Danger peptide receptor signaling in plants ensures basal immunity upon pathogen‐induced depletion of BAK 1
2015; Springer Nature; Volume: 35; Issue: 1 Linguagem: Inglês
10.15252/embj.201591807
ISSN1460-2075
AutoresKohji Yamada, Misuzu Yamashita‐Yamada, Taishi Hirase, Tadashi Fujiwara, Kenichi Tsuda, Kei Hiruma, Yusuke Saijo,
Tópico(s)Legume Nitrogen Fixing Symbiosis
ResumoArticle16 November 2015free access Source Data Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK1 Kohji Yamada Kohji Yamada Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Graduate School of Agriculture, Kyoto University, Kyoto, Japan Search for more papers by this author Misuzu Yamashita-Yamada Misuzu Yamashita-Yamada Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Taishi Hirase Taishi Hirase Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Tadashi Fujiwara Tadashi Fujiwara Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Kenichi Tsuda Kenichi Tsuda Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Kei Hiruma Kei Hiruma Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Yusuke Saijo Corresponding Author Yusuke Saijo Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan JST, PRESTO, Kawaguchi, Japan Search for more papers by this author Kohji Yamada Kohji Yamada Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Graduate School of Agriculture, Kyoto University, Kyoto, Japan Search for more papers by this author Misuzu Yamashita-Yamada Misuzu Yamashita-Yamada Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Taishi Hirase Taishi Hirase Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Tadashi Fujiwara Tadashi Fujiwara Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Kenichi Tsuda Kenichi Tsuda Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Search for more papers by this author Kei Hiruma Kei Hiruma Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan Search for more papers by this author Yusuke Saijo Corresponding Author Yusuke Saijo Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan JST, PRESTO, Kawaguchi, Japan Search for more papers by this author Author Information Kohji Yamada1,2, Misuzu Yamashita-Yamada1,5,‡, Taishi Hirase3,‡, Tadashi Fujiwara3, Kenichi Tsuda1, Kei Hiruma3 and Yusuke Saijo 1,3,4 1Department of Plant-Microbe Interactions, Max Planck Institute for Plant Breeding Research, Cologne, Germany 2Graduate School of Agriculture, Kyoto University, Kyoto, Japan 3Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Japan 4JST, PRESTO, Kawaguchi, Japan 5Present address: Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 743 72 5690; E-mail: [email protected] The EMBO Journal (2016)35:46-61https://doi.org/10.15252/embj.201591807 See also: D Tang & J-M Zhou (January 2016) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Pathogens infect a host by suppressing defense responses induced upon recognition of microbe-associated molecular patterns (MAMPs). Despite this suppression, MAMP receptors mediate basal resistance to limit host susceptibility, via a process that is poorly understood. The Arabidopsis leucine-rich repeat (LRR) receptor kinase BAK1 associates and functions with different cell surface LRR receptors for a wide range of ligands, including MAMPs. We report that BAK1 depletion is linked to defense activation through the endogenous PROPEP peptides (Pep epitopes) and their LRR receptor kinases PEPR1/PEPR2, despite critical defects in MAMP signaling. In bak1-knockout plants, PEPR elicitation results in extensive cell death and the prioritization of salicylate-based defenses over jasmonate-based defenses, in addition to elevated proligand and receptor accumulation. BAK1 disruption stimulates the release of PROPEP3, produced in response to Pep application and during pathogen challenge, and renders PEPRs necessary for basal resistance. These findings are biologically relevant, since specific BAK1 depletion coincides with PEPR-dependent resistance to the fungal pathogen Colletotrichum higginsianum. Thus, the PEPR pathway ensures basal resistance when MAMP-triggered defenses are compromised by BAK1 depletion. Synopsis LRR signaling co-receptor BAK1 is a central mediator of plant immunity, but when absent—as seen upon certain fungal invasions—its function is compensated by signaling through danger peptide receptors PEPRs. Loss of BAK1 leads to the sensitization of PEPR pro-death signaling. PEPRs are required for basal resistance against pathogens in absence of BAK1. PEPR-mediated signaling is rewired to reinforce salicylate-based defenses instead of jasmonate-based defenses in absence of BAK1. Pathogen effectors and BAK1 depletion additively enhance the generation and release of the PEPR pro-ligand PROPEP3. Fungal pathogen C. higginsianum invasion leads to depletion of BAK1. Introduction Innate immunity based on a limited set of germ line-encoded receptors is fundamental for both plants and animals to recognize and respond to diverse microbes (Ronald & Beutler, 2010). Plants rely solely on innate immunity, which involves two tiers of functionally interlinked immune responses. The first is mediated by cell surface-localized pattern recognition receptors (PRRs) that sense molecular signatures typical of microbes, termed microbe-associated molecular patterns (MAMPs), including bacterial flagellin, elongation factor (EF)-Tu, peptidoglycans, and fungal chitin (Boller & Felix, 2009; Macho & Zipfel, 2014). MAMP-triggered immunity (MTI) is typically insufficient to prevent infection by host-adapted pathogens that employ an array of virulence effectors to subvert PRR-mediated defenses. However, the second tier of plant immunity is triggered when these effectors are recognized. Effector-triggered immunity (ETI) leads to a robust and high-amplitude activation of immune responses that terminate pathogen growth, which is often accompanied by localized cell death. A large overlap in the defense outputs and signaling components leads to the notion that ETI is a magnified form of MTI (Jones & Dangl, 2006; Cui et al, 2015). ETI is often induced upon recognition of effector-mediated modifications of a host target (Cui et al, 2015). Animal studies have also described pathogen effectors that elicit immune responses to protect the host (Stuart et al, 2013). Defense activation upon sensing pathogen effectors seems to represent a key principle in both plant immunity and animal immunity. In plants, substantial defenses are activated when susceptible hosts are challenged with virulent pathogens, and thereby limit host susceptibility. This response, known as basal resistance (Jones & Dangl, 2006), seems not to require the recognition of a specific pathogen effector, but is often enhanced when plants deficient in MAMP responses are exposed to pathogen effectors (Laluk et al, 2011; Li et al, 2014). These observations imply a link between basal resistance and pathogen effector actions. On the other hand, loss of individual MAMP receptors increases host susceptibility to virulent pathogens having a complete effector assembly (Zipfel et al, 2004, 2006; Willmann et al, 2011), indicating a critical role for MAMP recognition in basal resistance. However, it remains poorly understood how MAMP receptors mediate host resistance despite effector-mediated suppression of MTI signaling. In Arabidopsis, the leucine-rich repeat (LRR) receptor kinases (RKs) FLS2 and EFR recognize the bacterial MAMPs flagellin (flg22 epitope) and EF-Tu (elf18 epitope), respectively, and induce anti-bacterial immunity (Zipfel et al, 2004, 2006). Immediately after ligand binding, FLS2/EFR physically associate with the LRR-RK coreceptor BAK1, thereby offering a platform for defense signaling (Chinchilla et al, 2007; Heese et al, 2007; Sun et al, 2013). The FLS2/EFR-BAK1 complexes mediate phosphorylation of the receptor-like cytoplasmic kinases (RLCKs) BIK1 and related PBL proteins, which in turn dissociate from the receptor complexes to regulate downstream signaling (Lu et al, 2010; Zhang et al, 2010; Liu et al, 2013; Lin et al, 2014). These events are followed by a stereotypic set of cellular responses, including a rapid burst of Ca2+ and reactive oxygen species (ROS), activation of Ca2+-dependent protein kinases (CDPKs) and mitogen-activated protein kinases (MAPKs), cell wall remodeling, production of the phytohormones ethylene (ET) and salicylate (SA), and extensive transcriptional reprogramming (Boller & Felix, 2009; Macho & Zipfel, 2014). Fine control of MAMP signaling is in part achieved through negative regulation within or in proximity to the PRR-BAK1 complexes. For instance, the LRR-RK BIR2 sequesters BAK1 from ligand-unbound FLS2 to avoid precocious signal activation (Halter et al, 2014). The BAK1-associated E3 ubiquitin ligases PUB12/PUB13 are recruited to the ligand-induced FLS2-BAK1 complex for ubiquitination and proteasomal degradation of the receptor (Lu et al, 2011). A ligand-induced decrease in FLS2 accumulation, apparently in association with receptor internalization, results in transient desensitization to flg22 before subsequent replenishment of the receptor (Robatzek et al, 2006; Smith et al, 2014). A subclass of protein phosphatase 2A dephosphorylates BAK1 to attenuate FLS2 signaling (Segonzac et al, 2014). However, it is less clear whether and how relief of negative regulation is linked to basal resistance during pathogen challenge. MAMP signaling induces a subset of the soluble pro-peptide (PROPEP) family (carrying an immunogenic Pep epitope in their C termini) and then involves the LRR-RK Pep receptors PEPR1/PEPR2 (Huffaker et al, 2006; Yamaguchi et al, 2006, 2010; Krol et al, 2010; Ma et al, 2012; Tintor et al, 2013). The lack of N-terminal signal sequences for canonical secretion led to a model in which PROPEP-derived elicitors provide danger-associated molecular patterns (DAMPs) following their release upon membrane disintegration (Yamaguchi & Huffaker, 2011). Pep perception by PEPRs leads to MTI-hallmark outputs, largely through the aforementioned scheme of MTI signaling (Yamaguchi & Huffaker, 2011; Liu et al, 2013). The PEPR pathway contributes to co-activation of SA and jasmonate (JA)/ET defenses (Ross et al, 2014) and to propagation of MAMP-triggered defense signaling (Ma et al, 2012; Flury et al, 2013; Tintor et al, 2013; Ross et al, 2014). These findings point to the importance of functional interactions between the FLS2/EFR and PEPR pathways as a critical step in MTI. However, despite increasing insight into the individual PRR pathways, the mechanisms underlying their functional interactions remain poorly understood. Of note, FLS2, EFR and PEPRs all function with BAK1 in signal initiation. It remains to be determined whether BAK1 provides a node of functional convergence or is simply a common component in separate PRR pathways. Nevertheless, either scenario predicts that MTI will be vulnerable to pathogen assaults on BAK1. Indeed, BAK1 is a recurrent target in different plant hosts for structurally and functionally unrelated virulence-promoting effectors (Xin & He, 2013; Macho & Zipfel, 2015). However, bak1-knockout (KO) plants display almost intact or even enhanced post-invasion resistance against (hemi)biotrophic pathogens (Kemmerling et al, 2007), despite critical defects in a major branch of MTI signaling (Liebrand et al, 2014). In addition to PRR signaling, BAK1 positively regulates brassinosteroid (BR) signaling and negatively regulates cell death (Liebrand et al, 2014). BAK1 acts as a co-receptor for the LRR-RK BR receptor BRI1 (Nam & Li, 2002; Li et al, 2002). BR signaling and MTI signaling antagonize each other (Albrecht et al, 2012; Belkhadir et al, 2012; Lin et al, 2013), but BAK1 is not rate-limiting between the two pathways (Albrecht et al, 2012). In cell death suppression, BAK1 acts together with the LRR-RK BIR1 and the membrane-associated copain-like BONZAI proteins BON1-BON3 (He et al, 2007; Kemmerling et al, 2007; Wang et al, 2011). Accordingly, bak1-KO plants exhibit enhanced cell death upon pathogen challenge, which is further enhanced by the loss of BKK1, the closest homolog of BAK1 (He et al, 2007; Kemmerling et al, 2007). Both bir1 and bon mutant plants display spontaneous cell death that is partially suppressed at high temperatures and by the loss of the lipase-like proteins EDS1/PAD4 or the nucleotide-binding LRR receptor (NLR) SNC1 (Yang et al, 2006; Gao et al, 2009). These findings suggest a link between cell death and resistance in bak1-KO plants, which remains to be explored. Here, we show that the loss of BAK1 sensitizes PEPR signaling toward cell death and results in reprogramming of PEPR-mediated defense outputs in favor of SA-related resistance. This is accompanied by increased extracellular release of PROPEP3 upon pathogen challenge. Notably, selective BAK1 depletion occurs during challenge with the fungal hemibiotrophic pathogen Colletotrichum higginsianum (Ch) and coincides with PEPR-dependent fungal resistance. Our findings indicate a critical role for PEPR-mediated DAMP signaling, which is stimulated and rewired when BAK1 is depleted, in plant immunity. Results Loss of BAK1 sensitizes PEPR-mediated signaling toward cell death Pursuing a molecular link between MAMP and PEPR pathways, we investigated a role of BAK1 in PEPR signaling. With transgenic plants expressing a functional PEPR1-Flag fusion in the pepr1 pepr2 background (Fig EV1A), co-immunoprecipitation (coIP) analyses confirmed ligand-induced association of PEPR1-Flag and BAK1 (Fig EV1B), as previously deduced (Schulze et al, 2010). Click here to expand this figure. Figure EV1. Functional significance of BAK1 and related SERK family members in PEPR signaling qRT–PCR analysis of PEPR1 in 10-day-old Arabidopsis wild-type (WT) seedlings and those expressing PEPR1-FLAG under the cauliflower mosaic virus 35S promoter in the pepr1 pepr2 background (PEPR1-FLAG pepr1 pepr2) (left). Immunoblot analysis of Pep2-induced MAPK activation in Arabidopsis pepr1 pepr2 plants (right). Equal loading was verified with Ponceau S staining (bottom). Data are averages (±SD) of three biological replicates. PEPR1-FLAG associates with BAK1 in Arabidopsis following Pep2 application for the indicated times. IB and IP denote immunoblotting and immunoprecipitation with the indicated antibodies, respectively. Immunoblot analysis with anti-phosphorylated p44/p42 MAPK antibody in 10-day-old seedlings exposed to 0.5 μM Pep2 or flg22 for the indicated times. Equal loading was verified with Ponceau S staining. Download figure Download PowerPoint Unexpectedly, however, Pep2-induced growth inhibition was greatly enhanced in bak1-3 and bak1-4 KO plants compared to the wild type (WT) (Figs 1A and EV2A). This phenotype was rescued by the introduction of a genomic BAK1 clone (Fig EV2B) and abolished in bak1 pepr1 pepr2 plants (Figs 1A and EV2A), confirming that Pep sensitization upon BAK1 disruption occurs through PEPRs. Of the BAK1-related SERK family members, single disruption of BAK1 specifically enhanced Pep2-induced growth inhibition (Fig EV2C), correlated with enhanced cell death (He et al, 2007; Kemmerling et al, 2007). Indeed, cell death staining revealed that Pep2, but not flg22, induced extensive cell death in the roots and cotyledons of bak1-4 plants, in a manner dependent on PEPRs (Figs 1B and EV3A). This demonstrates that PEPR-mediated pro-death signaling is sensitized upon BAK1 disruption. Moreover, loss of PEPRs substantially suppressed the dwarfism of bak1 bkk1 plants (Fig 1C), suggesting a contribution of endogenous PROPEP-PEPR signaling to cell death in the mutant. We thus conclude that PEPRs mediate one of the pro-death pathways that are suppressed by BAK1/BKK1. Figure 1. BAK1 disruption sensitizes PEPR signaling toward cell death A, B. Root length (averages ± SD, n ≥ 15) (A) and cell death staining of cotyledons and primary root tips (B) of Arabidopsis seedlings that were exposed to 100 nM Pep2 for 7 days, 2 days after germination. BAK1 (D416N) is a kinase-dead variant. *P < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. C. Overview of 3-week-old plants. D. Pep2 induces PEPR1-FLAG association with SERK1-, SERK2-, BKK1-, and BAK1-HA in N. benthamiana. Experiments were repeated at least three times, with the same conclusions. IP and IB denote immunoprecipitation and immunoblotting with the indicated antibodies, respectively. NT indicates non-transformed plants. Source data are available online for this figure. Source Data for Figure 1D [embj201591807-sup-0003-SDataFig1D.tif] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Pep-induced root growth inhibition in serk mutant plants bak1-4 plants are more sensitive to Pep2 than WT plants in root growth inhibition, in a manner dependent on PEPRs. Complementation of the Pep hypersensitive phenotype of bak1-4 plants by the introduction of a genomic BAK1 DNA clone. *P < 0.01 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-4 plants. Log2 values of two independent experiments were combined for statistical analysis. Of the SERK genes tested, single BAK1 disruption has the largest effect in increasing Pep sensitivity. *P < 0.01 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-4 plants. Log2 values were used for statistical analysis. Pep2-induced root growth inhibition in the presence of bri1-301. *P < 0.01 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bri1-301 plants. Log2 values of two independent experiments (n ≥ 15 each) were combined for statistical analysis. Pep2-induced root growth inhibition of serk1-3 bak1-3 plants in the presence or absence of a constitutive BR activator, BES1D-GFP. *P < 0.01 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of WT plants. Log2 values were used for statistical analysis. Simultaneous BKK1 disruption enhances Pep2 induction of PR1 and NHL10 in bak1-knockout plants. qRT–PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 μM Pep2 for 10 h. Results are averages ± SD. *P < 0.01 in two-tailed tests. Relative cycle threshold (Ct) values of two independent experiments with three biological replicates each were combined for statistical analysis. Data information: (A–E) Root length of 9-day-old seedlings was determined following 100 nM Pep2 application for 7 days. Results are averages ± SD (n ≥ 15). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Pep-induced cell death in bak1 mutant plants A. Evans blue staining for dead cells in primary root tips treated with flg22 at 100 nM. B–D. Pep2-induced root growth inhibition after treatment with Pep2 at 100 nM. (B) WT and lsd1c plants were indistinguishable in Pep2-induced root growth inhibition. (C) Characterization of bak1-3 eds1-2 plants. (D) Characterization of bak1-4 sid2-1 plants. Trypan blue staining for dead cells in cotyledons (left). E. Pep2-induced root growth inhibition was sensitized in bak1-null alleles. F. Pep2-induced root growth inhibition was suppressed in bak1 alleles with point substitutions in the kinase domain. Data information: Results are averages ± SD (n ≥ 15). Root length of 9-day-old seedlings was determined following 100 nM Pep2 application for 7 days. *P < 0.01 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of Aeq cyt (A) or Aeq vmd (B) plants. n.s. denotes non-significant differences (P > 0.05 in two-tailed tests). Two independent experiments were combined for statistical analysis. Download figure Download PowerPoint To assess the specificity of the bak1 effects, we tested Pep-induced growth inhibition in lsd1c plants, which display runaway cell death via EDS1 (Rustérucci et al, 2001). In contrast to bak1 plants, lsd1 plants retained WT-like Pep2 sensitivity (Fig EV3B). EDS1 disruption did not affect Pep2-induced growth arrest in bak1 plants (Fig EV3C). These results suggest that PEPR-mediated pro-death signaling occurs independently of EDS1 and is specifically sensitized upon BAK1 disruption. Enhanced cell death in bak1 plants was previously uncoupled from BR signaling (He et al, 2007; Kemmerling et al, 2007). Consistent with this, neither the reduction nor the sensitization of BR signaling in the presence of the bri1-301 allele (Nam & Li, 2002) or BES1-D (Albrecht et al, 2008), respectively, influenced the bak1 effects on Pep2-induced growth inhibition (Fig EV2D and E). Our results thus rule out antagonism between BR and PRR signaling as the cause for the sensitization of Pep-induced cell death. Disruption, but not catalytic inactivation, of BAK1 sensitizes PEPR signaling To determine whether loss of BAK1 accumulation or kinase activity sensitizes PEPR signaling, we characterized the previously described bak1 alleles (Schwessinger et al, 2011; Ranf et al, 2012). Pep2-induced root growth inhibition was enhanced in the tested bak1-null alleles relative to the corresponding WT controls, while, in contrast, that in the bak1 alleles with a point substitution in the kinase domain, including a kinase-dead variant, was suppressed (Figs 1A and EV3E and F). In the hypoactive bak1-5 allele (Schwessinger et al, 2011), Pep-induced growth inhibition and MAPK activation were almost abolished (Figs 1A and EV1C). These results are consistent with the established view that BAK1 positively regulates PEPR signaling (Roux et al, 2011). However, in contrast to catalytic inactivation of BAK1, our findings reveal that BAK1 depletion leads to the sensitization of PEPR-mediated pro-death signaling. It is conceivable that another SERK member compensates for the absence of BAK1 in PEPR signaling but is virtually, if not completely, blocked in the presence of a kinase-inactive BAK1 variant. This model also explains the retention of PEPR function leading to growth inhibition in bak1 bkk1 plants (Fig 1C). We thus tested pairwise associations between PEPR1 and SERK members by coIP analyses in Nicotiana benthamiana leaves, following Agrobacterium-mediated co-expression of PEPR1-FLAG with individual SERK-HA proteins. In contrast to the preferential FLS2-BAK1 association (Roux et al, 2011), PEPR1-FLAG associated with the four tested SERK members in response to Pep2 (Fig 1D). Therefore, promiscuous employment of SERK members seems to underlie the tolerance of PEPR signaling to BAK1/BKK1 disruption. PEPR signaling strictly requires BIK1 and PBL1 flg22, elf18 and Pep1 commonly induce phosphorylation of BIK1 and MAPKs, downstream of cognate PRR-BAK1 complexes (Lu et al, 2010; Zhang et al, 2010; Liu et al, 2013). PEPR1 directly phosphorylates BIK1, whereas FLS2 relies on BAK1 for BIK1 phosphorylation (Lu et al, 2010; Liu et al, 2013). Consistent with this, BIK1 phosphorylation and MAPK activation in response to Pep2 remain largely unaffected in bak1-KO plants, while those in response to flg22 were significantly reduced (Figs 2A and EV1C). By contrast, Pep responsiveness requires BIK1 and the closely related PBL1 (Liu et al, 2013). We verified that Pep2-induced MAPK activation and growth inhibition were reduced in bak1 bik1 pbl1 plants (Fig 2B and C), demonstrating that the authentic PEPR-SERK-RLCK module mediates sensitized Pep responses in the absence of BAK1. Figure 2. BIK1-dependent sensitization of PEPR signaling in the absence of BAK1 Immunoblot analysis for BIK1-HA in Arabidopsis protoplasts exposed to Pep2 or flg22 for 10 min. Arrowheads indicate phosphorylated BIK1-HA. Immunoblot analysis for Pep2-triggered MAPK activation. Ponceau S-stained loading controls are shown (bottom). Root length (averages ± SD, n ≥ 15) of Arabidopsis seedlings that were exposed to 100 nM Pep2. *P < 0.05 in two-tailed tests compared to the differences (± Pep2) from the corresponding values of bak1-3 plants. Two independent experiments were combined for statistical analysis. Download figure Download PowerPoint Reprogramming of the PEPR-regulated transcriptome upon BAK1 disruption To elucidate the bak1 effects on PEPR-regulated outputs, we performed a genome-wide microarray analysis for WT and bak1-3 seedlings exposed to Pep2. The loss of BAK1 had a larger effect on Pep2-regulated expression profiles at 10 h than at 2 h (Fig 3A). Cross-referencing Pep2-responsive genes in bak1 plants at 10 h with genes responsive to SA, methyl-JA and ET revealed an over-representation of SA- or JA-inducible genes among the Pep2 up- or down-regulated genes, respectively (Fig 3B; Table EV1). qRT–PCR analyses confirmed that Pep2 induction of SA-related and -unrelated defense markers, PR1 and NHL10, respectively, was enhanced, while in contrast that of a JA/ET marker, PDF1.2a, was suppressed in bak1-KO plants (Fig 3C and D). BIK1/PBL1 were again required for enhanced Pep2 induction of PR1 and NHL10 in bak1 plants (Fig 3D), indicating that the authentic PEPR-BIK1/PBL1 signaling is rewired upon BAK1 disruption. Together, our results suggest that BAK1 disruption leads to the prioritized activation of these SA-related and -unrelated defenses at the cost of the JA-dependent defenses. Figure 3. BAK1 disruption leads to reprogramming of PEPR-mediated defense outputs A. Scatter plots for log2 fold changes of gene expression in WT plants versus bak1-3 plants treated with Pep2 for 2 and 10 h. The regression of these scatter plots is indicated by the red lines. B. Hierarchical analysis of genes exhibiting a > two-fold change in expression level in bak1-3 plants after Pep2 application for 10 h in a whole-genome microarray analysis. Using Genevestigator v3, these genes were separately cross-referenced to public databases to show their expression responses to salicylate (SA), methyl jasmonate (JA), or ethylene (ET). C, D. qRT–PCR analysis of defense-related genes in 10-day-old seedlings exposed to 0.5 μM Pep2 for 10 h. Data are averages (± SD) of three biological replicates. *P < 0.05 in two-tailed tests compared to the corresponding bak1-4 and bak1-3 values in (C) and (D), respectively. Relative cycle threshold (Ct) values of two independent experiments were combined for statistical analysis. Download figure Download PowerPoint Of particular note, although SID2-mediated SA biosynthesis (Wildermuth et al, 2001) was required for PR1 induction in response to Pep2, it was dispensable for cell death, growth arrest, NHL10 induction, and suppression of PDF1.2a induction in bak1-KO plants (Figs 3C and EV3D). These results point to the SA independence of pro-death signaling and of suppressing JA/ET defense induction via PEPRs upon BAK1 disruption. Loss of BAK1 reinforces the PEPR pathway at both the proligand and receptor levels PEPR-mediated PROPEP2/PROPEP3 activation is thought to provide positive feedback for defense signal amplification (Yamaguchi & Huffaker, 2011). Pep2-induced PROPEP2/PROPEP3 activation was enhanced in bak1-KO plants (Fig 4A). With anti-GFP and anti-PROPEP3 antibodies (raised against both N- and C-terminal fragments of PROPEP3; Ross et al, 2014), we traced Pep2 induction of a functional PROPEP3-Venus fusion protein, driven by the native DNA regulatory sequences (Ross et al, 2014). To determine whether PROPEP3-Venus is released from the cell, we examined an extracellular protein fraction recovered from the surrounding liquid medium. Both antibodies detected a specific signal that is nearly of the predicted full-length size (~10.4 + 27 kDa) in both in planta and extracellular fractions (Fig 4B). The PROPEP3-Venus form was produced and released into the extracellular space in response to Pep2, to a greater degree in bak1-4 plants compared to WT plants (Fig 4B). Although differences were less pronounced in anti-PROPEP3 immunoblots, our results indicate that Pep-induced PROPEP3 release is increased in the absence of BAK1. Under our conditions, we failed to detect endogenous PROPEP3 or a small, possibly processed form of PROPEP3-Venus. These data suggest that PROPEP3 can be released without extensive processing. Figure 4. PROPEP proligand production and PEPR1 accumulation following Pep application are reinforced in the absence of BAK1 A. qRT–PCR analysis of PROPEP genes in 10-day-old seedlings exposed to 0.5 μM Pep2 for 10 h. *P < 0.05 in two-tailed tests compared to the corresp
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