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![Enhanced AtPep ROS is independent of previous ROS production . A, Leaf discs were treated with the indicated concentrations of flg22 either directly after punching (left) or after the standard 16-h incubation time floating on water (right). Graphs display averages of 12 replicates. Error bars show SE of the mean. B, Indicated pretreatment (1 mM flg22 or without any peptide [control]) was performed either directly after punching (0h+P/20h+E) or at 8 h after punching (0h-/8h+P/ 20h+E). Twenty hours after punching, all leaf discs were treated with indicated elicitors or without any peptide (control). Columns represent averages of the peak values of ROS production of eight biological replicates. Error bars show SE of the mean. C, Relative effect of pretreatments on ROS production. Leaf discs were pretreated as indicated for 16 h and then treated with either 1 mM AtPep1 or 1 mM elf18 (elicitation). Columns represent relative averages of the peak values of ROS production normalized to the respective control treatment. Error bars show SE of the mean of 12 biological replicates. Asterisks represent Student's t test results (**P , 0.01, ***P , 0.001). RLU, Relative light units.](profile/Thomas-Boller/publication/262100185/figure/fig6/AS:420387750989824@1477239658073/Enhanced-AtPep-ROS-is-independent-of-previous-ROS-production-A-Leaf-discs-were-treated.png)
Enhanced AtPep ROS is independent of previous ROS production . A, Leaf discs were treated with the indicated concentrations of flg22 either directly after punching (left) or after the standard 16-h incubation time floating on water (right). Graphs display averages of 12 replicates. Error bars show SE of the mean. B, Indicated pretreatment (1 mM flg22 or without any peptide [control]) was performed either directly after punching (0h+P/20h+E) or at 8 h after punching (0h-/8h+P/ 20h+E). Twenty hours after punching, all leaf discs were treated with indicated elicitors or without any peptide (control). Columns represent averages of the peak values of ROS production of eight biological replicates. Error bars show SE of the mean. C, Relative effect of pretreatments on ROS production. Leaf discs were pretreated as indicated for 16 h and then treated with either 1 mM AtPep1 or 1 mM elf18 (elicitation). Columns represent relative averages of the peak values of ROS production normalized to the respective control treatment. Error bars show SE of the mean of 12 biological replicates. Asterisks represent Student's t test results (**P , 0.01, ***P , 0.001). RLU, Relative light units.
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The endogenous Arabidopsis (Arabidopsis thaliana) peptides, AtPeps, elicit an innate immune response reminiscent of patterntriggered immunity. Detection of various danger signals, including microbe-associated molecular patterns (MAMPs), leads to elevated transcription of PROPEPs, the AtPep precursors, and PEPRs, the AtPep receptors. It has been hyp...
Citations
... We tested the hypothesis that external JA-Ile (primarily leaked out of cells on herbivory) activates a positive feedback mechanism to amplify the secondary Pep-PEPR pathway. AtPep leads to increased JA accumulation, and a functional JA signalling system is required for full-strength AtPep signalling (Flury et al., 2013;Huffaker et al., 2013). We find that external JA-Ile increases the expression of PROPEPs, and in absence of PEPRs, this activation is highly reduced. ...
Jasmonic acid‐isoleucine (JA‐Ile) is a plant defence hormone whose cellular levels are elevated upon herbivory and regulate defence signalling. Despite their pivotal role, our understanding of the rapid cellular perception of bioactive JA‐Ile is limited. This study identifies cell type‐specific JA‐Ile‐induced Ca ²⁺ signal and its role in self‐amplification and plant elicitor peptide receptor (PEPR)‐mediated signalling. Using the Ca ²⁺ reporter, R‐GECO1 in Arabidopsis , we have characterized a monophasic and sustained JA‐Ile‐dependent Ca ²⁺ signature in leaf epidermal cells. The rapid Ca ²⁺ signal is independent of positive feedback by the JA‐Ile receptor, COI1 and the transporter, JAT1. Microarray analysis identified up‐regulation of receptors, PEPR1 and PEPR2 upon JA‐Ile treatment. The pepr1 pepr2 double mutant in R‐GECO1 background exhibits impaired external JA‐Ile induced Ca ²⁺ cyt elevation and impacts the canonical JA‐Ile responsive genes. JA responsive transcription factor, MYC2 binds to the G‐Box motif of PEPR1 and PEPR2 promoter and activates their expression upon JA‐Ile treatment and in myc2 mutant, this is reduced. External JA‐Ile amplifies At Pep‐PEPR pathway by increasing the At Pep precursor, PROPEP expression. Our work shows a previously unknown non‐canonical PEPR‐JA‐Ile‐Ca ²⁺ ‐MYC2 signalling module through which plants sense JA‐Ile rapidly to amplify both At Pep‐PEPR and jasmonate signalling in undamaged cells.
... Many DAMPs/phytocytokines amplify the immunity initiated by PAMPs, and vice versa. For instance, pretreatment with the PAMP flg22 or elf18 leads to an enhanced DAMP/phytocytokine AtPep1-triggered ROS production (Flury et al., 2013). The DAMPs/phytocytokines PIPs enhance PAMP flg22-triggered immunity (Hou et al., 2014). ...
Rapid alkalinization factors (RALFs) in plants have been reported to dampen pathogen‐associated molecular pattern (PAMP)‐triggered immunity via suppressing PAMP‐induced complex formation between the pattern recognition receptor (PRR) and its co‐receptor BAK1. However, the direct and positive role of RALFs in plant immunity remains largely unknown. Herein, we report the direct and positive roles of a typical RALF, RALF22, in plant immunity. RALF22 alone directly elicited a variety of typical immune responses and triggered resistance against the devastating necrotrophic fungal pathogen Sclerotinia sclerotiorum in a FERONIA (FER)‐dependent manner. LORELEI (LRE)‐like glycosylphosphatidylinositol (GPI)‐anchored protein 1 (LLG1) and NADPH oxidase RBOHD were required for RALF22‐elicited reactive oxygen species (ROS) generation. The mutation of cysteines conserved in the C terminus of RALFs abolished, while the constitutive formation of two disulfide bridges between these cysteines promoted the RALF22‐elicited ROS production and resistance against S. sclerotiorum, demonstrating the requirement of these cysteines in the functions of RALF22 in plant immunity. Furthermore, RALF22 amplified the Pep3‐induced immune signal by dramatically increasing the abundance of PROPEP3 transcript and protein. Supply with RALF22 induced resistance against S. sclerotiorum in Brassica crop plants. Collectively, our results reveal that RALF22 triggers immune responses and augments the Pep3‐induced immune signal in a FER‐dependent manner, and exhibits the potential to be exploited as an immune elicitor in crop protection.
... Plant Elicitor Peptides (PEPs) are involved in starvation stress and biotic stress. PEPs crosstalk with salicylic acid (SA), jasmonic acid (JA), and ethylene during abiotic stress (Huffaker et al. 2006;Flury et al. 2013;Liu et al. 2013;Tintor et al. 2013). AtPROPEP3 recognized by the PEP RECEPTOR 1 (PEPR1) receptor, induced salinity stress tolerance and biotic stress tolerance in plants in response to exogenous application with 13-synthetic AtPROPEP3/AT13 peptide fragments (KPT-PSSGKGGKHN) in a culture-based salinity stress assay by regulating salinity-induced genes and activating Na + influx (Nakaminami et al. 2018). ...
... MAPK activity was determined using crude protein extracts from 8-day-old seedlings treated with 1 µM flg22 for 15-60 min as previously described [82]. Crude extracts were separated using 12% SDS-PAGE, and proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, www.bio-rad.com) ...
The importance of E3 ubiquitin ligases from different families for plant immune signaling has been confirmed. Plant RING-type E3 ubiquitin ligases are members of the E3 ligase superfamily and have been shown to play positive or negative roles during the regulation of various steps of plant immunity. Here, we present Arabidopsis RING-type E3 ubiquitin ligases AtRDUF1 and AtRDUF2 which act as positive regulators of flg22- and SA-mediated defense signaling. Expression of AtRDUF1 and AtRDUF2 is induced by pathogen-associated molecular patterns (PAMPs) and pathogens. The atrduf1 and atrduf2 mutants displayed weakened responses when triggered by PAMPs. Immune responses, including oxidative burst, mitogen-activated protein kinase (MAPK) activity, and transcriptional activation of marker genes, were attenuated in the atrduf1 and atrduf2 mutants. The suppressed activation of PTI responses also resulted in enhanced susceptibility to bacterial pathogens. Interestingly, atrduf1 and atrduf2 mutants showed defects in SA-mediated or pathogen-mediated PR1 expression; however, avirulent Pseudomonas syringae pv. tomato DC3000-induced cell death was unaffected. Our findings suggest that AtRDUF1 and AtRDUF2 are not just PTI-positive regulators but are also involved in SA-mediated PR1 gene expression, which is important for resistance to P. syringae.
... Yet, exogenous GLV4 peptide application does not elicit PTI, indicating that GLV4, as we demonstrate for GLV2 here, is rather an immune responses modulator. PIP1 and PEP perception leads to the upregulation of FLS2 transcription to cooperatively contribute to PTI against bacterial infection (Flury et al, 2013;Tintor et al, 2013;Hou et al, 2014). In contrast, we discovered that GLV2 signaling through RGIs posttranscriptionally increases FLS2 and EFR abundance. ...
Plant immune responses must be tightly controlled for proper allocation of resources for growth and development. In plants, endogenous signaling peptides regulate developmental and growth-related processes. Recent research indicates that some of these peptides also have regulatory functions in the control of plant immune responses. This classifies these peptides as phytocytokines as they show analogies with metazoan cytokines. However, the mechanistic basis for phytocytokine-mediated regulation of plant immunity remains largely elusive. Here, we identify GOLVEN2 (GLV2) peptides as phytocytokines in Arabidopsis thaliana. GLV2 signaling enhances sensitivity of plants to elicitation with immunogenic bacterial elicitors and contributes to resistance against virulent bacterial pathogens. GLV2 is perceived by ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors. RGI mutants show reduced elicitor sensitivity and enhanced susceptibility to bacterial infection. RGI3 forms ligand-induced complexes with the pattern recognition receptor (PRR) FLAGELLIN SENSITIVE 2 (FLS2), suggesting that RGIs are part of PRR signaling platforms. GLV2-RGI signaling promotes PRR abundance independent of transcriptional regulation and controls plant immunity via a previously undescribed mechanism of phytocytokine activity.
... Reduced expression of defense-related gene PDF1.2 and decreased ROS accumulation are reported in the mutants compared with wild-type plants (Flury et al., 2013;Krol et al., 2010;Ma et al., 2012). This suggests that PEPR1 and its close homolog PEPR2 activate defense-related genes for innate immune response. ...
Plants adjust amplitude and duration of immune responses via different strategies to maintain growth, development, and resistance to pathogens. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) play vital roles. Pattern recognition receptors, comprising a large number of receptor-like protein kinases and receptor-like proteins, recognize related ligands and trigger immunity. PTI is the first layer of the innate immune system, and it recognizes PAMPs at the plasma membrane to prevent infection. However, pathogens exploit effector proteins to bypass or directly inhibit the PTI immune pathway. Consistently, plants have evolved intracellular nucleotide-binding domain and leucine-rich repeat-containing proteins to detect pathogenic effectors and trigger a hypersensitive response to activate ETI. PTI and ETI work together to protect plants from infection by viruses and other pathogens. Diverse receptors and the corresponding ligands, especially several pairs of well-studied receptors and ligands in PTI immunity, are reviewed to illustrate the dynamic process of PTI response here.
[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
... However, to date, out of the PEP family, only PEP1 has been studied more completely. PEP1 is derived from the C-terminus of the 96 aa precursor protein PROPEP1, and the addition of even low nanomolar concentration of synthetic PEP1 to seedlings or plant parts is able to activate many PTI-related responses, such as mediator alkalinization, ethylene, nitric oxide (NO) and ROS production, calcium influx, MAPK activation, cGMP production, increased JA levels, and numerous gene expression changes (Huffaker et al., 2006;Krol et al., 2010;Flury et al., 2013;Ma et al., 2013;Gully et al., 2015;Klauser et al., 2015). Binding of PEP1 to its receptors inhibits root growth by affecting ROS formation and PIN-dependent auxin distribution through interaction with the plasma membrane proton ATPase (Jing et al., 2019;Jing et al., 2020;Shen et al., 2020). ...
Receptor kinases constitute the largest protein family in regulating various responses to external and internal biotic and abiotic signals. Functional characterization of this large protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified SIRK1 and QSK1 as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic changes induced by sucrose. Here, we now identify a member of the Elicitor Peptide (PEP) family, namely PEP7, as a ligand to receptor kinase SIRK1. PEP7 was shown to bind to the extracellular domain of SIRK1 with a binding constant of 19 µM. PEP7 was secreted to the apoplasm specifically in response to sucrose. Formation of a signaling complex involving SIRK1, QSK1 as well as aquaporins as substrates was induced by sucrose or external PEP7 treatment. PEP7 induced aquaporin phosphorylation and water influx activity. The knock-out mutant of receptor SIRK1 was not responsive to external PEP7 treatment. Binding to receptor SIRK1 and induction of physiological responses was specific to PEP7, neither other members of the PEP-family (PEP6, PEP4), nor other small signaling peptides (CLEs, IDA, RALFs) induced SIRK1 kinase activity, aquaporin phosphorylation, or protoplast water influx activity.
... Leaf tissue (100 mg per sample) was frozen and ground into powder. Each sample was added 100 µL extraction buffer containing 0.35 M Tris-HCl pH 6.8, 30% (v/v) glycerol, 10% (v/v) SDS, 0.6 M DTT and 0.012% (w/v) bromphenol blue and boiled for 10 min (Flury et al., 2013). The extracted total proteins were separated using 10% (w/v) SDS-PAGE gel and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes. ...
PevD1, a fungal effector secreted by Verticillium dahliae, could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco plants. PevD1 could drastically induce the expression of Nbnrp1, which is an asparagine-rich protein (NRP) of Nicotiana benthamiana. Our previous research indicated that Nbnrp1 positively regulated PevD1-induced cell necrosis and disease resistance. In this study, we further investigated PevD1-induced immune responses in both wild-type (WT) and Nbnrp1-RNAi lines through RNA-seq, in order to reveal the underlying mechanism of Nbnrp1-modulated PevD1-induced disease resistance in N. benthamiana. Results showed that Nbnrp1-RNAi lines exhibited reduced PevD1-induced immune responses, like inhibiting H2O2 accumulation and MAPK phosphorylation. To silence Nbnrp1 inhibited the expression of PevD1-induced differential expression genes (DEGs) involved in pathways associated with sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction and phenylpropanoid biosynthesis, etc. It is worth noting that sesquiterpene phytoalexin capsidiol accumulation were obviously decreased in Nbnrp1-RNAi plants after PevD1 treatment, accompanied with the down-expression of EAS and EAH, which were two key genes related to capsidiol biosynthesis. These results suggested that Nbnrp1 mediates PevD1-induced defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway.
... The RBOH members function differently in response to environmental stimuli or cell growth signals. RBOHD and RBOHF have been reported to function vitally in leaves to mediate ROS production under the induction of pathogens, PAMPs, or DAMPs [21,24,37]. In the current study, we found that the activation of Pep1-PEPR triggered a strong ROS accumulation in the roots. ...
Plant elicitor peptides (Peps) are damage/danger-associated molecular patterns (DAMPs) that are perceived by a pair of receptor-like kinases, PEPR1 and PEPR2, to enhance innate immunity and induce the growth inhibition of root in Arabidopsis thaliana. In this study, we show that PEPR1 and PEPR2 function vitally in roots to regulate the root immune responses when treating the roots with bacterial pathogen Pst DC3000. PEPR2, rather than PEPR1, played a predominant role in the perception of Pep1 in the roots and further triggered a strong ROS accumulation—the substance acts as an antimicrobial agent or as a secondary messenger in plant cells. Consistently, seedlings mutating two major ROS-generating enzyme genes, respiratory burst oxidase homologs D and F (RBOHD and RBOHF), abolished the root ROS accumulation and impaired the growth inhibition of the roots induced by Pep1. Furthermore, we revealed that botrytis-induced kinase 1 (BIK1) physically interacted with PEPRs and RBOHD/F, respectively, and served downstream of the Pep1-PEPRs signaling pathway to regulate Pep1-induced ROS production and root growth inhibition. In conclusion, this study demonstrates a previously unrecognized signaling crosstalk between Pep1 and ROS signaling to regulate root immune response and root growth.
... Leaf discs from 5-6-wk-old Arabidopsis plants were floated on MQ water overnight and then exposed to 100 nM flg22 or 10 µM chitin hexaose for 0, 5 or 15 min. Proteins were isolated from 10 mg plant material as described (Flury et al., 2013) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels, followed by Western blotting. The primary antibody (anti-p44/p42 MAPK, monoclonal D13.14.4E; Cell Signaling Technology, Leiden, Netherlands) was used at a 1 : 6000 dilution, and the secondary antibody (goat-anti-rabbit; Pierce, Appleton, WI, USA, 31460) at a 1 : 10 000 dilution. ...
Plant pathogens use effector proteins to promote host colonisation. The mode of action of effectors from root‐invading pathogens, such as Fusarium oxysporum (Fo), is poorly understood. Here, we investigated whether Fo effectors suppress pattern‐triggered immunity (PTI), and whether they enter host cells during infection.
Eight candidate effectors of an Arabidopsis‐infecting Fo strain were expressed with and without signal peptide for secretion in Nicotiana benthamiana and their effect on flg22‐triggered and chitin‐triggered reactive oxidative species (ROS) burst was monitored. To detect uptake, effector biotinylation by an intracellular Arabidopsis‐produced biotin ligase was examined following root infection.
Four effectors suppressed PTI signalling; two acted intracellularly and two apoplastically. Heterologous expression of a PTI‐suppressing effector in Arabidopsis enhanced bacterial susceptibility. Consistent with an intracellular activity, host cell uptake of five effectors, but not of the apoplastically acting ones, was detected in Fo‐infected Arabidopsis roots.
Multiple Fo effectors targeted PTI signalling, uncovering a surprising overlap in infection strategies between foliar and root pathogens. Extracellular targeting of flg22 signalling by a microbial effector provides a new mechanism on how plant pathogens manipulate their host. Effector translocation appears independent of protein size, charge, presence of conserved motifs or the promoter driving its expression.
