Author response: Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis
2014-05-23

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites. https://doi.org/10.7554/eLife.01990.001 eLife digest The immune response of plants and animals is triggered when cells detect small molecules that are present on the surface of the bacteria or fragments of peptidoglycans—the polymers that are a major component of the bacterial cell wall. The mechanisms by which small molecules trigger the immune response in plants have been widely studied in the model plant Arabidopsis thaliana, but less is known about the ways in which peptidoglycan fragments can initiate an immune response. Proteins called lysozymes are known to break peptidoglycans into smaller pieces in animals. Plants do not produce lysozymes, but they do produce other enzymes such as chitinases that have similar properties. Now Liu, Grabherr, et al. have shown that a chitinase called LYS1 acts as an enzyme that catalyzes the breakdown of peptidoglycans and has a central role in triggering the immune response of Arabidopsis. Plants that were genetically engineered to produce little or no LYS1 were highly susceptible to bacterial infection because there were no enzymes that could break the peptidoglycans into smaller fragments. However, plants that were engineered to produce very high levels of LYS1 also had a compromised immune response because the peptidoglycans were broken into fragments that were too small to be detected. The findings of Liu, Grabherr et al. demonstrate that animals and plants employ similar strategies to break down bacterial peptidoglycans to allow them to be detected by the immune system. However, as the enzymes responsible have different structures, they are likely to have evolved separately in plants and animals. https://doi.org/10.7554/eLife.01990.002 Introduction Activation of antibacterial defenses in multicellular eukaryotic organisms requires recognition of bacterial surface patterns through host-encoded pattern recognition receptors (PRRs) (Chisholm et al., 2006; Jones and Dangl, 2006; Ishii et al., 2008; Boller and Felix, 2009; Vance et al., 2009; Segonzac and Zipfel, 2011; Monaghan and Zipfel, 2012; Broz and Monack, 2013; Stuart et al., 2013). Immunogenic microbial signatures are collectively referred to as pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) (Janeway and Medzhitov, 2002). Bacteria-derived PAMPs such as lipopolysaccharides (LPS) or flagellins possess immunity-stimulating activities in metazoans and plants, suggesting that the ability to sense bacterial surface structures and mount immunity is conserved across lineage borders (Nürnberger et al., 2004; Boller and Felix, 2009). Likewise, peptidoglycans (PGNs) are major building blocks of the cell walls of Gram-positive and Gram-negative bacteria that have been shown to trigger host immune responses in mammalians, insects, and plants (Dziarski and Gupta, 2005; Gust et al., 2007; Erbs et al., 2008; Kurata, 2014). Structurally, PGNs are heteroglycan chains that are composed of polymeric alternating β(1,4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues (Schleifer and Kandler, 1972; Glauner et al., 1988). Such chains are interconnected by oligopeptide bridges which form a coordinate meshwork, thereby providing structural integrity to the bacterial envelope. Recognition of different PGN substructures in animal hosts is brought about by structurally diverse PRRs such as nucleotide-binding oligomerization domain-containing proteins (NODs), peptidoglycan recognition proteins (PGRPs/PGLYRPs), scavenger receptors, or Toll-like receptor TLR2 (Strober et al., 2006; Royet and Dziarski, 2007; Dziarski and Gupta, 2010; Müller-Anstett et al., 2010; Magalhaes et al., 2011; Kurata, 2014). In plants, a tripartite PGN recognition system at the plasma membrane of Arabidopsis thaliana with shared functions in PGN sensing and transmembrane signaling was recently described (Willmann et al., 2011). This system comprises Lysin motif (LysM) domain proteins LYM1 and LYM3 for PGN ligand binding and the transmembrane LysM receptor kinase CERK1 that is likely required for conveying the extracellular signal across the plasma membrane and for initiating intracellular signal transduction. All three proteins were shown to be indispensable for PGN sensitivity and to contribute to immunity to bacterial infection (Willmann et al., 2011), which is in agreement with their proposed role as a PGN sensor system. More recently, a similar PGN perception system made of LysM domain proteins LYP4 and LYP6 has been reported from rice (Liu et al., 2012a). Microbial patterns such as bacterial PGN, LPS, flagellin, or fungal chitin harbor immunogenic epitopes that are parts of supramolecular structures building microbial surfaces (Boller and Felix, 2009; Kumar et al., 2013; Newman et al., 2013; Pel and Pieterse, 2013). It is therefore assumed that recognition by host PRRs most likely requires the presence of soluble, randomly structured ligands derived from a complex matrix. X-ray structure-based insight into the binding of bacterial flagellin to the Arabidopsis receptor complex FLS2/BAK1 or of fungal chitin to the Arabidopsis receptor CERK1 supports this view (Willmann and Nürnberger, 2012; Liu et al., 2012b; Sun et al., 2013). Moreover, the existence of fungal LysM effector proteins that scavenge soluble chitin fragments, thus preventing recognition by plant PRRs, suggests that mechanisms releasing these soluble fragments from fungal cell walls must exist (de Jonge et al., 2010). Most often, however, it is an open question whether soluble ligand presentation to eukaryotic host PRRs is the result of spontaneous decomposition of the microbial extracellular matrix during infection or, alternatively, whether host-derived factors contribute to the generation of immunogenic ligands for PRR activation. For example, only monomers of bacterial flagellin induce immune responses through human TLR5 whereas filamentous flagella, in which the immunogenic flagellin structure is buried and thus is not accessible to TLR5, do not (Smith et al., 2003). It was proposed that a number of circumstances cause flagellin monomer release from intact flagella. For instance, Caulobacter crescentus deliberately ejects its flagellum once it is no longer required for the bacterial life cycle (Jenal and Stephens, 2002). Moreover, during infection, Pseudomonas aeruginosa produces rhamnolipids which act as surfactants and cause flagellin shedding from intact flagella, resulting in a more pronounced immune response (Gerstel et al., 2009). Alternatively, host factors such as proteases or environmental conditions such as pH, temperature, or bile salts have been proposed to mediate shearing of flagella from bacterial surfaces (Ramos et al., 2004). Likewise, recognition of PGN by intracellular receptors, such as mammalian NOD1 and NOD2, or by plasma membrane receptors, such as mammalian TLR2 or plant LYM1, LYM3 and CERK1 (Müller-Anstett et al., 2010; Sorbara and Philpott, 2011; Willmann et al., 2011), is facilitated by soluble ligands. Animal lysozymes have been implicated in PGN hydrolysis, bacterial lysis, and host immunity (Callewaert and Michiels, 2010), probably through partial PGN degradation and generation of soluble ligands for PGN sensors (Cho et al., 2005; Dziarski and Gupta, 2010; Davis et al., 2011). In plants, knowledge of the mode of release of immunogenic fragments from microbial extracellular structures and their contribution to plant immunity is lacking. We here describe a plant enzyme activity (LYS1) that hydrolyzes β(1,4) linkages between N-acetylmuramic acid and N-acetylglucosamine residues in PGN and between N-acetylglucosamine residues in chitooligosaccharides, thus closely resembling metazoan lysozymes (EC 3.2.1.17). Importantly, PGN breakdown products produced by LYS1 are immunogenic in plants, and LYS1 mutant genotypes were immunocompromised upon bacterial infection. Our findings suggest that plant enzymatic activities, such as LYS1, are capable of generating soluble PRR ligands that might contribute to the activation of immune responses in cells at and surrounding the site of their generation. We also infer that eukaryotic hosts more generally make concerted use of PGN hydrolytic activities and of PRRs in order to cope with bacterial infections. Results Arabidopsis PGN binding proteins LYM1 and LYM3 are devoid of PGN hydrolytic activity Soluble oligomeric PGN fragments have previously been shown to stimulate plant immune responses in Arabidopsis (Gust et al., 2007; Erbs et al., 2008; Willmann et al., 2011). As some metazoan PGRPs harbor PGN-degrading enzyme activities (Gelius et al., 2003; Wang et al., 2003; Bischoff et al., 2006; Dziarski and Gupta, 2010; Kurata, 2010), we tested whether recombinant Arabidopsis PGN binding proteins LYM1 and LYM3 were able to catalyze PGN degradation. For this, we have employed a standard lysozyme assay (Park et al., 2002) that is based on reduced turbidity in suspensions of Gram-positive Micrococcus luteus cell wall preparations due to PGN degradation. PGN-degrading activity of hen egg-white lysozyme served as a positive control in these assays. As shown in Figure 1A, lysozyme, but not recombinant LYM1 or LYM3, displayed cell wall-degrading lytic activity, suggesting that the latter are unable to release PGN fragments from bacterial cell walls. This is in agreement with a lack of sequence similarities between LYM1 or LYM3 and known metazoan PGN hydrolytic activities. We therefore conclude that LYM1 and LYM3 constitute plant PGN sensors that appear to be functionally related to non-enzymatic mammalian or Drosophila PGRPs (Cho et al., 2005; Bischoff et al., 2006; Dziarski and Gupta, 2010; Kurata, 2010). Figure 1 Download asset Open asset The Arabidopsis lysozyme 1 (LYS1) gene is transcriptionally activated upon pathogen-infection. (A) LYM1 and LYM3 do not possess peptidoglycan (PGN) hydrolytic activity. Micrococcus luteus cell wall preparations were incubated with 20 μg affinity-purified His6-tagged LYM1 or LYM3 or 0.5 μg hen egg-white lysozyme and PGN hydrolytic activity was assayed in a turbidity assay at the indicated time points. As negative control (nc), non-induced His6-tagged LYM3 bacterial lysates were used for affinity purification and eluates were subjected to turbidity assays. Means ± SD of three replicates per sample are given. Statistical significance compared with the negative control (**p<0.001, ***p<0.0001, Student’s t test) is indicated by asterisks. (B) Multiple sequence alignment of the 24 Arabidopsis chitinases using the ClustalW2 algorithm. Full length amino acid sequences were aligned and subgroups were classified according to Passarinho and de Vries (2002). Arabidopsis lysozyme 1 (LYS1, At5g24090) represents the only member of class III. (C) The expression of LYS1 in transgenic pLYS1::GUS reporter plants. Leaf halves of transgenic pLYS1::GUS or pPR1::GUS reporter plants were infiltrated with the virulent Pseudomonas syringae pv. tomato (Pto) DC3000, the type III secretion system-deficient Pto DC3000 hrcC- or the avirulent Pseudomonas syringae pv. phaseolicola (Pph) strain (108 cfu/ml) or 10 mM MgCl2 as control. After 24 hr the leaves were harvested and stained for β-glucuronidase (GUS) activity. (D) Leaves of wild-type plants were treated for 3 or 24 hr with 1 μM flg22, 100 μg/ml PGN from Pto or 100 μg/ml lipopolysaccharide (LPS). Total RNA was subjected to RT-PCR using LYS1 or Flagellin-responsive kinase 1 (FRK1) specific primers. EF1α transcript was used for normalization. All experiments shown in panels (A), (C) and (D) were repeated once with similar results. https://doi.org/10.7554/eLife.01990.003 LYS1 expression is activated upon bacterial infection Lysozymes (EC 3.2.1.17) hydrolyze β(1,4) linkages between N-acetylmuramic acid and N-acetylglucosamine residues in PGNs and between N-acetylglucosamine residues in chitodextrins (http://enzyme.expasy.org/EC/3.2.1.17). Plant genomes do not encode lysozyme-like proteins, but many plant species produce lysozyme-like enzyme activities such as chitinases (EC 3.2.1.14) (Audy et al., 1988; Sakthivel et al., 2010). Plant chitinases fall into five classes (I–V, Figure 1B) (Passarinho and de Vries, 2002) and are grouped into structurally unrelated families 18 and 19 of glycosyl hydrolases, respectively (Henrissat, 1991). Chitinases belonging to family 18 of glycosyl hydrolases are ubiquitously found in all organisms whereas chitinases of glycosyl hydrolase family 19 are found almost exclusively in plants. Class III chitinases (glycosyl hydrolase family 18) represent bifunctional plant enzymes with lysozyme-like activities. One such enzyme, hevamine from the rubber tree Hevea brasiliensis (Beintema et al., 1991), has been shown to hydrolyze PGN and the structurally closely related β(1,4)-linked GlcNAc homopolymer chitin in vitro (Bokma et al., 1997). To explore host-mediated PGN degradation and its possible implication in plant immune activation, we have addressed the only class III chitinase (which we named LYS1, At5g24090) encoded by the Arabidopsis genome (Passarinho and de Vries, 2002; Figure 1B). Bacterial infection of Arabidopsis plants stably expressing a pLYS1::GUS construct revealed that LYS1 gene expression is enhanced upon infection with host non-adapted Pseudomonas syringae pv. phaseolicola (Pph) or disarmed host adapted P. syringae pv. tomato (Pto) DC3000 hrcC−. Likewise, expression of the immune response marker pathogenesis-related protein 1 (PR1) was enhanced by the same treatment (Figure 1C). Failure to detect LYS1 expression in plants infected with virulent host adapted Pto DC3000 suggests bacterial effector-mediated suppression that is reminiscent of that observed for PGN receptor proteins LYM1 and LYM3 (Willmann et al., 2011) as well as numerous other immunity-associated genes (Kemmerling et al., 2007; Postel et al., 2010). LYS1 gene expression is not only triggered upon bacterial infection, but was also observed upon treatment with different MAMPs including bacterial flagellin, LPS, or PGN preparations (Figure 1D), similar to the immune marker gene Flagellin-responsive kinase 1 (FRK1). Altogether, infection-induced LYS1 transcriptional activation suggests that the LYS1 protein is implicated in immunity to bacterial infection. LYS1 is a plant lysozyme To analyze the enzymatic properties of LYS1, recombinant protein production was attempted. Overexpression in Escherichia coli failed to produce active enzyme and LYS1 production in eukaryotic Pichia pastoris entirely failed to produce recombinant protein (not shown). Therefore, we resorted to generate p35S::LYS1-GFP-overexpressing (LYS1OE) plants (Figure 2A,B). Notably, LYS1-GFP was glycosylated (Figure 2C), possibly explaining the failure to produce enzymatically active LYS1 protein in E. coli. Expression of the green fluorescent protein (GFP) fusion protein in Arabidopsis plants was accompanied by substantial proteolytic cleavage resulting in the predominant release of a protein with an approximate molecular mass of 35 kDa, most likely representing untagged LYS1 (Figure 2B). Analysis of this major cleavage product by liquid chromatography-mass spectrometry (LC-MS/MS) after tryptic in-gel digestion and by peptide mass fingerprint not only confirmed the identity of LYS1 in this band but also yielded peptides spanning the whole protein sequence, except for the first 53 amino acids (data not shown), thus indicating cleavage of the LYS1-GFP fusion protein between LYS1 and GFP. Figure 2 Download asset Open asset Analysis of LYS1 overexpression lines. (A) RT-qPCR analyses of transcript levels in mature leaves of two independent transgenic lines expressing p35S::LYS1-GFP (LYS1OE-1, LYS1OE-2) relative to expression levels in wild-type. EF1α transcript was used for normalization. Error bars, SD (n = 3). Statistical significance compared with wild-type (***p<0.001, Student’s t test) is indicated by asterisks. (B) Immunoblot analysis of protein extracts from leaves of two independent LYS1OE lines, a LYS1 knock-down line (LYS1KD-1, see Figure 3) and wild-type plants. Total leaf protein was separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Immunodetection was carried out using α-tobacco class III chitinase (α-Chit) or green fluorescent protein (α-GFP) (both from rabbit) and an anti-rabbit HRP-coupled secondary antibody. Ponceau S red staining of the large subunit of RuBisCO served as loading control. (C) Total protein extracts from leaves of LYS1OE-1 plants were subjected to deglycosylation with a deglycosylation kit (NEB). The negative control (−) was treated as the deglycosylation sample (+) but without addition of the deglycosylation enzyme mix. Immunoblot analysis was carried out as described in (B). All experiments shown were repeated at least once. https://doi.org/10.7554/eLife.01990.004 Three mutant lines with T-DNA insertions in the LYS1 gene were available from the Nottingham Arabidopsis Stock Centre. However, neither the insertion in the 5' untranslated region nor the insertions in the first intron and at the end of the last exon of the coding region abolished formation of the LYS1 transcript (Figure 3—figure supplement 1). As an alternative to knock-out lines, LYS1 knock-down lines (LYS1KD) were produced by artificial micro RNA technology (Schwab et al., 2006; Figure 3). As proven by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), we obtained two genetically independent LYS1KD lines with residual transcript levels not exceeding 10% of those detected in wild-type plants (Figure 3C). In contrast, the transcription of potential off-target genes was not affected (Figure 3C). Protein extracts derived from transgenic plants were tested for chitinolytic activity by employing 4-methylumbelliferyl β-D-N, N′, N′′-triacetylchitotriose (4-MUCT) as substrate. Leaf protein extracts from LYS1OE plants exhibited significant chitinase activity when compared with a Streptomyces griseus chitinase control (Figure 4A). In contrast, wild-type and LYS1KD plants exhibited only marginal chitinase activities. Likewise, using 4-MUCT in a gel electrophoretic separation-based chitinase assay produced a zymogram in which enzyme activity was solely detectable in protein extracts obtained from LYS1OE plants, but not in those from control plants expressing secreted GFP (secGFP) (Figure 4B). Thus, LYS1 indeed harbors the predicted chitinase activity. As 4-MUCT is also a typical substrate for lysozymes (Brunner et al., 1998), this was the first indication that LYS1 might also harbor lysozyme activity. Next, leaf protein extracts from LYS1OE plants were tested for their ability to solubilize complex PGN presented by intact Gram-positive M. luteus cells and to cleave preparations of complex, insoluble Bacillus subtilis PGN. Again, protein extracts from LYS1OE plants exhibited significant PGN-degrading activity whereas wild-type and LYS1KD plants showed basal activity levels only (Figure 4C,D). Likewise, PGN-solubilizing activity profiles of protoplast suspensions derived from these transgenics confirmed significant PGN-degrading activity of LYS1OE plants (Figure 4E). Figure 3 with 1 supplement see all Download asset Open asset Analysis of LYS1 amiRNA lines. (A) Predicted LYS1 gene structure (exons, black bars; introns, black lines; untranslated The region by the amiRNA construct is indicated by an (B) genes for the construct were using the The region by the amiRNA is for are indicated in possess more at or have at 10 which amiRNA (C) levels of the shown in (B) were by RT-qPCR in of two independent transgenic knock-down lines (LYS1KD-1, using for LYS1 and EF1α transcript was used for normalization. Error bars, SD (n = 3). Statistical significance compared with the wild-type control (which was to 1 for is indicated by (***p<0.001, Student’s t The was repeated once with similar results. Figure with 2 see all Download asset Open asset LYS1 is a Protein extracts from wild-type or and lines were assayed for hydrolytic activity Plants expressing secreted green fluorescent protein (GFP) (secGFP) served to control the of GFP. (A) Leaf protein extracts from indicated transgenic plants were assayed for chitinolytic activity using the 4-methylumbelliferyl β-D-N, N′, N′′-triacetylchitotriose (4-MUCT) substrate. activities hr after treatment were using Streptomyces griseus chitinase as positive control (B) Protein extracts from LYS1OE-1 or plants were separated on a gel and hydrolytic activity was assayed by the gel with the substrate are of substrate The the of and Micrococcus luteus cells (C) or Bacillus subtilis peptidoglycan (PGN) (D) were subjected to by leaf protein extracts and PGN hydrolytic activity was after hr using hen egg-white lysozyme as positive control compared with the control are indicated by Student’s t of transgenic lines were and protein extracts of the protoplast or were subjected to the PGN assay as described in As or protoplast was Means ± SD of two replicates per sample are with different are different based on of M. luteus cells was in a turbidity assay with LYS1OE leaf protein extracts as described in (C) at the indicated Means ± SD of two replicates per sample are given. All experiments shown were repeated at least once. To specific enzyme activities, untagged LYS1 was from LYS1OE Arabidopsis lines by protein liquid and used for enzyme assays. The 4-MUCT assay yielded a of ± μM and a of ± μM for LYS1, and a of 53 ± μM and a of ± μM for griseus the turbidity assay with M. luteus cell wall a of ± and of ± were obtained for LYS1, and a of ± and of ± for hen egg-white The for LYS1 are thus to the As shown in Figure the of LYS1 activity was found in the of the suggesting an of To this we from LYS1OE Arabidopsis lines. the LYS1-GFP fusion protein as well as LYS1 was detectable in whereas the protein kinase was only present in the leaf protein (Figure supplement Moreover, expression in the plant system of the p35S::LYS1-GFP construct in of the cell whereas expression of a construct the LYS1 signal sequence yielded of intracellular structures (Figure supplement 1B). of the fluorescent a plasma membrane and marker et al., revealed that LYS1 to a large with the plasma membrane (Figure supplement 1B). Thus, LYS1 likely in of the plant the Arabidopsis cell wall et al., suggests that LYS1 acts in the plant the plant is an et al., we whether LYS1 is active at For this, the M. luteus cell wall-degrading activity of an LYS1OE leaf was at different active at from to a pronounced of LYS1 activity was detected which with the of plant cells (Figure To LYS1 hydrolytic activity, an LYS1 fusion construct was in (Figure to the Arabidopsis LYS1OE leaf extracts from expressing leaves also displayed in-gel chitinolytic activity (Figure compared with extracts from control leaves expressing the Likewise, protein extracts were able to cleave preparations of complex insoluble subtilis PGN (Figure Figure Download asset Open asset LYS1 in hydrolytic activity. (A) Protein extracts from leaves expressing LYS1 to the control of the were separated on an gel and by using the As plants were infiltrated with the of construct Protein are indicated on the (B) protein extracts from leaves expressing or were separated on a gel and hydrolytic activity was assayed by the gel with the substrate 4-methylumbelliferyl β-D-N, N′, are of substrate the of (C) Protein extracts from leaves expressing or were assayed for peptidoglycan (PGN) hydrolytic activity in a turbidity assay using Bacillus subtilis PGN. activities hr were using hen egg-white lysozyme as Statistical significance compared with the control Student’s t test) is indicated by asterisks. All experiments shown were repeated at least once. In we that LYS1 harbors hydrolytic activity for chitin as well as for PGN of the and Importantly, LYS1 failed to exhibit activity on as a indicating it might have no activity (Figure supplement Thus, LYS1 enzymatic activities reported for metazoan lysozymes and be classified as lysozyme (EC 3.2.1.17) of chitinase (EC LYS1 plant immunogenic PGN fragments To analyze immunogenic activities of PGN cleavage products by LYS1, untagged LYS1 was from LYS1OE Arabidopsis lines by and used for degradation of subtilis PGN. PGN fragments found in the of PGN were by high liquid (Figure a could be detected in the of PGN incubated with a control or with In contrast, PGN produced by LYS1 yielded that were also detectable in the of PGN preparations treated with which has been shown to cleave between GlcNAc and residues in complex PGN et al., PGN fragments were tested for their ability to trigger plant immunity-associated responses (Figure