The unique cellular interaction between the leaf pathogen Cymadothea trifolii and Trifolium repens

Cellular interactions between the ascomycete Cymadothea trifolii and Trifolium repens (white clover) were analyzed using high-pressure freezing and freeze substitution. Cymadothea trifolii, a biotrophic leaf pathogen, forms a unique structure within its own hyphae, presumably for nutrient uptake from its host. This structure, called an interaction apparatus, consists of long, thin, often net-like cisternae surrounded by a membrane continuous with the fungal plasma membrane. The plant plasmalemma opposite the interaction apparatus invaginates to produce a host bubble. The interaction apparatus and host bubble are apoplastic and are linked by a tube with an electron dense sheath that may channel nutrients from the host to the pathogen. Within the tube, the cell walls of host and parasite appear altered. The interaction apparatus and host bubble may be analogous to haustoria in other obligately biotrophic fungi while the electron dense sheath of the tube may be equivalent to the haustorial neckband.     

PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans

PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and beta-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between beta-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in beta-1,3-glucan-associated beta-1, 6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between beta-1,6- and beta-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process beta-1,3-glucans and make available acceptor sites for the attachment of beta-1,6-glucans.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea . α-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α-1,3-glucan and chitosan, but after enzymatic digestion of α-1,3-glucan, β-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed α-1,3-glucan and β-1,3-glucan intermixed in the cell wall of infectious hyphae; however, α-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α-1,3-glucan. Accumulation of α-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, α-1,3-glucan spatially and functionally masks β-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea .     

GPI7-mediated glycosylphosphatidylinositol anchoring regulates appressorial penetration and immune evasion during infection of Magnaporthe oryzae

Glycosylphosphatidylinositol (GPI) anchoring plays key roles in many biological processes by targeting proteins to the cell wall; however, its roles are largely unknown in plant pathogenic fungi. Here, we reveal the roles of the GPI anchoring in Magnaporthe oryzae during plant infection. The GPI-anchored proteins were found to highly accumulate in appressoria and invasive hyphae. Disruption of GPI7, a GPI anchor-pathway gene, led to a significant reduction in virulence. The Δgpi7 mutant showed significant defects in penetration and invasive growth. This mutant also displayed defects of the cell wall architecture, suggesting GPI7 is required for cell wall biogenesis. Removal of GPI-anchored proteins in the wild-type strain by hydrofluoric acid (HF) pyridine treatment exposed both the chitin and β-1,3-glucans to the host immune system. Exposure of the chitin and β-1,3-glucans was also observed in the Δgpi7 mutant, indicating GPI-anchored proteins are required for immune evasion. The GPI anchoring can regulate subcellular localization of the Gel proteins in the cell wall for appressorial penetration and abundance of which for invasive growth. Our results indicate the GPI anchoring facilitates the penetration of M. oryzae into host cells by affecting the cell wall integrity and the evasion of host immune recognition.     

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging β-glucan decasaccharide to subvert immune responses

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) β-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved β-1,3;1,6-glucan decasaccharide (β-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight β-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by β-1,3-endoglucanases due to the presence of three β-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of β-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant–microbe interface.

A β-1,3;1,6-glucan decasaccharide released from the fungal matrix by an apoplastic host hydrolase contributes to plant immune suppression and fungal accommodation.

IN A NUTSHELL Background: Plants secrete various hydrolytic enzymes into the apoplastic space to protect themselves against invading microbes. Some of these enzymes target the fungal cell wall polymer chitin. This enzymatic attack leads to the release of chitin oligomers, which can be perceived by the plant immune system, informing the plant to activate its defense machinery. However, chitin accounts for only a small part of most fungal cell walls. Recent studies have highlighted a largely uncharacterized, β-glucan-rich extracellular polysaccharide matrix (EPS) surrounding the cell wall of various plant-colonizing fungi. Question: This EPS matrix is made of glucose and abundantly produced during colonization. As its secretion into the extracellular environment is costly for the fungus, we explored how this EPS matrix affects plant immunity and fungal colonization. Findings: We demonstrated that EPS matrices from a symbiotic and pathogenic plant-colonizing fungus are distinct from the nonsoluble fungal cell walls with respect to their protein and carbohydrate composition. Enzymatic digests revealed that a secreted plant hydrolase from barley (HvBGLUII) acts on these EPS matrices and releases a highly branched β-glucan decasaccharide (β-GD) fragment. This fragment is not perceived by the plant immune system but instead detoxifies reactive oxygen species produced by the plant host as a defense mechanism and contributes to host colonization. We thus have shown that the outermost fungal EPS layer represents a protective shield against oxidative stress. Next steps: The diversity of linkage types and branching patterns of β-glucans not only accounts for their different biochemical properties, but also makes them important messengers for the plant, potentially encoding specific information on the approaching fungal invader. Future studies should aim to identify other plant hydrolases and the elusive glucan receptors, to disentangle the contribution of β-glucans to the communication between plant hosts and fungi.     

The fungal cell wall and the mechanism of action of anidulafungin

The fungal cell wall is a structure with a high plasticity that protects the cell from different types of environmental stresses including changes in osmotic pressure. In addition to that, the cell wall allows the fungal cell to interact with its environment, since some of its proteins are adhesins and receptors. Some of its components are highly immunogenic. The structure of the fungal cell wall is unique to the fungi, and it is composed of glucan, chitin and glycoproteins. Since humans lack the components present in the cell walls of fungi, this structure is an excellent target for the development of antifungal drugs. Anidulafungin, like the rest of echinocandins acts on beta-1,3-D-glucan synthase inhibiting the formation of beta-1,3-D-glucan and causing, depending on the type of fungus, a fungicidal or either a fungistatic effect.     

Characterization of Aspergillus nidulans mutants deficient in cell wall chitin or glucan.

By screening for the osmotically remediable phenotype, mutations in two genes (orlA and orlB) affecting the cell wall chitin content of Aspergillus nidulans were identified. Strains carrying temperature-sensitive alleles of these genes produce conidia which swell excessively and lyse when germinated at restrictive temperatures. Growth under these conditions is remedied by osmotic stabilizers and by N-acetylglucosamine (GlcNAc). Remediation by GlcNAc suggests that the mutations affect early steps in the synthesis of chitin. Temperature and medium shift experiments indicate that the phenotype is the result of decreased synthesis rather than increased chitin degradation and that osmotic stabilizers act to stabilize a defective wall rather than to stabilize the gene product. Two genes, orlC and orlD, which affect cell wall beta-1,3-glucan content were also identified. Walls from strains carrying mutations in these genes exhibit normal amounts of alpha-1,3-glucan and chitin but reduced amounts of beta-1,3-glucan. As for the chitin-deficient mutants, orlC and orlD mutants spontaneously lyse on conventional media but are remedied by osmotic stabilizers. These results indicate that both chitin and beta-1,3-glucan are likely to contribute to the structural rigidity of the cell wall.     

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.     

Chemical Organization of the Cell Wall Polysaccharide Core of Malassezia restricta

Malassezia species are ubiquitous residents of human skin and are associated with several diseases such as seborrheic dermatitis, tinea versicolor, folliculitis, atopic dermatitis, and scalp conditions such as dandruff. Host-Malassezia interactions and mechanisms to evade local immune responses remain largely unknown. Malassezia restricta is one of the most predominant yeasts of the healthy human skin, its cell wall has been investigated in this paper. Polysaccharides in the M. restricta cell wall are almost exclusively alkali-insoluble, showing that they play an essential role in the organization and rigidity of the M. restricta cell wall. Fractionation of cell wall polymers and carbohydrate analyses showed that the polysaccharide core of the cell wall of M. restricta contained an average of 5% chitin, 20% chitosan, 5% β-(1,3)-glucan, and 70% β-(1,6)-glucan. In contrast to other yeasts, chitin and chitosan are relatively abundant, and β-(1,3)-glucans constitute a minor cell wall component. The most abundant polymer is β-(1,6)-glucans, which are large molecules composed of a linear β-(1,6)-glucan chains with β-(1,3)-glucosyl side chain with an average of 1 branch point every 3.8 glucose unit. Both β-glucans are cross-linked, forming a huge alkali-insoluble complex with chitin and chitosan polymers. Data presented here show that M. restricta has a polysaccharide organization very different of all fungal species analyzed to date.     

A structural model for the mechanisms of elicitor release from fungal cell walls by plant beta-1,3-endoglucanase.

The release of elicitor-active carbohydrates from fungal cell walls by beta-1,3-endoglucanase contained in host tissues has been implicated as one of the earliest processes in the interaction between soybean (Glycine max) and the fungal pathogen Phytophthora megasperma f. sp. glycinea leading to host defense responses such as phytoalexin production. The present study was conducted to evaluate the primary structure of the glucanase-released elicitor (RE). Gel-filtration chromatography of carbohydrates released from mycelial walls by purified soybean beta-1,3-endoglucanase resolved them into the four fractions (elicitor-active RE-I, -II, and -III and elicitor-inactive RE-IV). Sugar composition analysis indicated that all of the fractions were composed almost entirely of glucose. 1H- and 13C-nuclear magnetic resonance analysis indicated the presence of both beta-1,3- and beta-1,6-linkages for the elicitor-active RE-I, -II, and -III fractions and only beta-1,3 linkage for the elicitor-inactive RE-IV fraction. Methylation analysis and degradation studies employing beta-1,3-endo- and beta-1,3-exoglucanase further suggested that the basic structure of elicitor-active RE consists of beta-1,6-linked glucan backbone chains of various lengths with frequent side branches composed of beta-1,3-linked one or two glucose moieties. From these structural analyses of RE, a structural model of how RE is originally present in fungal cell walls and released by host beta-1,3-endoglucanase is also proposed.     

A Cytochemical Study of Extracellular Sheaths Associated with Rigidoporus lignosus during Wood Decay

An ultrastructural and cytochemical investigation of the development of Rigidoporus lignosus, a white-rot fungus inoculated into wood blocks, was carried out to gain better insight into the structure and role of the extracellular sheaths produced by this fungus during wood degradation. Fungal sheaths had a dense or loose fibrillar appearance and were differentiated from the fungal cell wall early after wood inoculation. Close association between extracellular fibrils and wood cell walls was observed at both early and advanced stages of wood alteration. Fungal sheaths were often seen deep in host cell walls, sometimes enclosing residual wood fragments. Specific gold probes were used to investigate the chemical nature of R. lignosus sheaths. While labeling of chitin, pectin, β-1,4- and β-1,3-glucans, β-glucosides, galactosamine, mannose, sialic acid, RNA, fucose, and fimbrial proteins over fungal sheaths did not succeed, galactose residues and laccase (a fungal phenoloxidase) were found to be present. The positive reaction of sheaths with the PATAg test indicates that polysaccharides such as β-1,6-glucans are important components. Our data suggest that extracellular sheaths produced by R. lignosus during host cell colonization play an important role in wood degradation. Transportation of lignin-degrading enzymes by extracellular fibrils indicates that alteration of plant polymers may occur within fungal sheaths. It is also proposed that R. lignosus sheaths may be involved in recognition mechanisms in fungal cell-wood surface interactions.     

Irregular growth forms and cell wall modifications,polygalacturonase detection,and endocell formation in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">radicis-lycopersici</Emphasis> infecting tomato plants,as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.     

Cell wall α-1,3-glucans from a biocontrol isolate of Rhizoctonia: immunocytolocalization and relationship with α-glucanase activity from potato sprouts

The interface between plants and pathogens plays an important role in their interaction. Studies of fungal cell walls are scarce and previous results show the existence of α-1,3-glucans in addition to ß-glucans. In addition, α-1,3-glucans are not present in plant cell walls, and α-glucanase activity in plants has not been described before. In a previous work, we purified and characterized an α-1,3-glucan from a binucleated, non-pathogenic Rhizoctonia isolate, which induces plant defence responses. Therefore, in order to study the architecture of the fungal cell wall, and the accessibility and localization of the α-glucan elicitor, we prepared an antibody against the α-1,3-glucan and analysed its localization by TEM. Immunolocalization showed the presence of the α-1,3-glucan in the intercellular spaces and along the cell walls, mainly on the inner layers. This result, and the presence of the α-1,3-glucan in the liquid culture medium in which binucleated non-pathogenic Rhizoctonia was grown, confirmed that the α-glucan had been secreted. The α-1,3-glucan was also immunocytolocalized on potato sprouts tissue elicited with the glucan; gold particles were observed in vacuoles and close to the plasmalemma. In addition, α-glucanase activity in potato sprouts was detected using cell wall glucans from the pathogenic isolate R. solani AG-3 as substrates; whereas, when cell wall glucans from non-pathogenic isolates were used, no α-glucanase activity was detected. Our results suggest that the presence of α-1,3-glucans could be associated with the formation and integrity of the cell wall and also with plant–fungi interactions. This is the first report to describe α-glucanolytic activity in plants.     

Tasting the fungal cell wall

The search for common host mechanisms that recognize human fungal pathogens as non‐self has led to an increased interest in cell wall polysaccharides since they are absent from mammals and at least for some of them, common to all fungal species. Even though the receptors recognizing mannans and β‐1,3‐glucans have been extensively studied to date, the epitope of the polysaccharide ligand is often not well defined. In addition, receptors recognizing other cell wall major components such as chitin, α‐1,3‐glucan or galactose polymers remain to be identified. Moreover, the fungal adhesins playing a role in adhesion to host have been only explored in yeasts. Eventhough progresses have been made in the last 10 years, a comprehensive understanding of the interactions between the host membrane receptors and the fungal cell wall components is still lacking.     

Enzyme Production by the Mycoparasite Verticillium biguttatum against Rhizoctonia solani

Verticillium biguttatum, a mycoparasite of the ubiquitous soil-borne plant pathogen Rhizoctonia solani, excreted chitinase and beta-1,3-glucanase into liquid medium when grown on laminarin and chitin, respectively. Neither chitinase nor beta-1,3-glucanase was produced by the mycoparasite when grown on cell walls of two isolates of R. solani representing anastomosis groups (AG)-3 and AG-8. Extracellular protease was induced by growth on cell walls of the pathogen, whereas beta-1,3-glucanase and chitinase were produced bound to the cell wall of V. biguttatum. This is the first report of chitinase, beta-1,3-glucanase and protease production by V. biguttatum. These enzymes may play a previously unforeseen role in dissolving and penetrating the cell walls of R. solani.     

Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria

Powdery mildew fungi are biotrophic pathogens that require living plant cells for their growth and reproduction. Elaboration of a specialized cell called a haustorium is essential for their pathogenesis, providing a portal into host cells for nutrient uptake and delivery of virulence effectors. Haustoria are enveloped by a modified plant plasma membrane, the extrahaustorial membrane (EHM), and an extrahaustorial matrix (EHMx), across which molecular exchange must occur, but the origin and composition of this interfacial zone remains obscure. Here we present a method for isolating Golovinomyces orontii haustoria from Arabidopsis leaves and an ultrastructural characterization of the haustorial interface. Haustoria were progressively encased by deposits of plant cell wall polymers, delivered by secretory vesicles and multivesicular bodies (MVBs) that ultimately become entrapped within the encasement. The EHM and EHMx were not labelled by antibodies recognizing eight plant cell wall and plasma membrane antigens. However, plant resistance protein RPW8.2 was specifically recruited to the EHMs of mature haustoria. Fungal cell wall-associated molecular patterns such as chitin and β-1,3-glucans were exposed at the surface of haustoria. Fungal MVBs were abundant in haustoria and putative exosome vesicles were detected in the paramural space and EHMx, suggesting the existence of an exosome-mediated secretion pathway.     

Fungal chitin from asthma-associated home environments induces eosinophilic lung infiltration

Development of asthma and allergic inflammation involves innate immunity, but the environmental contributions remain incompletely defined. Analysis of dust collected from the homes of asthmatic individuals revealed that the polysaccharide chitin is environmentally widespread and associated with β-glucans, possibly from ubiquitous fungi. Cell wall preparations of Aspergillus isolated from house dust induced robust recruitment of eosinophils into mouse lung, an effect that was attenuated by enzymatic degradation of cell wall chitin and β-glucans. Mice expressing constitutively active acidic mammalian chitinase in the lungs demonstrated a significant reduction in eosinophil infiltration after fungal challenge. Conversely, chitinase inhibition prolonged the duration of tissue eosinophilia. Thus, fungal chitin derived from home environments associated with asthma induces eosinophilic allergic inflammation in the lung, and mammalian chitinases, including acidic mammalian chitinase, limit this process.     

Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds

Chitin, a beta-1,4-linked polysaccharide of N-acetylglucosamine, is a major structural component of fungal cell walls. Fungi have multiple classes of chitin synthases that catalyse N-acetylglucosamine polymerization. Here, we demonstrate the requirement for a class V chitin synthase during host infection by the vascular wilt pathogen Fusarium oxysporum. The chsV gene was identified in an insertional mutagenesis screen for pathogenicity mutants. ChsV has a putative myosin motor and a chitin synthase domain characteristic of class V chitin synthases. The chsV insertional mutant and a gene replacement mutant of F. oxysporum display morphological abnormalities such as hyphal swellings that are indicative of alterations in cell wall structure and can be partially restored by osmotic stabilizer. The mutants are unable to infect and colonize tomato plants or to grow invasively on tomato fruit tissue. They are also hypersensitive to plant antimicrobial defence compounds such as the tomato phytoanticipin alpha-tomatine or H2O2. Reintroduction of a functional chsV copy into the mutant restored the growth phenotype of the wild-type strain. These data suggest that F. oxysporum requires a specific class V chitin synthase for pathogenesis, most probably to protect itself against plant defence mechanisms.