Immune responses induced by oligogalacturonides are differentially affected by AvrPto and loss of BAK1/BKK1 and PEPR1/PEPR2

Plants possess an innate immune system capable of restricting invasion by most potential pathogens. At the cell surface, the recognition of microbe‐associated molecular patterns (MAMPs) and/or damage‐associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) represents the first event for the prompt mounting of an effective immune response. Pathogens have evolved effectors that block MAMP‐triggered immunity. The Pseudomonas syringae effector AvrPto abolishes immunity triggered by the peptide MAMPs flg22 and elf18, derived from the bacterial flagellin and elongation factor Tu, respectively, by inhibiting the kinase function of the corresponding receptors FLS2 and EFR, as well as their co‐receptors BAK1 and BKK1. Oligogalacturonides (OGs), a well‐known class of DAMPs, are oligomers of α‐1,4‐linked galacturonosyl residues, released on partial degradation of the plant cell wall homogalacturonan. We show here that AvrPto affects only a subset of the OG‐triggered immune responses and that, among these responses, only a subset is affected by the concomitant loss of BAK1 and BKK1. However, the antagonistic effect on auxin‐related responses is not affected by either AvrPto or the loss of BAK1/BKK1. These observations reveal an unprecedented complexity among the MAMP/DAMP response cascades. We also show that the signalling system mediated by Peps, another class of DAMPs, and their receptors PEPRs, contributes to OG‐activated immunity. We hypothesize that OGs are sensed through multiple and partially redundant perception/transduction complexes, some targeted by AvrPto, but not necessarily comprising BAK1 and BKK1.     

The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe‐associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP‐activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs–MKK4/5–MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1–MKK1/2–MPK4 cascade and the plasma membrane‐localized receptor‐like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane‐localized receptor‐like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild‐type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.     

Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases

Plants use receptor kinases, such as FLS2 and EFR, to perceive bacterial pathogens and initiate innate immunity. This immunity is often suppressed by bacterial effectors, allowing pathogen propagation. To counteract, plants have evolved disease resistance genes that detect the bacterial effectors and reinstate resistance. The Pseudomonas syringae effector AvrPto promotes infection in susceptible plants but triggers resistance in plants carrying the protein kinase Pto and the associated resistance protein Prf. Here we show that AvrPto binds receptor kinases, including Arabidopsis FLS2 and EFR and tomato LeFLS2, to block plant immune responses in the plant cell. The ability to target receptor kinases is required for the virulence function of AvrPto in plants. The FLS2-AvrPto interaction and Pto-AvrPto interaction appear to share similar sequence requirements, and Pto competes with FLS2 for AvrPto binding. The results suggest that the mechanism by which AvrPto recognizes virulence targets is linked to the evolution of Pto, which, in association with Prf, recognizes the bacterium and triggers strong resistance.     

BAK1 is not a target of the Pseudomonas syringae effector AvrPto

Plant cell surface-localized receptor kinases such as FLS2, EFR, and CERK1 play a crucial role in detecting invading pathogenic bacteria. Upon stimulation by bacterium-derived ligands, FLS2 and EFR interact with BAK1, a receptor-like kinase, to activate immune responses. A number of Pseudomonas syringae effector proteins are known to block immune responses mediated by these receptors. Previous reports suggested that both FLS2 and BAK1 could be targeted by the P. syringae effector AvrPto to inhibit plant defenses. Here, we provide new evidence further supporting that FLS2 but not BAK1 is targeted by AvrPto in plants. The AvrPto-FLS2 interaction prevented the phosphorylation of BIK1, a downstream component of the FLS2 pathway.     

Two distinct Pseudomonas effector proteins interact with the Pto kinase and activate plant immunity

The Pto serine/threonine kinase of tomato confers resistance to speck disease by recognizing strains of Pseudomonas syringae that express the protein AvrPto. Pto and AvrPto physically interact, and this interaction is required for activation of host resistance. We identified a second Pseudomonas protein, AvrPtoB, that interacts specifically with Pto and is widely distributed among plant pathogens. AvrPtoB is delivered into the plant cell by the bacterial type III secretion system, and it elicits Pto-specific defenses. AvrPtoB has little overall sequence similarity with AvrPto. However, AvrPto amino acids, which are required for interaction with Pto, are present in AvrPtoB and required for its interaction with Pto. Thus, two distinct bacterial effectors activate plant immunity by interacting with the same host protein kinase through a similar structural mechanism.     

The Arabidopsis leucine-rich repeat receptor-like kinases BAK1/SERK3 and BKK1/SERK4 are required for innate immunity to hemibiotrophic and biotrophic pathogens

Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.     

Engineering plants to secrete affinity‐tagged pathogen elicitors for deciphering immune receptor complex or inducing enhanced immunity

Plant cells mount plenty of pattern-recognition receptors(PRRs) to detect the microbe-associated molecular patterns(MAMPs) from potential microbial pathogens.MAMPs are overrepresented by proteinaneous patterns,such as the flg22 peptide from bacterial flagellin. Identification of PRR receptor complex components by forward or reverse genetics can be time/labor-consuming, and be confounded by functional redundancies. Here, we present a strategy for identifying PRR complex components by engineering plants to inducibly secrete affinity-tagged proteinaneous MAMPs to the apoplast. The PRR protein complexes bound to self-secreted MAMPs are enriched through affinity purification and dissected by mass spectrometry. As a proof of principle, we could capture the flg22 receptor FLS2 and co-receptor BAK1 using Arabidopsis plants secreting FLAG-tagged flg22 under estradiolinduction. Moreover, we identified receptor-like kinases LIK1 and PEPR1/PEPR2 as potential components in the FLS2 receptor complex, which were further validated by protein–protein interaction assays and the reverse genetics approach. Our study showcases a simple way to biochemically identify endogenous PRR complex components without overexpressing the PRR or using chemical crosslinkers, and suggests a possible crosstalk between different immune receptors in plants. A modest dose of estradiol can also be applied to inducing enhanced immunity in engineered plants to both bacterial and fungal pathogens.     

Phosphorylation of the Pseudomonas syringae effector AvrPto is required for FLS2/BAK1-independent virulence activity and recognition by tobacco

The type III effector protein AvrPto from Pseudomonas syringae pv. tomato is secreted into plant cells where it promotes bacterial growth and enhances symptoms of speck disease on susceptible tomato plants. The virulence activity of AvrPto is due, in part, to its interaction with components of host pattern recognition receptor complexes, which disrupts pathogen-associated molecular pattern-triggered immunity. This disruption mechanism requires a structural element of the AvrPto protein, the CD loop, which is also required for triggering Pto/Prf-mediated resistance in tomato. We have shown previously that the carboxyl-terminal domain (CTD) of AvrPto is phosphorylated and also contributes to bacterial virulence. Here we report that phosphorylation of the CTD on S147 and S149 promotes bacterial virulence in an FLS2/BAK1-independent manner, which is mechanistically distinct from the CD loop. In a striking corollary with Pto recognition of the CD loop in tomato, the tobacco species Nicotiana sylvestris and Nicotiana tabacum have a recognition mechanism that specifically detects the phosphorylation status of the CTD. Thus different species in the Solanaceae family have evolved distinct recognition mechanisms to monitor the same type III effector.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

Phosphorylation of receptor-like cytoplasmic kinases by bacterial Flagellin

Molecular mechanisms that distinguish self and non-self are fundamental in innate immunity to prevent infections in plants and animals. Recognition of the conserved microbial components triggers immune responses against a broad spectrum of potential pathogens. In Arabidopsis, bacterial flagellin was perceived by a leucine-rich repeat-receptor-like kinase (LRR-RLK) FLS2. Upon flagellin perception, FLS2 forms a complex with another LRR-RLK BAK1. The intracellular signaling events downstream of FLS2/BAK1 receptor complex are still poorly understood. We recently identified a receptor-like cytoplasmic kinase (RLCK) BIK1 that associates with flagellin receptor complex to initiate plant innate immunity. BIK1 is rapidly phosphorylated upon flagellin perception in an FLS2- and BAK1-dependent manner. BAK1 directly phosphorylates BIK1 with an in vitro kinase assay. Plants have evolved a large number of RLCK genes involved in a wide range of biological processes. We provided evidence here that additional RLCKs could also be phosphorylated by flagellin and may play redundant role with BIK1 in plant innate immunity.     

The multifaceted function of BAK1/SERK3: Plant immunity to pathogens and responses to insect herbivores

Almost a decade ago BRI1-associated kinase 1 (BAK1) was identified as a co-receptor of brassinosteroid (BR) insensitive 1 (BRI1), the receptor for BRs, which plays an essential role in transducing BR signaling to regulate plant development. BAK1 is also critical in resistance to various pathogens. BAK1 rapidly binds to certain receptors for pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) after the perception of pathogen elicitors and is required for the full elicitation of pathogen-induced defense responses, such as the activation of the mitogen-activated protein kinase 6 (MPK6) and production of reactive oxygen species. Thus, BAK1 functions in both BR signaling and PAMP-triggered immunity (PTI). Recently BAK1 was also found to play an important role in mediating defense responses against an insect herbivore (Manduca sexta) of Nicotiana attenuata. In this interaction, BAK1 positively modulates wound- or herbivore feeding-induced accumulation of jasmonic acid (JA) and JA-isoleucine (JA-Ile). This mini-review summarizes recent advances in our understanding of the functions of BAK1 in resistance to pathogens and herbivores.Key words: BAK1, defense, herbivore, immunity, insect, jasmonate, pathogen, wounding     

Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis

Plants possess two distinct types of immune receptor. The first type, pattern recognition receptors (PRRs), recognizes microbe-associated molecular patterns (MAMPs) and initiates pattern-triggered immunity (PTI) on recognition. FLS2 is a PRR, which recognizes a part of bacterial flagellin. The second type, resistance (R) proteins, recognizes pathogen effectors and initiates effector-triggered immunity (ETI) on recognition. RPM1, RPS2 and RPS5 are R proteins. Here, we provide evidence that FLS2 is physically associated with all three R proteins. Our findings suggest that signalling interactions occur between PTI and ETI at very early stages and/or that FLS2 forms a PTI signalling complex, some components of which are guarded by R proteins.     

Danger peptide receptor signaling in plants ensures basal immunity upon pathogen‐induced depletion of BAK1

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.     

The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.     

Stabbing in the BAK--an original target for avirulence genes of plant pathogens

Paradoxically, plant pathogens possess avirulence genes that render them avirulent in resistant hosts. In Cell Host & Microbe, Shan et al. (2008) show that the original role of the Pseudomonas syringae avirulence genes AvrPto and AvrPtoB is to target BAK1, a protein kinase important in hormone and innate immunity signaling.     

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors

Pseudomonas syringae pv. tomato DC3000 and its derivatives cause disease in tomato, Arabidopsis and Nicotiana benthamiana. The primary virulence factors include a repertoire of 29 effector proteins injected into plant cells by the type III secretion system and the phytotoxin coronatine. The complete repertoire of effector genes and key coronatine biosynthesis genes have been progressively deleted and minimally reassembled to reconstitute basic pathogenic ability in N. benthamiana, and in Arabidopsis plants that have mutations in target genes that mimic effector actions. This approach and molecular studies of effector activities and plant immune system targets have highlighted a small subset of effectors that contribute to essential processes in pathogenesis. Most notably, HopM1 and AvrE1 redundantly promote an aqueous apoplastic environment, and AvrPtoB and AvrPto redundantly block early immune responses, two conditions that are sufficient for substantial bacterial growth in planta. In addition, disarmed DC3000 polymutants have been used to identify the individual effectors responsible for specific activities of the complete repertoire and to more effectively study effector domains, effector interplay and effector actions on host targets. Such work has revealed that AvrPtoB suppresses cell death elicitation in N. benthamiana that is triggered by another effector in the DC3000 repertoire, highlighting an important aspect of effector interplay in native repertoires. Disarmed DC3000 polymutants support the natural delivery of test effectors and infection readouts that more accurately reveal effector functions in key pathogenesis processes, and enable the identification of effectors with similar activities from a broad range of other pathogens that also defeat plants with cytoplasmic effectors.