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

Fungal Defenses One of the major driving forces of evolution is the constant arms race between plants and animals and the microbial pathogens that infect them. The fungus Cladosporium fulvum causes leaf mold on tomato plants. One of the ways tomato plants sense infections by C. fulvum is by detecting chitin, a component of fungal cell walls. In response, the fungus has evolved strategies to evade detection. De Jonge et al. (p. 953) have now identified one such mechanism in C. fulvum, mediated by the effector protein Ecp6. Secreted Ecp6 is able to bind to chitin oligosaccharides that are released upon degradation of the fungal cell wall and sequester them so that they are not detected by tomato chitin receptors. Proteins with domain structure similar to Ecp6 are conserved throughout the fungal kingdom, which suggests that chitin sequestration may represent a general mechanism used by fungi to evade immune detection. A fungal protein binds to a host cell wall component to allow the fungus to escape immune responses. 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.

Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease

This work shows that the rice blast fungus secretes a protein that can suppress plant defenses by affecting the way in which chitin, a component of fungal cell walls, is perceived by the rice plant. Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.

Analysis of Two in Planta Expressed LysM Effector Homologs from the Fungus Mycosphaerella graminicola Reveals Novel Functional Properties and Varying Contributions to Virulence on Wheat

Secreted effector proteins enable plant pathogenic fungi to manipulate host defenses for successful infection. Mycosphaerella graminicola causes Septoria tritici blotch disease of wheat (Triticum aestivum) leaves. Leaf infection involves a long (approximately 7 d) period of symptomless intercellular colonization prior to the appearance of necrotic disease lesions. Therefore, M. graminicola is considered as a hemibiotrophic (or necrotrophic) pathogen. Here, we describe the molecular and functional characterization of M. graminicola homologs of Ecp6 (for extracellular protein 6), the Lysin (LysM) domain-containing effector from the biotrophic tomato (Solanum lycopersicum) leaf mold fungus Cladosporium fulvum, which interferes with chitin-triggered immunity in plants. Three LysM effector homologs are present in the M. graminicola genome, referred to as Mg3LysM, Mg1LysM, and MgxLysM. Mg3LysM and Mg1LysM genes were strongly transcriptionally up-regulated specifically during symptomless leaf infection. Both proteins bind chitin; however, only Mg3LysM blocked the elicitation of chitin-induced plant defenses. In contrast to C. fulvum Ecp6, both Mg1LysM and Mg3LysM also protected fungal hyphae against plant-derived hydrolytic enzymes, and both genes show significantly more nucleotide polymorphism giving rise to nonsynonymous amino acid changes. While Mg1LysM deletion mutant strains of M. graminicola were fully pathogenic toward wheat leaves, Mg3LysM mutant strains were severely impaired in leaf colonization, did not trigger lesion formation, and were unable to undergo asexual sporulation. This virulence defect correlated with more rapid and pronounced expression of wheat defense genes during the symptomless phase of leaf colonization. These data highlight different functions for MgLysM effector homologs during plant infection, including novel activities that distinguish these proteins from C. fulvum Ecp6.

Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen

Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms.

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization

While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex. DOI: http://dx.doi.org/10.7554/eLife.00790.001

LysM Effectors: Secreted Proteins Supporting Fungal Life

Fungi occupy a plethora of niches and play essential roles indiverse environments through decomposition of organic materialas saprophytes or through establishment of symbiotic relationshipswith plants and animals that range from mutually beneficial topathogenic. During colonization of their niches, fungi secreteproteins that include carbohydrate-degrading enzymes to feed oncomplex molecules and effectors that mediate the establishment ofinteractions with host organisms [1]. Although effectors aretypically thought to be species- or even lineage-specific, someeffectors are widespread among pathogens, such as the necrosis-and ethylene-inducing-like proteins (NLPs) that are widely spreadin bacteria, fungi, and oomycetes [2,3]. Several studies have shownthat NLPs contribute to pathogen virulence through phytotoxicactivity, but more recent work has revealed that some NLPs act inprocesses other than pathogenicity, such as fungal growth andsporulation [4]. A more recently identified class of conservedeffectors are LysM effectors: fungal effectors that carry norecognizable protein domains other than lysin motifs (LysMs)[5]. Intriguingly, like NLPs, LysM effectors occur in bothpathogenic and in nonpathogenic fungi.

Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes

Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants.

The role of chitin detection in plant–pathogen interactions

Despite the deployment of antifungal defence strategies, fungal diseases occur in all types of multicellular organisms. In plants, the role of fungal chitin as pathogen-associated molecular pattern that activates host defence is well established. Interestingly, plants employ homologs of the chitin immune receptors to initiate microbial symbiosis. Accumulating evidence shows that fungal pathogens developed secreted effectors to disarm chitin-triggered host immunity.

Chaperones of the endoplasmic reticulum are required for Ve1-mediated resistance to Verticillium

The tomato receptor-like protein (RLP) Ve1 mediates resistance to the vascular fungal pathogen Verticillium dahliae. To identify the proteins required for Ve1 function, we transiently expressed and immunopurified functional Ve1-enhanced green fluorescent protein (eGFP) from Nicotiana benthamiana leaves, followed by mass spectrometry. This resulted in the identification of peptides originating from the endoplasmic reticulum (ER)-resident chaperones HSP70 binding proteins (BiPs) and a lectin-type calreticulin (CRT). Knock-down of the different BiPs and CRTs in tomato resulted in compromised Ve1-mediated resistance to V. dahliae in most cases, showing that these chaperones play an important role in Ve1 functionality. Recently, it has been shown that one particular CRT is required for the biogenesis of the RLP-type Cladosporium fulvum resistance protein Cf-4 of tomato, as silencing of CRT3a resulted in a reduced pool of complex glycosylated Cf-4 protein. In contrast, knock-down of the various CRTs in N. benthamiana or N. tabacum did not result in reduced accumulation of mature complex glycosylated Ve1 protein. Together, this study shows that the BiP and CRT ER chaperones differentially contribute to Cf-4- and Ve1-mediated immunity.

Verticillium dahliae LysM effectors differentially contribute to virulence on plant hosts

Summary Chitin‐binding lysin motif (LysM) effectors contribute to the virulence of various plant‐pathogenic fungi that are causal agents of foliar diseases. Here, we report the LysM effectors of the soil‐borne fungal vascular wilt pathogen Verticillium dahliae. Comparative genomics revealed three core LysM effectors that are conserved in a collection of V. dahliae strains. Remarkably, and in contrast with the previously studied LysM effectors of other plant pathogens, no expression of core LysM effectors was monitored in planta in a taxonomically diverse panel of host plants. Moreover, targeted deletion of the individual LysM effector genes in V. dahliae strain JR2 did not compromise virulence in infections on Arabidopsis, tomato or Nicotiana benthamiana. Interestingly, an additional lineage‐specific LysM effector is encoded in the genome of V. dahliae strain VdLs17, but not in any other V. dahliae strain sequenced to date. Remarkably, this lineage‐specific effector is expressed in planta and contributes to the virulence of V. dahliae strain VdLs17 on tomato, but not on Arabidopsis or N. benthamiana. Functional analysis revealed that this LysM effector binds chitin, is able to suppress chitin‐induced immune responses and protects fungal hyphae against hydrolysis by plant hydrolytic enzymes. Thus, in contrast with the core LysM effectors of V. dahliae, this lineage‐specific LysM effector of strain VdLs17 contributes to virulence in planta.