LysM Proteins Research Articles

LysM protein BdLM1 of Botryosphaeria dothidea plays an important role in full virulence and inhibits plant immunity by binding chitin and protecting hyphae from hydrolysis.

Botryosphaeria dothidea infects hundreds of woody plants and causes a severe economic loss to apple production. In this study, we characterized BdLM1, a protein from B. dothidea that contains one LysM domain. BdLM1 expression was dramatically induced at 6 h post-inoculation in wounded apple fruit, strongly increased at 7 d post-inoculation (dpi), and peaked at 20 dpi in intact shoots. The knockout mutants of BdLM1 had significantly reduced virulence on intact apple shoots (20%), wounded apple shoots (40%), and wounded apple fruit (40%). BdLM1 suppressed programmed cell death caused by the mouse protein BAX through Agrobacterium-mediated transient expression in Nicotiana benthamiana, reduced H2O2 accumulation and callose deposition, downregulated resistance gene expression, and promoted Phytophthora nicotianae infection in N. benthamiana. Moreover, BdLM1 inhibited the active oxygen burst induced by chitin and flg22, bound chitin, and protected fungal hyphae against degradation by hydrolytic enzymes. Taken together, our results indicate that BdLM1 is an essential LysM effector required for the full virulence of B. dothidea and that it inhibits plant immunity. Moreover, BdLM1 could inhibit chitin-triggered plant immunity through a dual role, i.e., binding chitin and protecting fungal hyphae against chitinase hydrolysis.

LysM Proteins TaCEBiP and TaLYK5 are Involved in Immune Responses Mediated by Chitin Coreceptor TaCERK1 in Wheat.

Plant lysin motif (LysM) ectodomain receptors interact with pathogen-associated molecular patterns (PAMPs) and have critical functions in plant-microbe interactions. In this study, 65 LysM family genes were identified using the recent version of the reference sequence of bread wheat (Triticum aestivum), in which 23, 16, 20, and 6 members belonged to LysM-containing receptor-like kinases (LYKs), LysM-containing receptor-like proteins (LYPs), extracellular LysM proteins (LysMes), and intracellular nonsecretory LysM proteins (LysMns), respectively. The study found that TaCEBiP, TaLYK5, and TaCERK1 were highly responsive to PAMP elicitors and phytopathogens, with TaCEBiP and TaLYK5 binding directly to chitin. TaCERK1 acted as a coreceptor with TaCEBiP and TaLYK5 at the plasma membrane. Overexpression of TaCEBiP, TaLYK5, and TaCERK1 in Nicotiana benthamiana leaves exhibited enhanced resistance to Sclerotinia sclerotiorum. Subsequently, knocking down TaCEBiP, TaLYK5, and TaCERK1 genes with barley stripe mosaic virus-VIGS compromised the wheat defense response to an avirulent strain of Puccinia striiformis. The study concluded that wheat has two synergistic chitin perception systems for detecting pathogen elicitors, with the activated CERK1 intracellular kinase domain leading to signaling transduction. This research provides valuable insights into the functional roles and regulatory mechanisms of wheat LysM members under biotic stress.

Hallmarks of Comparative Transcriptome between Rhizomorphs and Hyphae of Armillaria sp. 541 Participating in Fungal Symbiosis with Emphasis on LysM Domains.

Armillaria sp. 541, a genus of root-infecting fungi, forms a symbiosis with traditional Chinese medicine Gastrodia elata (Orchid) and Polyporus umbellatus via extensive networks of durable rhizomorphs. It is not clear the hallmarks of comparative transcriptome between the rhizomorphs and hyphae of Armillaria sp. 541. In the present study, transcriptomic analysis of Armillaria sp. 541 identified 475 differentially expressed genes (DEGs) between Armillaria rhizomorphs (AR) and hyphae (AH). Of them, 285 genes were upregulated and 190 were downregulated. Bioinformatics analyses and tests demonstrated DEGs involved in oxidoreductase activity and peptidoglycan binding were significantly enriched in this process when rhizomorph formed from hyphae. We accordingly obtained 14 gene-encoding proteins containing the LysM domain, and further consensus pattern and phylogenetic analysis indicated that their amino acid sequences were conserved and their biological functions may be peptidoglycan binding for recognition between the fungus and host. Among these genes, one, named Armillaria LysM domain recognition gene (aLDRG), was expressed significantly when rhizomorphs were differentiated from hyphae. It was located in the cortical cells of the rhizomorph by in situ hybridization. Furthermore, biolayer interferometry (BLI) assay demonstrated that aLDRG can bind specifically to chitin oligosaccharide of the fungal cell wall, including N,N',N″-Triacetylchitotriose (CO3) and N,N',N″,N'″,N″″-Pentaacetylchitopentaose (CO5). Therefore, we deduced that Armillaria sp. 541 expressed higher levels of LysM protein aLDRG for better binding of oligosaccharide after rhizomorphs were generated. This study provides functional genes for further studies on the interaction between Armillaria sp. 541 and its host.

Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Genome-wide analysis of LysM gene family members and their expression in response to Colletotrichum fructicola infection in Octoploid strawberry(Fragaria × ananassa).

The cultivated octoploid strawberry (Fragaria × ananassa) is an economically important fruit that is planted worldwide. The lysin motif (LysM) protein family is composed of the major class of plant pattern recognition receptors, which play important roles in sensing pathogen-associated molecular patterns (PAMPs), and subsequently triggers downstream plant immunity. In the present study, a comprehensive, genome-wide analysis of F. × ananassa LysM (FaLysM) genes was performed to investigate gene structures, phylogenic relationships, chromosome location, collinear relationships, transcription factor binding sites, and protein model analysis. We aimed to identify the LysM genes involved in the defense against plant pathogens. A total of 14 FaLysM genes were identified in the F. × ananassa genome and divided into 2 subgroups (LYP and LYK) on the basis of the phylogenetic analysis. The Ka/Ks ratio for the duplicated pair of most FaLysM genes was less than 1, which indicates that the selection pressure was mostly subject to the purifying selection during evolution. The protein model analysis revealed that FaLysM2-10 contain conserved mode of chitin binding, which suggest the potential role of FaLysM2-10 in pathogen perception and plant immunity. The RNA-Seq results showed the differential regulation of 14 FaLysM genes in response to Colletotrichum fructicola infection, implying the complex interaction between C. fructicola and strawberry. Knockout of candidate effector gene CfLysM2, which was previously proved to be highly expressed during C. fructicola infection, resulted in the up-regulation of six FaLysM genes (FaLysM1, FaLysM2, FaLysM3, FaLysM7, FaLysM8, and FaLysM12), indicating the competitive relations between CfLysM2 and FaLysM genes. Overall, this study provides fundamental information on the roles of LysM proteins in octoploid strawberry and its interaction with C. fructicola, laying useful information for further investigation on the C. fructicola-strawberry interaction and strawberry resistance breeding.

Transcriptome analysis of maize pathogen Fusarium verticillioides revealed FvLcp1, a secreted protein with type-D fungal LysM and chitin-binding domains, that plays important roles in pathogenesis and mycotoxin production

Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.

Lasiodiplodia theobromae protein LtScp1 contributes to fungal virulence and protects fungal mycelia against hydrolysis by grapevine chitinase.

The LysM proteins have been reported to be important for the virulence and host immunity suppression in herbaceous plant pathogens, whereas far less information is documented in the woody plant pathogen Lasiodiplodia theobromae. To investigate the functional mechanism of LysM protein in L. theobromae, one gene LtScp1 was cloned and characterized detailedly in the current study. Transcription profiling revealed that LtScp1 was highly expressed at the infectious stages. Compared to wild type, overexpression and silencing of LtScp1 in L. theobromae led to significantly increased and decreased lesion areas, respectively. Moreover, LtScp1 was determined to be a secreted protein via a yeast signal peptide trapping system. Interestingly, LtScp1 was confirmed to be modified by the N-glycosylation, which is necessary for the homodimerization of LtScp1 molecules. Furthermore, it was found that LtScp1 interacted with the grapevine chitinase VvChi4 and interfered the ability of VvChi4 to bind chitin. Collectively, these results suggest that LtScp1 functions as a virulence factor to protect the fungus from degradation during the infection.

Comparative genomics reveals a dynamic genome evolution in the ectomycorrhizal milk-cap (Lactarius) mushrooms.

Ectomycorrhizal fungi play a key role in forests by establishing mutualistic symbioses with woody plants. Genome analyses have identified conserved symbiosis-related traits among ectomycorrhizal fungal species, but the molecular mechanisms underlying host specificity remain poorly known. We sequenced and compared the genomes of seven species of milk-cap fungi (Lactarius, Russulales) with contrasting host specificity. We also compared these genomes with those of symbiotic and saprotrophic Russulales species, aiming to identify genes involved in their ecology and host specificity. The size of Lactarius genomes is significantly larger than other Russulales species, owing to a massive accumulation of transposable elements and duplication of dispensable genes. As expected, their repertoire of genes coding for plant cell wall-degrading enzymes is restricted, but they retained a substantial set of genes involved in microbial cell wall degradation. Notably, Lactarius species showed a striking expansion of genes encoding proteases, such as secreted ectomycorrhiza-induced sedolisins. A high copy number of genes coding for small secreted LysM proteins and Lactarius-specific lectins were detected, which may be linked to host specificity. This study revealed a large diversity in the genome landscapes and gene repertoires within Russulaceae. The known host specificity of Lactarius symbionts may be related to mycorrhiza-induced species-specific genes, including secreted sedolisins.

Heterologous expression of the Haynaldia villosa pattern-recognition receptor CERK1-V in wheat increases resistance to three fungal diseases

Wheat production is under continuous threat by various fungal pathogens. Identification of multiple-disease resistance genes may lead to effective disease control via the development of cultivars with broad-spectrum resistance. Plant Lysin-motif (LysM)-type pattern-recognition receptors, which elicit innate immunity by recognizing fungal pathogen associated molecular patterns such as chitin, are potential candidates for such resistance. In this study, we cloned a LysM receptor-like kinase gene, CERK1-V, from the diploid wheat relative Haynaldia villosa. CERK1-V expression was induced by chitin and Blumeria graminis f. sp. tritici, the causal agent of wheat powdery mildew. Heterologous overexpression of CERK1-V in wheat inhibited the development of three fungal pathogens, thereby increased resistance to powdery mildew, yellow rust, and Fusarium head blight. CERK1-V physically interacted with the wheat LysM protein TaCEBiPs. CERK1-V/TaCEBiPs interaction promoted chitin recognition and activated chitin signal transduction in wheat. Transgenic plants with excessively high CERK1-V expression showed high resistance but abnormal plant growth, whereas plants with moderate expression level showed adequate resistance level with no marked impairment of plant growth. In transgenic lines, RNA-seq showed that gene expression involved in plant innate immunity was activated. Expression of genes involved in photosynthesis, ER stress and multiple phytohormone pathways was also activated. Optimized expression of CERK1-V in wheat can confer disease resistance without compromising growth or defense fitness.

Lysin Motif (LysM) Proteins: Interlinking Manipulation of Plant Immunity and Fungi.

The proteins with lysin motif (LysM) are carbohydrate-binding protein modules that play a critical role in the host-pathogen interactions. The plant LysM proteins mostly function as pattern recognition receptors (PRRs) that sense chitin to induce the plant’s immunity. In contrast, fungal LysM blocks chitin sensing or signaling to inhibit chitin-induced host immunity. In this review, we provide historical perspectives on plant and fungal LysMs to demonstrate how these proteins are involved in the regulation of plant’s immune response by microbes. Plants employ LysM proteins to recognize fungal chitins that are then degraded by plant chitinases to induce immunity. In contrast, fungal pathogens recruit LysM proteins to protect their cell wall from hydrolysis by plant chitinase to prevent activation of chitin-induced immunity. Uncovering this coevolutionary arms race in which LysM plays a pivotal role in manipulating facilitates a greater understanding of the mechanisms governing plant-fungus interactions.

Genome-Wide Analysis of LysM-Containing Gene Family in Wheat: Structural and Phylogenetic Analysis during Development and Defense.

The lysin motif (LysM) family comprise a number of defense proteins that play important roles in plant immunity. The LysM family includes LysM-containing receptor-like proteins (LYP) and LysM-containing receptor-like kinase (LYK). LysM generally recognizes the chitin and peptidoglycan derived from bacteria and fungi. Approximately 4000 proteins with the lysin motif (Pfam PF01476) are found in prokaryotes and eukaryotes. Our study identified 57 LysM genes and 60 LysM proteins in wheat and renamed these genes and proteins based on chromosome distribution. According to the phylogenetic and gene structure of intron–exon distribution analysis, the 60 LysM proteins were classified into seven groups. Gene duplication events had occurred among the LysM family members during the evolution process, resulting in an increase in the LysM gene family. Synteny analysis suggested the characteristics of evolution of the LysM family in wheat and other species. Systematic analysis of these species provided a foundation of LysM genes in crop defense. A comprehensive analysis of the expression and cis-elements of LysM gene family members suggested that they play an essential role in defending against plant pathogens. The present study provides an overview of the LysM family in the wheat genome as well as information on systematic, phylogenetic, gene duplication, and intron–exon distribution analyses that will be helpful for future functional analysis of this important protein family, especially in Gramineae species.

A LysM Domain-Containing Protein LtLysM1 Is Important for Vegetative Growth and Pathogenesis in Woody Plant Pathogen Lasiodiplodia theobromae.

Lysin motif (LysM) proteins are reported to be necessary for the virulence and immune response suppression in many herbaceous plant pathogens, while far less is documented in woody plant pathogens. In this study, we preliminarily characterized the molecular function of a LysM protein LtLysM1 in woody plant pathogen Lasiodiplodia theobromae. Transcriptional profiles revealed that LtLysM1 is highly expressed at infectious stages, especially at 36 and 48 hours post inoculation. Amino acid sequence analyses revealed that LtLysM1 was a putative glycoprotein with 10 predicted N-glycosylation sites and one LysM domain. Pathogenicity tests showed that overexpressed transformants of LtLysM1 displayed increased virulence on grapevine shoots in comparison with that of wild type CSS-01s, and RNAi transformants of LtLysM1 exhibited significantly decreased lesion length when compared with that of wild type CSS-01s. Moreover, LtLysM1 was confirmed to be a secreted protein by a yeast signal peptide trap assay. Transient expression in Nicotiana benthamiana together with protein immunoblotting confirmed that LtLysM1 was an N-glycosylated protein. In contrast to previously reported LysM protein Slp1 and OsCEBiP, LtLysM1 molecule did not interact with itself based on yeast two hybrid and co-immunoprecipitation assays. These results indicate that LtLysM1 is a secreted protein and functions as a critical virulence factor during the disease symptom development in woody plants.

LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea.

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Δlysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Δlysm2 and Δlysm1Δlysm2 strains displayed reduced ability to colonize wheat roots, while only Δlysm1Δlysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol.

A lysin motif-containing protein (SpLysMD3) functions as a PRR involved in the antibacterial responses of mud crab, Scylla paramamosain

Lysin motif (LysM)-containing proteins function as pattern-recognition receptors in plants to recognize different N-acetylglucosamine-containing ligands, thereby triggering specific defense responses against pathogens. However, the biological functions of these proteins in animals remain unclear. In this study, we characterized a novel LysM protein, designated as SpLysMD3, in mud crab Scylla paramamosain. The cDNA sequence of SpLysMD3 had 1058 bp with an open reading frame of 840 bp encoding a protein with 279 amino acid residues. The deduced protein contained a LysM domain and a transmembrane region. SpLysMD3 was highly expressed in gills, intestine, muscle, and hemocytes and upregulated after challenges with bacteria, suggesting that it may be involved in antibacterial defense. Binding assay showed that SpLysMD3 possessed specific binding activities to all tested microorganisms as well as bacterial cell wall components lipopolysaccharide (LPS) and peptidoglycan (PGN), indicating that SpLysMD3 was an important LPS- and PGN-binding protein in mud crab. Bacterial clearance assay revealed that coating bacteria with SpLysMD3 accelerated bacterial clearance in vivo. The promotion of bacterial clearance by SpLysMD3 was further determined by using SpLysMD3-silenced crabs injected with S. aureus or V. parahemolyticus. Silencing SpLysMD3 dramatically suppressed the bacterial clearance. Meanwhile, knockdown of SpLysMD3 also severely impaired the expression of a specific set of antimicrobial peptides (AMPs); moreover, SpLysMD3 overexpression can enhance the promoter activity of SpALF2. These results suggested that SpLysMD3 affected bacterial clearance by regulating AMPs. Collectively, all the results demonstrated that SpLysMD3 may function as a potential receptor involved in innate immunity by binding to LPS and PGN and by regulating AMPs to eliminate invading pathogen. This study provided new insights into the biological functions of LysM proteins in animals and the mechanisms underlying the antibacterial activity of crustaceans.

Tal6 From Trichoderma atroviride Is a LysM Effector Involved in Mycoparasitism and Plant Association.

LysM effectors play a relevant role during the plant colonization by successful phytopathogenic fungi, since they enable them to avoid either the triggering of plant defense mechanisms or their attack effects. Tal6, a LysM protein from Trichoderma atroviride, is capable of binding to complex chitin. However, until now its biological function is not completely known, particularly its participation in plant–Trichoderma interactions. We obtained T. atroviride Tal6 null mutant and Tal6 overexpressing strains and determined the role played by this protein during Trichoderma-plant interaction and mycoparasitism. LysM effector Tal6 from T. atroviride protects the hyphae from chitinases by binding to chitin of the fungal cell wall, increases the fungus mycoparasitic capacity, and modulates the activation of the plant defense system. These results show that beneficial fungi also employ LysM effectors to improve their association with plants.

Comparative genomics of chytrid fungi reveal insights into the obligate biotrophic and pathogenic lifestyle of Synchytrium endobioticum

Synchytrium endobioticum is an obligate biotrophic soilborne Chytridiomycota (chytrid) species that causes potato wart disease, and represents the most basal lineage among the fungal plant pathogens. We have chosen a functional genomics approach exploiting knowledge acquired from other fungal taxa and compared this to several saprobic and pathogenic chytrid species. Observations linked to obligate biotrophy, genome plasticity and pathogenicity are reported. Essential purine pathway genes were found uniquely absent in S. endobioticum, suggesting that it relies on scavenging guanine from its host for survival. The small gene-dense and intron-rich chytrid genomes were not protected for genome duplications by repeat-induced point mutation. Both pathogenic chytrids Batrachochytrium dendrobatidis and S. endobioticum contained the largest amounts of repeats, and we identified S. endobioticum specific candidate effectors that are associated with repeat-rich regions. These candidate effectors share a highly conserved motif, and show isolate specific duplications. A reduced set of cell wall degrading enzymes, and LysM protein expansions were found in S. endobioticum, which may prevent triggering plant defense responses. Our study underlines the high diversity in chytrids compared to the well-studied Ascomycota and Basidiomycota, reflects characteristic biological differences between the phyla, and shows commonalities in genomic features among pathogenic fungi.

Trichophyton rubrum LysM proteins bind to fungal cell wall chitin and to the N-linked oligosaccharides present on human skin glycoproteins.

The Trichophyton rubrum genome contains six proteins containing two or more lysin M (LysM) domains. We have characterized two of these proteins, LysM1 and LysM2, and demonstrated that these proteins have the capacity to bind two substrates, chitin and N-linked oligosaccharides associated with human skin glycoproteins. We have characterized the individual LysM domains in LysM1, and shown that the protein contains two functional LysM domains. Each of these domains can bind to chitin, to N-linked oligosaccharides in human skin glycoproteins, and to N-linked oligosaccharides on fungal glycoproteins. We hypothesize that LysM proteins could provide the pathogen with three important functions. First, the T. rubrum LysM proteins could shield host cell wall chitin from the human immune system. Second, the LysM proteins could shield the pathogen’s glycoproteins from host degradation and immune surveillance. Third, the LysM proteins could help facilitate pathogen adhesion to human skin.

Characterisation of novel-cell-wall LysM-domain proteins LdpA and LdpB from the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus, a filamentous fungus that is ubiquitous in the environment, causes several human pulmonary disorders, including chronic and acute invasive infections and allergic diseases. Lysin motif (LysM) is a small protein domain that binds chitin, a major component of fungal cell wall polysaccharides. Several secreted LysM-domain proteins without catalytic function (LysM effectors) have been identified. They act as virulence factors in plant pathogenic fungi by preventing the immune response induced by chitin; however, LysM proteins in mammalian pathogenic fungi remain largely unexplored. We describe two novel LysM-domain proteins, LdpA and LdpB, in A. fumigatus. Functional analyses of single and double knockouts revealed no significant effects on cell wall chitin content, cell wall integrity, fungal morphology and fungal growth. Fluorescent signals from LdpA-green fluorescent protein (GFP) and LdpB-GFP were observed in cell wall and extracellular matrix. In a mouse model of invasive pulmonary aspergillosis, survival did not differ between ΔldpA/B and wild-type infection; however, further studies are required to reveal their functions in fungal−host interactions.

A short LysM protein with high molecular diversity from an arbuscular mycorrhizal fungus, Rhizophagus irregularis

Abstract Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.

Expression profiling in a mammalian host reveals the strong induction of genes encoding LysM domain-containing proteins in Enterococcus faecium

Enterococcus faecium is an important health care-associated pathogen that is difficult to treat due to the high level of antibiotic resistance of clinical isolates. The identification of new potential therapeutic targets or vaccination strategies is therefore urgently needed. In this regard, we carried out a transcriptomic analysis of the E. faecium vancomycin-resistant strain AUS0004, comparing the gene expression of bacteria grown under laboratory conditions and bacteria isolated from an infection site. This analysis highlighted more than 360 genes potentially induced under infection conditions. Owing to their expression profiles, four LysM domain-containing proteins were characterized in more detail. The EFAU004_01059, 1150 and 494 proteins are highly homologous, whereas EFAU004_01209 has a unique domain-architecture and sequence. The analysis of corresponding mutants showed that all LysM proteins played relevant roles in the infection process of E. faecium in mice. The EFAU004_01209 mutant also displayed profound morphological modifications, suggesting it has a role in cell wall synthesis or cell division. Furthermore, the adhesion to kidney cells and growth of the mutant was affected in human urine. All these phenotypes and the surface exposure of EFAU004_01209 identify this protein as an interesting new drug target in E. faecium.