Important Pathogen-associated Molecular Pattern Research Articles

Guanylate-binding protein-5 is involved in inflammasome activation by bacterial DNA but only the cooperation of multiple GBPs accounts for control of Brucella abortus infection.

Guanylate-binding proteins (GBPs) are produced in response to pro-inflammatory signals, mainly interferons. The most studied cluster of GBPs in mice is on chromosome 3. It comprises the genes for GBP1-to-3, GBP5 and GBP7. In humans, all GBPs are present in a single cluster on chromosome 1. Brucella abortus is a Gram-negative bacterium known to cause brucellosis, a debilitating disease that affects both humans and animals. Our group demonstrated previously that GBPs present on murine chromosome 3 (GBPchr3) is important to disrupt Brucella-containing vacuole and GBP5 itself is important to Brucella intracellular LPS recognition. In this work, we investigated further the role of GBPs during B. abortus infection. We observed that all GBPs from murine chromosome 3 are significantly upregulated in response to B. abortus infection in mouse bone marrow-derived macrophages. Of note, GBP5 presents the highest expression level in all time points evaluated. However, only GBPchr3-/- cells presented increased bacterial burden compared to wild-type macrophages. Brucella DNA is an important Pathogen-Associated Molecular Pattern that could be available for inflammasome activation after BCV disruption mediated by GBPs. In this regard, we observed reduced IL-1β production in the absence of GBP2 or GBP5, as well as in GBPchr3-/- murine macrophages. Similar result was showed by THP-1 macrophages with downregulation of GBP2 and GBP5 mediated by siRNA. Furthermore, significant reduction on caspase-1 p20 levels, LDH release and Gasdermin-D conversion into its mature form (p30 N-terminal subunit) was observed only in GBPchr3-/- macrophages. In an in vivo perspective, we found that GBPchr3-/- mice had increased B. abortus burden and higher number of granulomas per area of liver tissue, indicating increased disease severity. Altogether, these results demonstrate that although GBP5 presents a high expression pattern and is involved in inflammasome activation by bacterial DNA in macrophages, the cooperation of multiple GBPs from murine chromosome 3 is necessary for full control of Brucella abortus infection.

Chemiluminescence-Based Assay to Monitor Early Oxidative Bursts in Soybean (Glycine max) Lateral Roots.

The reactive oxygen species (ROS) burst assay is a valuable tool for studying pattern-triggered immunity (PTI) in plants. During PTI, the interaction between pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) leads to the rapid production of ROS in the apoplastic space. The resultant ROS can be measured using a chemiluminescent approach that involves the usage of horseradish peroxidase and luminol. Although several methods and protocols are available to detect early ROS bursts in leaf tissues, no dedicated method is available for root tissues. Here, we have established a reliable method to measure the PAMP-triggered ROS burst response in soybean lateral roots. In plants, lateral roots are the potential entry and colonization sites for pathogens in the rhizosphere. We have used important PAMPs such as chitohexaose, flagellin 22 peptide fragment, and laminarin to validate our method. In addition, we provide a detailed methodology for the isolation and application of fungal cell wall components to monitor the oxidative burst in soybean lateral roots. Furthermore, we provide methodology for performing ROS burst assays in soybean leaf discs with laminarin and fungal cell walls. This approach could also be applied to leaf and root tissues of other plant species to study the PTI response upon elicitor treatment. © 2023 Wiley Periodicals LLC. Basic Protocol: Reactive oxygen species (ROS) burst assay in soybean lateral root tissues Alternate Protocol: ROS burst assay in soybean leaf discs Support Protocol: Isolating fungal cell wall fractions.

A Staphylococcal Glucosaminidase Drives Inflammatory Responses by Processing Peptidoglycan Chains to Physiological Lengths.

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of proinflammatory cytokines, including interleukin-1β (IL-1β). However, how bacterial processing of peptidoglycan drives IL-1β production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified SagB as a mediator of IL-1β production. A ΔsagB mutant fails to promote IL-1β production by macrophages and dendritic cells, and processing of peptidoglycan by SagB is essential for this response. SagB-dependent IL-1β production by macrophages is independent of canonical pattern recognition receptor engagement and NLRP3 inflammasome-mediated caspase activity. Instead, treatment of macrophages with heat-killed cells from a ΔsagB mutant leads to reduced caspase-independent cleavage of pro-IL-1β, resulting in accumulation of the pro form in the macrophage cytosol. Furthermore, SagB is required for virulence in systemic infection and promotes IL-1β production in a skin and soft tissue infection model. Taken together, our results suggest that the length of S. aureus cell wall glycan chains can drive IL-1β production by innate immune cells through a previously undescribed mechanism related to IL-1β maturation.

Innate immune sensing of nucleic acid in endodontic infection.

Innate immunity is the first line of defence and fights against microorganisms. Nucleic acids are important pathogen-associated molecular patterns to be recognized in innate immunity. There are three types of nucleic acid sensors, including endosomal sensors (NA-sensing TLRs), cytosolic DNA sensors (cGAS and AIM2) and cytosolic RNA sensors (RLG-I, MDA5 and LGP2). Recent studies have shown that nucleic acid sensors are expressed differently in a variety of dental pulp cells and mediate inflammation through complex pathways. Nucleic acid sensing may play a vital role in the development of endodontic infection. This review aims to summarize and analyse the possible roles of various nucleic acid sensors in endodontic infection. It may help better understand the pathogenesis of these diseases and find new strategies for prevention and treatment.

α- and β-1,3-Glucan Synthesis and Remodeling.

Glucans are characteristic and major constituents of fungal cell walls. Depending on the species, different glucan polysaccharides can be found. These differ in the linkage of the D-glucose monomers which can be either in α- or β-conformation and form 1,3, 1,4 or 1,6 O-glycosidic bonds. The linkages and polymer lengths define the physical properties of the glucan macromolecules, which may form a scaffold for other cell wall structures and influence the rigidity and elasticity of the wall. β-1,3-glucan is essential for the viability of many fungal pathogens. Therefore, the β-1,3-glucan synthase complex represents an excellent and primary target structure for antifungal drugs. Fungal cell wall β-glucan is also an important pathogen-associated molecular pattern (PAMP). To hide from innate immunity, many fungal pathogens depend on the synthesis of cell wall α-glucan, which functions as a stealth molecule to mask the β-glucans itself or links other masking structures to the cell wall. Here, we review the current knowledge about the biosynthetic machineries that synthesize β-1,3-glucan, β-1,6-glucan, and α-1,3-glucan. We summarize the discovery of the synthases, major regulatory traits, and the impact of glucan synthesis deficiencies on the fungal organisms. Despite all efforts, many aspects of glucan synthesis remain yet unresolved, keeping research directed toward cell wall biogenesis an exciting and continuously challenging topic.

Bactericidal/Permeability-Increasing Protein Is an Enhancer of Bacterial Lipoprotein Recognition.

Adequate perception of immunologically important pathogen-associated molecular patterns like lipopolysaccharide and bacterial lipoproteins is essential for efficient innate and adaptive immune responses. In the context of Gram-negative infection, bactericidal/permeability-increasing protein (BPI) neutralizes endotoxic activity of lipopolysaccharides, and thus prohibits hyperactivation. So far, no immunological function of BPI has been described in Gram-positive infections. Here, we show a significant elevation of BPI in Gram-positive meningitis and, surprisingly, a positive correlation between BPI and pro-inflammatory markers like TNFα. To clarify the underlying mechanisms, we identify BPI ligands of Gram-positive origin, specifically bacterial lipopeptides and lipoteichoic acids, and determine essential structural motifs for this interaction. Importantly, the interaction of BPI with these newly defined ligands significantly enhances the immune response in peripheral blood mononuclear cells (PBMCs) mediated by Gram-positive bacteria, and thereby ensures their sensitive perception. In conclusion, we define BPI as an immune enhancing pattern recognition molecule in Gram-positive infections.

Mutational analysis of vaccinia virus E3 protein: the biological functions do not correlate with its biochemical capacity to bind double-stranded RNA.

Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related antiviral immune responses. However, no direct experimental data confirm such a model. In this study of vaccinia E3 protein, we found that the biological functions of the E3 protein are not necessarily linked to its biochemical capacity of dsRNA binding. Thus, our data strongly point to a new concept of virus modulation of cellular antiviral responses triggered by dsRNA PAMPs.

Recognition of Streptococcus pneumoniae and muramyl dipeptide by NOD2 results in potent induction of MMP-9, which can be controlled by lipopolysaccharide stimulation.

Matrix metallopeptidase 9 (MMP-9) is a protease involved in the degradation of extracellular matrix collagen. Evidence suggests that MMP-9 is involved in pathogenesis during Streptococcus pneumoniae infection. However, not much is known about the induction of MMP-9 and the regulatory processes involved. We show here that the Gram-positive bacteria used in this study induced large amounts of MMP-9, in contrast to the Gram-negative bacteria that were used. An important pathogen-associated molecular pattern (PAMP) for Gram-positive bacteria is muramyl dipeptide (MDP). MDP is a very potent inducer of MMP-9 and showed a dose-dependent MMP-9 induction. Experiments using peripheral blood mononuclear cells (PBMCs) from Crohn's disease patients with nonfunctional NOD2 showed that MMP-9 induction by Streptococcus pneumoniae and MDP is NOD2 dependent. Increasing amounts of lipopolysaccharide (LPS), an important PAMP for Gram-negative bacteria, resulted in decreasing amounts of MMP-9. Moreover, the induction of MMP-9 by MDP could be counteracted by simultaneously adding LPS. The inhibition of MMP-9 expression by LPS was found to be regulated posttranscriptionally, independently of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endogenous inhibitor of MMP-9. Collectively, these data show that Streptococcus pneumoniae is able to induce large amounts of MMP-9. These high MMP-9 levels are potentially involved in Streptococcus pneumoniae pathogenesis.

Steered molecular dynamics identifies critical residues of the Nodamura virus B2 suppressor of RNAi

Nearly all RNA viruses produce double-stranded RNA (dsRNA) during their replication cycles--an important pathogen-associated molecular pattern recognized by the RNA interference (RNAi) pathway in invertebrates and plants. Nodamura virus (NoV) encodes a suppressor of RNA silencing termed B2, which binds to dsRNA and prevents the initiation of RNAi as well as the loading of silencing complexes. Using the published crystal structure of NoV-B2, we performed a series of molecular dynamics (MD) simulations to determine the relative electrostatic and van der Waals contributions of various residues in binding dsRNA, identifying four novel potential interactors: R56, E48, P68 and R69. Additionally, steered MD was used to simulate the binding affinity of NoV-B2 sequences bearing substitutions at positions F49, R56 or R59 to dsRNA, with F49S and R56L/R59L substitutions found to have a significant negative impact on the ability of NoV-B2 to bind dsRNA. NoV RNA1 variants were tested for self-directed replication in both vertebrate (RNAi⁻) and invertebrate (RNAi⁺) cultured cells. Consistent with a role in dsRNA binding, NoV replication in F49C and F49S variant constructs was affected negatively only in RNAi⁺ cells. Thus, we used a combination of MD simulations and experimental mutagenesis to further characterize residues important for NoV-dsRNA interactions.

Genetic variations in MyD88 adaptor-like are associated with atopic dermatitis

Toll-like receptors (TLRs) are important pathogen-associated molecular pattern recognition receptors involved in initiating immune responses. The adaptor protein MyD88 adaptor-like (Mal), involved in signaling downstream of TLRs, plays a crucial role in mediating NF-κB activation. The association of Mal polymorphisms with allergic diseases has not previously been defined. The objective of this study was to detect polymorphisms in the Mal gene and to investigate their association with allergic diseases. Mal gene polymorphisms were genotyped in 310 subjects. The functional effects of Mal variants were analyzed in vitro. One Mal polymorphism, c.303 G>A (Q101Q), was found at a significantly lower frequency in atopic dermatitis patients (p=0.016). Q101Q is in linkage disequilibrium with -103 A>G (rs1893352) and c.539 C>T (S180L) (rs8177374) in the HapMap database. The A allele of -103 A>G showed significantly reduced transcription of Mal compared with the G allele. In addition, three rare variants were identified in this study, c.394 G>A (E132K), c.428 G>A (R143Q) and c.570 G>C (E190D), and were shown to lead to loss-of-function of Mal. It is possible that gene polymorphisms in Mal could affect atopic dermatitis by influencing the innate immune system. We show that Q101Q, which is in linkage disequilibrium with -103 A>G and S180L, may play a protective role against atopic dermatitis. Furthermore, we propose that loss-of-function variants of Mal could predispose individuals to atopic dermatitis or other immunological disorders.

Recognition of Cytoplasmic RNA Results in Cathepsin-Dependent Inflammasome Activation and Apoptosis in Human Macrophages

dsRNA is an important pathogen-associated molecular pattern that is primarily recognized by cytosolic pattern-recognition receptors of the innate-immune system during virus infection. This recognition results in the activation of inflammasome-associated caspase-1 and apoptosis of infected cells. In this study, we used high-throughput proteomics to identify secretome, the global pattern of secreted proteins, in human primary macrophages that had been activated through the cytoplasmic dsRNA-recognition pathway. The secretome analysis revealed cytoplasmic dsRNA-recognition pathway-induced secretion of several exosome-associated proteins, as well as basal and dsRNA-activated secretion of lysosomal protease cathepsins and cysteine protease inhibitors (cystatins). Inflammasome activation was almost completely abolished by cathepsin inhibitors in response to dsRNA stimulation, as well as encephalomyocarditis virus and vesicular stomatitis virus infections. Interestingly, Western blot analysis showed that the mature form of cathepsin D, but not cathepsin B, was secreted simultaneously with IL-18 and inflammasome components ASC and caspase-1 in cytoplasmic dsRNA-stimulated cells. Furthermore, small interfering RNA-mediated silencing experiments confirmed that cathepsin D has a role in inflammasome activation. Caspase-1 activation was followed by proteolytic processing of caspase-3, indicating that inflammasome activation precedes apoptosis in macrophages that had recognized cytoplasmic RNA. Like inflammasome activation, apoptosis triggered by dsRNA stimulation and virus infection was effectively blocked by cathepsin inhibition. In conclusion, our results emphasize the importance of cathepsins in the innate immune response to virus infection.

Influence of the lipopolysaccharide structure of Salmonella enterica serovar Enteritidis on interactions with pig neutrophils

The key process for immune response development is the recognition of bacteria by the immune system of the host based on the sensing of pathogen-associated molecular patterns (PAMP). One of the most important PAMP is the lipopolysaccharide (LPS) molecule, a complex molecule present in the outer membrane of Gram negative bacteria. In this study we were interested in how different parts of the LPS of Salmonella enterica serovar Enteritidis are recognized by porcine neutrophils. To this aim, we constructed S. Enteritidis mutants with rfaL and rfaC genes disabled in the attachment of the O-antigen and in the synthesis of the inner oligosaccharide core of LPS, respectively. We found that in the absence of serum, both the rfa mutants associated with neutrophils and stimulated them for reactive oxygen species (ROS) production significantly more than the wild-type strain. Addition of polymyxin B, which neutralized lipid A, the endotoxic moiety of LPS, effectively decreased the association of the wild-type strain and the rfaC mutant with neutrophils, but not the rfaL mutant. This indicates that the oligosaccharide core newly exposed on the surface in the rfaL mutant, protected from interaction in the wild-type strain by the O-antigen but completely absent in the rfaC mutant, may represent a new ligand for porcine neutrophils that cannot be neutralized by polymyxin B.

Human Serum Mannose-binding Lectin Senses Wall Teichoic Acid Glycopolymer of Staphylococcus aureus, Which Is Restricted in Infancy

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

The mitA gene of Aspergillus fumigatus is required for mannosylation of inositol-phosphorylceramide, but is dispensable for pathogenicity

GDP-mannose:inositol-phosphorylceramide (MIPC)-derived glycosphingolipids are important pathogen-associated molecular patterns (PAMP) of Candida albicans and according to recently published data also of Aspergillus fumigatus. MIPC transferases are essential for the synthesis of MIPC, but have so far been studied only in Saccharomyces cerevisiae and C. albicans. Here, we have identified MitA as the only MIPC transferase in A. fumigatus. The ΔmitA mutant lacks MIPC and MIPC-derived glycosphingolipids and accumulates the precursor IPC. The mutant grows normally, shows no defects in cell wall or membrane organization and a normal resistance to different stressors. It is, however, sensitive to high Ca2+ concentrations, especially during germination. Germination of ΔmitA mutant conidia is also decelerated under normal growth conditions, but neither the virulence of this mutant in a systemic model of infection nor its ability to trigger a cytokine response in macrophages is impaired, arguing against a role of MIPC-derived glycosphingolipids as important A. fumigatus PAMPs.

Global gene expression patterns and induction of innate immune response in human laryngeal epithelial cells in response toAcinetobacter baumanniiouter membrane protein A

The outer membrane protein A of Acinetobacter baumannii (AbOmpA) is an important pathogen-associated molecular pattern that induces host cell death. We determined the gene expression profiles of human laryngeal epithelial HEp-2 cells in response to the sublethal concentration of recombinant AbOmpA (rAbOmpA) and investigated the molecular mechanisms by which rAbOmpA induces an innate immune response. The microarray analysis showed that rAbOmpA sequentially regulated a relatively small set of genes, including those associated with signal transductions and molecules involved in immune response. Among the differentially expressed genes involved in innate immune responses, the surface expression of Toll-like receptor 2 and the production of inducible nitric oxide synthase (iNOS) were prominently observed. However, rAbOmpA did not induce the production of proinflammatory cytokines and chemokines. rAbOmpA activated c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase mitogen-activated protein kinases (MAPKs). Inhibition of JNK MAPK suppressed iNOS production in the rAbOmpA-treated HEp-2 cells. These results suggest that interaction of laryngeal epithelial cells with AbOmpA has a significant impact on the induction of innate immunity during the early stages of A. baumannii infection.

Anti-tumor activity of Acinetobacter baumannii outer membrane protein A on dendritic cell-based immunotherapy against murine melanoma

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-gamma+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens.

A novel ML protein from Manduca sexta may function as a key accessory protein for lipopolysaccharide signaling

Lipopolysaccharide (LPS) present on the outer membrane of Gram-negative bacteria is one of the most important pathogen-associated molecular patterns and a potent elicitor in innate immunity. In human, TLR4 (Toll-like receptor 4) and MD-2 (myeloid differiation-2) form a receptor complex to transduce the LPS signal into cells. However, in invertebrates, receptors that recognize LPS have not been determined. Here we report the purification, characterization and cDNA cloning of an ML (MD-2-related lipid-recognition) protein from the tobacco hornworm Manduca sexta. The full-length cDNA of this M. sexta ML protein, named MsML-1, is 532 bp with an open reading frame of 456 bp that encodes a polypeptide of 151 amino acids containing an ML domain. MsML-1 is a secreted glycoprotein and its mRNA is expressed in fat body and hemocytes. The expression level of MsML-1 mRNA in fat body and hemocytes as well as MsML-1 protein in hemolymph are not induced by immune challenge. Recombinant MsML-1 protein specifically binds to LPS from several Gram-negative bacteria and LPS Re mutant, as well as to lipid A, but not to KDO (2-keto-3-deoxyoctonate). Our results suggest that MsML-1 may function as a key accessory protein for LPS signaling in M. sexta against Gram-negative bacterial infection.

Early events in the pathogenicity of Pseudomonas syringae on Nicotiana benthamiana

Conserved microbial molecules known as PAMPs (pathogen-associated molecular patterns) elicit defence responses in plants through extracellular receptor proteins. One important PAMP is the flagellin protein derived from motile bacteria. We show here that the solanaceous species Nicotiana benthamiana perceives the flagellin proteins of both pathogenic and non-host species of Pseudomonas syringae. The response to flagellin required a gene closely related to that encoding the Arabidopsis thaliana flagellin receptor that we designated NbFls2. In addition, silencing of NbFls2 led to increased growth of compatible, non-host and non-pathogenic strains of P. syringae. Thus, flagellin perception restricts growth of P. syringae strains on N. benthamiana. Pathogenic bacteria secrete effector proteins into the plant cell to enhance virulence. We tested the ability of several unrelated effectors to suppress PAMP-mediated defences. The effector proteins AvrPto and AvrPtoB, but not AvrRps4, suppressed all responses tested including the hypersensitive response induced by non-host flagellins and the oomycete elicitor INF1. Strikingly, transient expression of avrPto or avrPtoB stimulated the growth of non-pathogenic Agrobacterium tumefaciensin planta, suggesting that multiplication of this species is also restricted by PAMP perception. Unexpectedly, AvrPtoB but not AvrPto required the defence-associated genes Rar1, Sgt1 and Eds1 for suppression. This observation separates the respective mechanisms of the two effectors, and suggests that AvrPtoB may target the defence machinery directly for its suppressive effect.