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Conserved Fungal LysM Effector Ecp6 Prevents Chitin-Triggered Immunity in Plants

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Science  20 Aug 2010:
Vol. 329, Issue 5994, pp. 953-955
DOI: 10.1126/science.1190859

Abstract

Multicellular organisms activate immunity upon recognition of pathogen-associated molecular patterns (PAMPs). Chitin is the major component of fungal cell walls, and chitin oligosaccharides act as PAMPs in plant and mammalian cells. Microbial pathogens deliver effector proteins to suppress PAMP-triggered host immunity and to establish infection. Here, we show that the LysM domain–containing effector protein Ecp6 of the fungal plant pathogen Cladosporium fulvum mediates virulence through perturbation of chitin-triggered host immunity. During infection, Ecp6 sequesters chitin oligosaccharides that are released from the cell walls of invading hyphae to prevent elicitation of host immunity. This may represent a common strategy of host immune suppression by fungal pathogens, because LysM effectors are widely conserved in the fungal kingdom.

Multicellular organisms activate immune responses upon recognition of microbe-derived nonself components. These responses are mediated by pattern recognition receptors (PRRs), cell surface receptors that recognize invariant structures, usually originating from microbial surfaces that are essential for microbial survival and not present in the host. These microbial structures are known as pathogen-associated molecular patterns (PAMPs) (14). Well-known PAMPs include bacterial lipopolysaccharides, peptidoglycans, flagellin, and fungal cell wall–derived glucans and mannans (46). Also chitin, an unbranched β-1-4–linked N-acetyl-glucosamine (GlcNAc) homopolymer that is the major structural component of fungal cell walls, acts as a PAMP in many organisms (68). In the plant species rice and Arabidopsis, single PRRs were shown to be required for the activation of host immunity upon chitin perception (912). Mutants in these receptors are compromised in their response to chitin and are impaired in their defense against chitin-containing fungal pathogens, which indicates that perception of chitin fragments plays a pivotal role in resistance of plants to fungal pathogens. Both chitin receptors of rice and Arabidopsis were shown to contain extracellular LysM domains that generally occur in glycan-binding proteins (913).

Cladosporium fulvum is a fungal pathogen that causes leaf mold of tomato (Solanum lycopersicum) (14). During colonization of the intercellular spaces of the leaves, the fungus secretes effector proteins to establish disease, one of which, Avr4, is a chitin-binding lectin that protects fungal cell walls against hydrolysis by plant chitinases (15, 16). Recently, the in planta abundantly secreted C. fulvum LysM domain–containing effector Ecp6 was identified and shown to be required for full virulence (17, 18).

We first examined the affinity of Ecp6 for insoluble polysaccharides because the presence of three LysM domains in Ecp6 suggested that it has glycan-binding activity. Ecp6 showed specific affinity for chitin, as it coprecipitated with insoluble chitin (chitin beads and crab shell chitin), but not with chitosan (i.e., deacytelated chitin) or the plant cell wall polysaccharides xylan and cellulose (19) (Fig. 1A). To further examine Ecp6 substrate specificity, a glycan array was used to test the affinity of Ecp6 for more than 400 different glycan substrates (20). Ecp6 only bound to the three chitin oligosaccharides present on the array, (GlcNAc)3, (GlcNAc)5, and (GlcNAc)6, but not to any other glycan, including the N-linked glycan chitobiose (Fig. 1B and table S1). Therefore, we conclude that Ecp6 is a highly specific chitin-binding LysM effector.

Fig. 1

C. fulvum Ecp6 binds chitin. (A) Affinityprecipitation (19) ofEcp6 with insoluble chitin. Ecp6 protein remaining in concentrated supernatant(S) and the insoluble polysaccharide pellet (P) after SDS-polyacrylamide gel electrophoresis and Coomassie staining. Ecp6 is specifically precipitated withchitin (beads) and chitin from crab shells, but not with other insoluble polysaccharides of plants (xylan, cellulose) and fungi (chitosan). The figure is representative of three independent experiments. (B) Glycan array analysis of Ecp6. Relative fluorescence upon scanning of a glycan array that contains probes for 406 glycans (see table S1 for identities) after Ecp6 hybridization. Ecp6 only hybridizes to probes 170 to 172 representing (GlcNAc)6, (GlcNAc)5, and (GlcNAc)3,respectively. (C) Thermodynamics of binding of chitin oligosaccharides with different degrees of polymerization to Ecp6. The number of calculated binding sites per protein molecule is represented byn.

The affinity of Ecp6 for soluble chitin oligosaccharides was determined by isothermal titration calorimetry (ITC). The binding curves for chitin tetra-, penta- and hexamer oligosaccharides [(GlcNAc)4, (GlcNAc)5, and (GlcNAc)6, respectively] obeyed a “one binding site” model, revealing three binding sites for these oligosaccharides per Ecp6 molecule, which matches with the three LysM domains in Ecp6 (fig. S1, A to C). The (GlcNAc)8 binding curve deviated from this model, which suggested that the size of this octamer allows it to interact with multiple LysM domains simultaneously (fig. S1D). The dissociation constant (Kd) for the various GlcNAc oligosaccharides was similar and decreased from 11.5 to 3.7 μM between (GlcNAc)4 and (GlcNAc)8 (Fig. 1C), which showed that Ecp6 had high affinity for chitin oligosaccharides of various lengths. It was previously determined that the invertebrate (CBM14) chitin-binding domain of Avr4 exclusively interacts with (GlcNAc)3 repeats and that the Avr4 Kd decreased from 1.3mM [mu]M to 6.3 μM between (GlcNAc)4 and (GlcNAc)6 (21). This shows that, in contrast to Ecp6, Avr4 has low affinity for short-chain chitin oligosaccharides.

Avr4 fully protects fungal cell walls against hydrolysis by plant chitinases at a concentration of 10 μM (15, 16). Despite its chitin-binding activity, however, 10 μM or 100 μM Ecp6 failed to protect the fungus Trichoderma viride against hydrolysis by crude extracts of tomato leaves containing intracellular basic chitinases (Fig. 2A and fig. S2). We conclude that Ecp6 effector function did not involve prevention of the hydrolysis of fungal cell walls by plant chitinases.

Fig. 2

Ecp6 cannot protect fungal hyphae from hydrolysis by tomato chitinases but inhibits chitin-induced medium alkalinization of tomato cell suspensions.(A) Micrographs of Trichoderma viride taken 24hours after the addition of water (w), crude extract of tomato leaves containing intracellular, basic chitinases (ChiB), pretreatment with 10 μM Ecp6followed by addition of tomato extract (Ecp6 & ChiB), and pretreatment with 10μM Avr4 followed by addition of tomato extract (Avr4 & ChiB). Scalebars, 50 μm. The figure is representative of three independent experiments. (B) Medium alkalinization of tomato cell suspensions induced by chitin oligosaccharides is inhibited by Ecp6. TheΔpHmax determined after treatment of tomato cell suspensions with mixtures of chitin oligosaccharides (GlcNAc)6 andEcp6, after normalization to the response upon treatment with 10 nM(GlcNAc)6 only, is indicated (19). Bars represent means ± standard error of at least three replicate experiments. Statistically significant differences when compared to treatment with 10 nM (GlcNAc)6 were determined using the Dunnett test (two-sided; *P <0.05).

Apart from hydrolyzing fungal cells, host chitinases cause the release of chitin oligosaccharide PAMPs from the cell walls of the invading fungus (6, 22). Suspension-cultured plant cells react with medium alkalinization to treatment with nanomolar concentrations of chitin oligosaccharides (8). We speculated that LysM effectors might affect chitin perception by the host (17, 18) and tested the effect of Ecp6 treatment on PAMP-triggered immunity by measuring chitin-induced pH shifts in tomato and tobacco cell suspensions. Treatment of the cells with nanomolar chitin oligosaccharide [(GlcNAc)6] concentrations resulted in medium alkalinization, whereas addition of equimolar amounts of Ecp6 indeed attenuated this response (Fig. 2B). The pH shift attenuation occurred in a dose-dependent manner and, similarly, occurred with various chitin oligosaccharides of different lengths and in both tomato and tobacco suspensions (Fig. 2B and fig. S3A). In contrast, even a 10-fold molar excess of Avr4 did not affect the chitin-induced pH shift (Fig. 2B and fig. S3B). Similarly, a 10-fold excess of the C. fulvum effectors Avr9, Ecp1, and Ecp4, which, like Ecp6, are small cysteine-rich proteins that are abundantly secreted in the apoplast during colonization of the host plant but that do not bind chitin (15) (fig. S3C), did not affect the chitin-induced pH shift (fig. S3B). These data show that only Ecp6 is able to suppress chitin-triggered immunity. The control oligosaccharides laminarihexaose (β-1,3-glucan), d-cellohexaose (β-1,4-glucan), chitosan hexamer (GlcN)6, and chitosan did not induce a pH shift in tomato cell suspensions (fig. S3D). As expected, Ecp6 did not inhibit alkalinization induced by the bacterial PAMP flg22, the epitope of bacterial flagellin, which suggested that suppression of chitin-triggered immunity by Ecp6 occurs through specific binding of chitin oligosaccharide PAMPs (fig. S3E).

These findings were further substantiated in experiments to assess whether, besides cell suspensions, Ecp6 also perturbs chitin-induced host immunity in tomato and tobacco leaves. Treatment of leaf discs with (GlcNAc)6 resulted in the production of reactive oxygen species (ROS), whereas this response was abolished in the presence of Ecp6 (Fig. 3). Furthermore, inhibition of the induction of chitin-responsive genes in the presence of Ecp6 was recorded in tomato leaves (fig. S4). Similar to the alkalinization response in cell suspensions, Avr4 and Avr9 did not affect the chitin-induced ROS production, and Ecp6 could not inhibit the flg22-induced ROS burst (fig. S5).

Fig. 3

The chitin-induced oxidative burst in (A) tomato, Solanum lycopersicum, and (B) tobacco, N. benthamiana, leaf discs is inhibited by Ecp6. Production of ROS was determined using luminol-dependent chemiluminescence (19). (Left) Integrated images of 5-minexposures were analyzed with ImageJ, and the relative luminescence was calculated by normalization to water-treated leaf discs and plotted. (Right) Representative image sequence of a single experiment showing the ROS-dependent luminescence over time in leaf discs treated with one of the following: water,10 μM (GlcNAc)6 (■), 1 μM (GlcNAc)6(○), a mixture of 10 μM (GlcNAc)6 and 10 μM Ecp6(◊), a mixture of 1 μM (GlcNAc)6 and 10 μM Ecp6(Δ), and 10 μM Ecp6 (□). The figure is representative of three independent experiments.

Finally, we tested whether Ecp6 is able to compete directly for chitin binding with a plant PRR. It has previously been demonstrated that the rice PRR CEBiP directly binds chitin oligosaccharides (9). As chitin binding was shown to this receptor, we performed competition assays in which a microsomal membrane preparation from suspension-cultured rice cells containing CEBiP was treated with biotinylated (GlcNAc)8 in the presence or absence of Ecp6 (23). Although incubation of microsomal membranes with 0.4 μM biotinylated (GlcNAc)8 resulted in labeling of the CEBiP receptor, incubation in the presence of a 100-fold molar excess of nonbiotinylated (GlcNAc)8 prevented receptor labeling (Fig. 4). Incubation of microsomal membranes with biotinylated (GlcNAc)8 in the presence of an equimolar amount of Ecp6 almost completely prevented receptor labeling while a signal at the height of Ecp6 was observed, which demonstrated that Ecp6 directly competes for chitin binding with the CEBiP chitin receptor (Fig. 4).

Fig. 4

Ecp6 competes for chitin binding with the rice chitin receptor and inhibits chitin-induced defense responses in rice cells. (A) Western blot using a biotin antibody showing affinity labeling of a microsomal membrane preparation (Rice MF) from suspension-cultured rice cells containing the PRR CEBiP, with biotinylated (GlcNAc)8 [(GlcNAc)8-BIO], in the absence or presence of Ecp6, nonbiotinylated (GlcNAc)8, and the control proteins concanavalin A, myoglobin, and trypsin inhibitor. The experiment was performed twice with similar results. (B) Ecp6 inhibits the chitin-induced oxidative burst in rice suspension cells. Production of ROS 20 min after induction with 1 nM (GlcNAc)8 was determined as described previously (9) in the absence or presence of Ecp6 (1and 10 nM). The experiment was performed twice with similar results.(C) Ecp6 inhibits chitin-induced PAL1 gene expression in rice suspension cells. The bars display the relative transcript level of the chitin-responsive gene PAL1 normalized to the constitutively expressed ubiquitin gene, and the relative transcript level of the suspension cells treated with 1 μM (GlcNAc)8 was set at100%. The mean with standard error of two replicate experiments is shown, and asterisks indicate significant differences (P < 0.05) when compared with the 1 μM (GlcNAc)8 treatment.

In conclusion, our data show that the abundantly secreted C. fulvum LysM effector Ecp6 is a chitin-binding lectin that inhibits activation of chitin-triggered host immunity. Thus, at present, two chitin-binding C. fulvum effectors have been identified; Avr4, which carries an invertebrate chitin-binding domain with high affinity for long-chain chitin oligosaccharides and which protects fungal hyphae against lysis by plant chitinases, and Ecp6, which carries LysM domains with high affinity for various short-chain chitin oligosaccharides and which prevents activation of chitin PAMP-triggered immunity. These distinct activities of both effectors corroborate the finding that Avr4 protects hyphae against hydrolysis by basic, vacuolar, endochitinases that are released by the host upon cellular collapse (15, 16), but not necessarily against exochitinases that are present in the apoplast and that are able to release short-chitin oligosaccharides from the fungal cell wall. Besides differing from Avr4, the scavenger function of Ecp6 also differs from the role of the effector Avr2, which is secreted by C. fulvum to inhibit extracellular tomato cysteine proteases that are required for host basal defense (2426). Ecp6 orthologs are present in many fungi, often occurring in multigene families (18). This suggests that scavenging of chitin oligosaccharides to avoid perception by other organisms may be an important survival strategy of fungi.

Supporting Online Material

www.sciencemag.org/cgi/content/full/329/5994/953/DC1

Materials and Methods

Figs. S1 to S5

Table S1

References

References and Notes

  1. Materials and Methods are available as supporting online material on Science Online.
  2. We are indebted to Yaizu Suisankagaku Industrial Co. Ltd. for the supply of chitosan oligosaccharides and to the Consortium for Functional Glycomics (CFG) for performing the glycan array analysis. This research was supported by a Vidi grant of the Research Council for Earth and Life Sciences (ALW) of the Netherlands Organization for Scientific Research (NWO), by the European Research Area–Network (ERA-NET) Plant Genomics and by the Centre for BioSystems Genomics (CBSG), which is part of the Netherlands Genomics Initiative and NWO. Part of this research was supported by the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN) and a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors thank P. de Wit, F. Govers, T. Bisseling, N. Talbot, and anonymous reviewers for critical evaluation of the manuscript and L. Verhage and L. Kaaij for technical assistance.
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