Title : Danger peptide receptor signaling in plants ensures basal immunity upon pathogen-induced depletion of BAK 1

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. Main Text 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 (Cui et al, 2015; Jones & Dangl, 2006). 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 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 Ca and reactive oxygen species (ROS), activation of Ca-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 dephosphorylate 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; Krol et al, 2010; Ma et al, 2012; Tintor et al, 2013; Yamaguchi et al, 2006, 2010). 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 coreceptor 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), coimmunoprecipitation (coIP) analyses confirmed ligand-induced