverse regulatory roles of BAK1 at various signalling steps may be the association of BAK1 with receptors of numerous signalling pathways (Postel et al., 2010), as well as several receptor-like cytoplasmic kinases (Lu et al., 2010; Zhang et al., 2010) which are connected with MAMP signalling pathways. For instance, BAK1 also interacts with the Pep receptors (PEPR1/2) and this is suggested to provide a feed-forward amplification loop of MAMP signals (Postel et al., 2010). Thus, specificity in MAMP and DAMP signalling is, in part, determined by BAK1, possibly via differential amplification at distinct steps.

The Ca2+/ROS feedback loop

Inhibitor studies suggest that MAMP- and DAMP-induced RbohD-mediated generation of ROS is strictly dependent on an initial upstream [Ca2+]cyt elevation (Figure S10). This is supported by the Ca2+-dependent regulation of RbohD, either direct Ca2+ activation by binding to the N-terminal EF-hand motifs in RbohD (Ogasawara et al., 2008) or indirectly via Ca2+-activated CPK-dependent phosphorylation (Kobayashi et al., 2007; Boudsocq et al., 2010). Nevertheless, ROS have a feedback effect on the [Ca2+]cyt response (Figure 6). Intriguingly, complete abrogation of MAMP-induced ROS generation in the rbohD mutant or by inhibition with DPI, did not result in an overall reduction of the [Ca2+]cyt elevations but rather affected solely the second peak or prolonged plateau. In tobacco cells, DPI similarly inhibited the second of two oligogalacturonic acid (OGA)-triggered [Ca2+]cyt peaks (Lecourieux et al., 2002). The timing of the second [Ca2+]cyt peak, with a maximum at around 5 min after flg22 elicitation (Figure 6a), correlates with the kinetics of MAMP-induced ROS accumulation, which is measurable starting from 3–4 min after flg22 elicitation (Figures 4c and 7a). Accordingly, the less pronounced second [Ca2+]cyt peaks for elf18 or Pep1, and particularly ch8, are generally associated with a lower, and in some cases delayed, production of ROS (Figures 4d, 7b and S12). In any case, the secondary ROS-induced [Ca2+]cyt elevation is transient, which is comparable to a direct H2O2-induced [Ca2+]cyt elevation (Rentel and Knight, 2004). This may be due to saturation and a refractory period of the H2O2 perception system; and may also explain why the prolonged ROS response in mpk3 mutants did not significantly alter the [Ca2+]cyt elevation induced by flg22 or elf18 (Figure 7c,d). The ‘double Ca2+peak’ reported here has so far not been reported by others for the Arabidopsis system (Jeworutzki et al., 2010; Krol et al., 2010; Qi et al., 2010), which could be due to a different experimental setup, in particular the integration intervals for luminescence measurement. Alternatively, in line with a possible different H2O2 refractory period speculated upon above, the use of wounded/excised plant material may lead to this second Ca2+ peak being overlooked.

Although H2O2 triggers [Ca2+]cyt elevations and H2O2-responsive plasma membrane Ca2+-permeable channels have been described (Pei et al., 2000; Rentel and Knight, 2004), it is unclear from the current data whether RbohD-derived inline image or its dismutation product H2O2 directly or indirectly activate Ca2+-permeable channels and if the ROS-triggered [Ca2+]cyt elevation involves Ca2+ influx from the apoplast or release from internal stores. Moreover, apoplastically generated and membrane-permeable ROS like H2O2 are able to diffuse to or into neighbouring cells to activate signalling. Indeed, RbohD-derived ROS have been implicated in long-distance signalling in abiotic stress reactions (Miller et al., 2009). In conclusion, although speculative, it appears plausible that the feedback effect between ROS and Ca2+ signalling observed here may similarly allow cell-to-cell propagation of MAMP/DAMP signals.

The role of MAPKs in early MAMP signalling

Loss of MPK3, but not MPK6, leads to substantially prolonged ROS accumulation upon elicitation with flg22 or elf18, while the maximum ROS levels were not increased (Figure 7a,b). Although ROS accumulation is strictly dependent on [Ca2+]cyt elevation, this does not result from a prolonged increase in [Ca2+]cyt. Surprisingly, a previous screen for components involved in the flg22-induced ROS response did not reveal a phenotype for mpk3 (Mersmann et al., 2010). This might result from the different conditions used: Mersmann et al., (2010) analysed ROS accumulation at lower flg22 concentrations in intact seedlings grown in liquid medium and pre-treated with 10 nm flg22. However, although such pre-treatment was designed to reduce variability of the ROS assay, it may pre-stimulate the ROS catabolic pathway and mask the prolonged ROS accumulation in the mpk3 mutant.

It has been proposed that MPK3 and MPK6 act upstream of the ROS production, as expression of constitutively active MKK5 leads to ROS-dependent callose formation without a MAMP stimulus and MPK3/6 inactivation via the effector protein HopAI1 diminishes ROS accumulation (Zhang et al., 2007). Such artificial over-expression of constitutively active MKKs or effector proteins lacks stimulus-specific regulation, and hence the observed ROS accumulation may be due to indirect effects. Several lines of evidence rather point to MPK3/6-independent MAMP/DAMP-induced ROS production (Figures 7 and S11): (i) loss of MPK3 is associated with a prolonged, rather than a reduced, ROS accumulation; (ii) loss of MPK6 has no impact on ROS accumulation; and (iii) activation of ROS production is strictly dependent on Ca2+, whereas MAPK-activation is only partially dependent on Ca2+. The prolonged ROS accumulation and the enhanced root growth inhibition phenotype in mpk3 (Figure 7), instead, point to a possible role for MPK3 in down-regulating MAMP- and DAMP-induced signalling. Generally, all activated signalling components need to be eventually attenuated to prevent over-stimulation.

In conclusion, different MAMP- and DAMP-induced signalling pathways converge at very early stages by sharing their main signalling components, like ion channels (Krol et al., 2010), NADPH oxidase, MAPK cascades, several defence genes (Navarro et al., 2004; Zipfel et al., 2006; Denoux et al., 2008) and, in some cases, the common signalling partner BAK1 (Chinchilla et al., 2007; Krol et al., 2010). While this is generally true, we show through kinetic analyses of [Ca2+]cyt elevations, MAPK activation, promoter activity and MAMP-induced growth arrest that there are specific differences between flg22- and elf18-induced responses. Moreover, BAK1 is differentially required in several signalling steps induced by different MAMPs but, in particular, in late as well as early elf18-induced responses. Hence, BAK1 participates in the maintenance of the MAMP-specific response.

Finally, together with published data, the observations here accentuate the role of Ca2+ as a second messenger in MAMP and DAMP signalling and collectively support a threshold-dependent ‘on–off switch’ function for Ca2+. [Ca2+]cyt elevations are crucial for downstream responses such as ROS accumulation, which, in turn, contributes to Ca2+ signalling in a positive-feedback loop. Thus, the interplay between components acting up- or downstream of Ca2+ forms the complex MAMP/DAMP signalling network that awaits further discovery.

Postscript

During revision of this work, Segonzac et al. (2011) reported that silencing of tobacco WIPK, the orthologue of Arabidopsis MPK3, led to similar results of prolonged flg22- induced ROS accumulation.

Experimental Procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material and growth conditions

All Arabidopsis thaliana lines were in the Col-0 background (Table S1). Seeds were surface-sterilized if required and stratified at 4°C for two or more days. Plants were grown on soil in climate chambers under short-day conditions (8h light, 16h darkness) or on Arabidopsis thaliana medium + sucrose (ATS) agar plates (Estelle and Somerville, 1987) or in liquid MS medium (0.5 × MS, 0.25% sucrose, 1 mm MES, pH 5.7) in 24-well plates (10 seedlings per well) under long-day conditions (16h light, 8h darkness) at 20–22°C.

Elicitors

Flg22, elf18 and AtPep1 (Felix et al., 1999; Kunze et al., 2004; Huffaker et al., 2006) were synthesized using an Abimed EPS221 (http://www.abimed.de) system. N-acetylchitooctaose (ch8) was provided by N. Shibuya (Albert et al., 2006).

Aequorin luminescence measurements

For aequorin luminescence measurements, Col-0 plants expressing cytosolic p35S-apoaequorin were used (pMAQ2; Knight et al., 1991). Mutant lines were generated by crossing or Agrobacterium tumefaciens (Strain GV3101)-mediated transformation (Table S1). Eight-day-old liquid-grown seedlings or roots of 10-day-old seedlings from agar plates were placed individually in 96-well plates in 10 μm coelenterazine/H2O (native coelenterazine; P.J.K., http://www.pjk-gmbh.com/) in the dark over night. Luminescence was recorded by scanning each row in 6-sec intervals (Luminoskan Ascent 2.1; Thermo Scientific, http://www.thermo.com/). The remaining aequorin was discharged and Ca2+ concentrations were calculated according to Rentel and Knight (2004). In the case of aequorin-transformation, for clarity only one of three or more independent lines (Table S1) is shown, if all lines behaved similarly. Statistical analysis was performed using GraphPad Prism 5.0 (http://www.graphpad.com/) as indicated in figure legends.

Protoplast transient expression assay

Protoplasts were isolated and transformed according to Yoo et al. (2007). pFRK1-, pNHL10- and pPHI1-promoter–luciferase constructs were used as reporters (Table S2; Asai et al., 2002; Boudsocq et al., 2010). pUBQ10-GUS was co-transfected for normalization (Sun and Callis, 1997). Luminescence of protoplast suspensions containing d-luciferin (200 μm; Invitrogen, http://www.invitrogen.com/) and treated with indicated MAMPs/DAMPs was recorded in 96-well plates at indicated intervals (Luminoskan Ascent 2.1). Results are expressed as LUC/GUS ratios relative to untreated controls. For statistical analysis a Kruskal–Wallis test with Dunn’s post-test (P < 0.05) was performed using GraphPad Prism 5.0 (http://www.graphpad.com/).

Immunoblot analysis

Fourteen-day-old liquid-grown seedlings were equilibrated in fresh MS for >2 h. The medium was discarded, and after 30 min recovery seedlings were elicited with 1 μm elicitor in MS. Seedlings were harvested at the indicated time points. Protein extraction and immunoblot with anti-pTEpY (α-phospho-p44/42-ERK; CST, http://www.cellsignal.com/) were performed as described (Saijo et al., 2009).

Detection of ROS in Arabidopsis leaves

Production of ROS was assayed as described (Gomez-Gomez et al., 1999) using 3-mm leaf discs in 96-well plates measured in 2-min intervals (Luminoskan Ascent 2.1). For statistical analysis a Kruskal–Wallis test with Dunn’s post-test (P < 0.05) was performed using GraphPad Prism 5.0.

Root growth inhibition

Seedlings were grown vertically on agar plates ± 1 μm flg22 for 14 days or 5-day-old seedlings were transferred to plates ± 10 μm flg22 for 20 days. Two-way anova was performed on log2-transformed root length data (genotype × treatment; P < 0.001; R statistical package; Delker et al., 2010). Data were depicted as percentage root growth inhibition compared with the control.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to Sacco de Vries, Birgit Kemmerling, Marc Knight, Naoto Shibuya, Gary Stacey and Cyril Zipfel for providing material, Frédéric Brunner and Malou Fraiture for help with protoplast assays and Carolin Delker, Yvonne Pöschl and Ivo Große for advice on statistics. We thank Christel Rülke and Nicole Bauer for technical assistance. This work is supported by a DFG grant (LE2321/1-2) within the priority project SPP1212. L.E.-L. and P.P. are financed by the BMBF project ProNet-T3 (03ISO2211B) and the DFG project SFB648-B1, respectively.

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  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental Procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Figure S1. Inactive forms of MAMPs do not evoke [Ca2+] elevations. cyt (a) The shorter flg15Δ5 peptide or the flg22-analogous peptide from Agrobacterium tumefaciens (A.  tum) do not induce [Ca2+] elevations in Arabidopsis seedlings in comparison to the categorical Pseudomonas aeruginosa cyt (P.  aer) flg22 peptide. (b) The shorter elf12 peptide is inactive in eliciting [Ca2+] elevations in comparison to the elf18 peptide. cyt (c) Ch6 does not induce [Ca2+] elevations in comparison to ch8. cyt Arrows mark time of MAMP application. Data represent mean ± SD of ≥3 independent experiments (≥ 12).

Figure S2. Pre-treatment with LaCl or BAPTA abolishes MAMP-induced [Ca2+] elevations. 3 cyt (a–c) Pre-treatment of Col-0 seedlings with 10 mM LaCl or 10 mM BAPTA for 30 min inhibits [Ca2+] elevations 3 cyt induced by the indicated MAMPs. 2+ (d) Both inhibitors slightly raise the [Ca ] resting level as observed in the water control. Arrows mark time of cyt MAMP application. Data represent mean ± SD of two independent experiments (≥ 3).

Figure S3. MAMP/DAMP-induced [Ca2+] elevations are dose-dependent. cyt [Ca2+] elevations in Arabidopsis seedlings induced by the indicated concentrations of (a) flg22, (b) elf18, (c) Pep1 cyt and (d) ch8. Nearly saturating concentrations reveal similar patterns for all the tested MAMPs/DAMPs but distinct [Ca2+] amplitudes. At lower concentrations, the [Ca2+] responses were also very similar in shape, but in general cyt cyt the maximum peak heights were lower and the maxima occurred at later times. Arrows mark time of MAMP/DAMP application. Data represent mean ± SD of ≥3 independent experiments (n ≥ 8).

Figure S4. Individual traces of MAMP/DAMP-induced [Ca2+] elevations in seedlings. cyt Five individual traces (illustrated by different colours) of [Ca2+] elevations in Col-0 seedlings upon application of cyt the indicated MAMPs/DAMPs. While individual traces illustrate the occurrence of distinct Ca peaks, these “fine structures” are not always visible in average graphs (dotted black line).

Figure S5. MAMPs/DAMPs induce comparable [Ca2+] elevations in intact and root-dissected Arabidopsis cyt seedlings. [Ca2+] elevations upon application (marked by arrow) of the MAMPs/DAMPs flg22, elf18, ch8 and Pep1 in root- cyt dissected Col-0 seedlings are comparable to intact seedlings (cf. fig. 1a). To facilitate comparison, an overlay of the [Ca2+] elevations in intact (shown in figure 1a) and root-dissected (w/o roots) seedlings is included. Thus, the cyt [Ca2+] response in intact seedlings mostly originates from the aerial parts, which under the growth conditions used cyt also comprise the bulk of the seedling fresh weight. Water was used as control. Data represent mean ± SD of ≥3 independent experiments (≥ 24).

Figure S6. MAMP/DAMP-induced [Ca2+] elevations in receptor mutants. cyt [Ca2+] elevations induced by flg22, elf18, Pep1 and ch8 in seedlings of the receptor mutants (a) fls2, (b) efr-1 and cyt (c) cerk1-2. Arrows mark time of MAMP/DAMP application. Data represent mean ± SD of ≥2 independent experiments (n ≥ 6).

Figure S7. Ch8-mediated [Ca2+] elevations are unaltered in serk mutants. cyt [Ca2+] elevations in serk mutant seedlings induced by 1 μ M ch8 compared to Col-0. For clarity, graphs are depicted cyt offset. Arrows mark time of MAMP application. Data represent mean ± SD (n ≥ 6). Letters indicate statistically significant differences between the [Ca2+] amplitude of the mutant compared to wild type, separately calculated for cyt each mutant-wild type pair, with the statistically significant groups categorized by different letters (Student’s t-test; < 0.001).

Figure S8. Pep1-mediated [Ca2+] elevations in serk mutants. cyt (a–d) [Ca2+] elevations in serk seedlings induced by 1 μM Pep1 compared to Col-0. Arrows mark time of cyt MAMP/DAMP application. Data represent mean ± SD of ≥3 independent experiments (n ≥ 18). Letters indicate statistically significant differences, separately calculated for each time point, with the statistically significant groups categorized by different letters (Student’s t-test; < 0.001).

Figure S9. Elf18-induced growth arrest in bak1 and serk mutants. Five-days-old seedlings from agar plates were transferred to liquid medium ± 1 μM elf18 and fresh weight was analysed after 15 days. Growth arrest of bak1, serk and efr (control) mutant seedlings (black bars) each compared to its respective wild-type control (shaded bars). Data are given as % inhibition compared to untreated control; mean ± SE of ≥ 7 seedlings; *Statistically significant difference; ns = not significant (2-way ANOVA genotype × treatment; < 0.001).

Figure S10. Flg22- and elf18-induced ROS accumulation is abolished by LaCl pre-treatment. (a,b) ROS (H O) production induced by the indicated MAMPs was monitored using a luminol-based assay in Col-0 leaf discs pre-treated with 10 mM LaCl for 30 min. Data are given as relative light units (RLU) and represent mean ± SE of ≥ 16. Two independent experiments revealed similar results.

Figure S11. Flg22-induced MAPK activation is reduced, but not abolished by pre-treatment with LaCl and BAPTA. (a,b) MAPK activation upon flg22 application in Arabidopsis suspension-cultured cells was analysed by antipTEpY Western blot at indicated time points. Cells were pre-treated with 10 mM LaCl, 10 mM BAPTA or water as control for 2 h. Anti-MPK6 Western blot shows equal amounts of MPK6 protein in untreated and inhibitor pretreated samples. Amido-black-stained membranes show equal loading. Three independent experiments revealed comparable results.

Figure S11. Flg22-induced MAPK activation is reduced, but not abolished by pre-treatment with LaCl and BAPTA. (a,b) MAPK activation upon flg22 application in Arabidopsis suspension-cultured cells was analysed by antipTEpY Western blot at indicated time points. Cells were pre-treated with 10 mM LaCl, 10 mM BAPTA or water as control for 2 h. Anti-MPK6 Western blot shows equal amounts of MPK6 protein in untreated and inhibitor pretreated samples. Amido-black-stained membranes show equal loading. Three independent experiments revealed comparable results.

Figure S12. Flg22- and ch8-induced ROS accumulation. ROS (H O) production induced by the indicated MAMPs was monitored using a luminol-based assay in Col-0 leaf discs. Data are given as relative light units (RLU) and represent mean ± SE of ≥ 16. Two independent experiments revealed similar results.

Table S1. Mutant lines used in the reverse genetic screen. Mutant lines used are listed below showing AGI code, identity of insertion line, primers used for genotyping, kind and number of aequorin transgenic lines (T = transformants, C = crossing) and references.

Table S2. Primers for cloning of promoters. To clone the NHL10 and PHI1 promoters into the pFRK1-LUC vector, BamHI and NcoI restriction sites were added to the 5’- and 3’- primers (underlined), respectively, allowing direct exchange of the original FRK1 promoter.

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