Disruption of Botrytis cinerea class I chitin synthase gene Bcchs1 results in cell wall weakening and reduced virulence

To get a better insight into the relationship between cell wall integrity and pathogenicity of the fungus Botrytis cinerea, we have constructed chitin synthase mutants. A 620 bp class I chitin synthase gene fragment (Bcchs1) obtained by PCR amplification was used to disrupt the corresponding gene in the genome. Disruption of Bcchs1 occurred at a frequency of 8%. Nine independent mutants were obtained and the Bcchs1 mutant phenotype compared to that of transformants in which the gene was not disrupted. These disruption mutants were dramatically reduced in their in vitro Mg2+, Mn2+, and Co2+-dependent chitin synthase activity. Chitin content was reduced by 30%, indicating that Bcchs1p contributes substantially to cell wall composition. Enzymatic degradation by a cocktail of glucanases revealed cell wall weakening in the mutant. Bcchs1 was transcribed at a constant level during vegetative exponential growth, suggesting that it was necessary throughout hyphal development. Bcchs1 mutant growth was identical to undisrupted control transformant growth, however, the mutant exhibited reduced pathogenicity on vine leaves. It can be assumed that disruption of Bcchs1 leads to cell wall weakening which might slow down in planta fungal progression.     

CHS8-a fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity, but is dispensable for growth

In silico analysis of the genome sequence of the human pathogenic fungus Candida albicans identified an open reading frame encoding a putative fourth member of the chitin synthase gene family. This gene, named CaCHS8, encodes an 1105 amino acid open reading frame with the conserved motifs characteristic of class I zymogenic chitin synthases with closest sequence similarity to the non-essential C. albicans class I CHS2 gene. Although the CaCHS8 gene was expressed in both yeast and hyphal cells, homozygous chs8 Delta null mutants had normal growth rates, cellular morphologies and chitin contents. The null mutant strains had a 25% reduction in chitin synthase activity and were hypersensitive to Calcofluor White. A chs2 Delta chs8 Delta double mutant had less than 3% of normal chitin synthase activity and had increased wall glucan and decreased mannan but was unaffected in growth or cell morphology. The C. albicans class I double mutant did not exhibit a bud-lysis phenotype as found in the class I chs1 Delta mutant of Saccharomyces cerevisiae. Therefore, C. albicans has four chitin synthases with two non-essential class I Chs isoenzymes that contribute collectively to more than 97% of the in vitro chitin synthase activity.     

Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.     

Relationships between phosphatidylcholine content, chitin synthesis, growth, and morphology of Aspergillus nidulans choC

The phosphatidylcholine (PC) content of Aspergillus nidulans choC was varied by growing the auxotroph in medium containing various concentrations of choline chloride. Direct linear correlations were observed between PC content and in vivo chitin synthase activity, between in vivo chitin synthase activity and mean hyphal extension rate, and between mean hyphal extension rate and hyphal growth unit length; hyphal growth unit length is a measure of hyphal branching. Further, there was a correlation between PC content and colony radial growth rate. Thus, membrane composition is an important determinant of both hyphal (and colony) extension rate and mycelial morphology.     

Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six euascomycetes genomes.

We describe a strategy for systematic amplification of chitin synthase genes (chs) in the filamentous ascomycetes plant-pathogen Botrytis cinerea using PCR with multiple degenerate primers designed on specific and conserved sequence motifs. Eight distinct chs genes were isolated, named Bcchs I, II, IIIa, IIIb, IV, V, VI and VII. They probably constitute the entire chs multigenic family of this fungus, as revealed by careful analysis of six euascomycetes genomes. Bcchs I, IIIa, IIIb, IV and VI genes were subjected to DNA walking and their deduced amino acid sequences were compared by hydrophobic cluster analysis (HCA) to localize putative residues critical for CHS activity. HCA also enabled us to highlight three different transmembrane topologies of the CHS membranous isoenzymes. We found that the N-terminal region of the BcCHSI isoenzyme, and its orthologues in other euascomycetes, probably contain folded peptide motifs with conserved tyrosine residues. Their putative role is discussed. The BcCHSVII isoenzyme appeared to belong to a new class of CHS orthologues that was demonstrated by phylogenetic study to branch apart from division 1 and 2 of CHS.     

Aspergillus fumigatus chsE: A Gene Related to CHS3 of Saccharomyces cerevisiae and Important for Hyphal Growth and Conidiophore Development but Not Pathogenicity

The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A. fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3). The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD). A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.     

Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune.

In the presence of MgSo4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (alpha-1,3-glucan), R-glucan (beta-1,3, beta-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion of hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 mug/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.     

An IQGAP-related protein,encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii

In filamentous ascomycetes hyphae are compartmentalized by septation in which the cytoplasm of the compartments are interconnected via septal pores. Thus, septation in filamentous fungi is different from cytokinesis in yeast like fungi. We have identified an Ashbya gossypii orthologue of the Saccharomyces cerevisiae CYK1 gene which belongs to the IQGAP-protein family. In contrast to S. cerevisiae disruption of AgCYK1 yields viable mutant strains that exhibit wildtype-like polarized hyphal growth rates. In the Agcyk1 mutant cortical actin patches localize to growing hyphal tips like wildtype, however, mutant hyphae are totally devoid of actin rings at presumptive septal sites. Septation in wildtype results in the formation of chitin rings. Agcyk1 mutant hyphae are aseptate and do not accumulate chitin in their cell walls. Agcyk1 mutant strains are completely asporogenous indicating that septation is essential for the formation of sporangia in A. gossypii. AgCyk1p-GFP localizes to sites of future septation as a ring prior to chitin depositioning. Furthermore, decrease in Cyk1p-ring diameter was found to be a prerequisite for the accumulation of chitin and septum formation.     

An essential gene for fruiting body initiation in the basidiomycete Coprinopsis cinerea is homologous to bacterial cyclopropane fatty acid synthase genes

The self-compatible Coprinopsis cinerea homokaryon AmutBmut produces fruiting bodies without prior mating to another strain. Early stages of fruiting body development include the dark-dependent formation of primary hyphal knots and their light-induced transition to the more compact secondary hyphal knots. The AmutBmut UV mutant 6-031 forms primary hyphal knots, but development arrests at the transition state by a recessive defect in the cfs1 gene, isolated from a cosmid library by mutant complementation. A normal primordia phenotype was achieved when cfs1+ was embedded at both sides in at least 4.0 kb of native flanking DNA. Truncations of the flanking DNA lead to reduction in transformation frequencies and faults in primordia tissue formation, suggesting that the gene is also acting at later stages of development. The cfs1 gene encodes a protein highly similar to cyclopropane fatty acid synthases, a class of enzymes shown in prokaryotes and recently in a plant to convert membrane-bound unsaturated fatty acids into cyclopropane fatty acids. In C. cinerea 6-031, the mutant cfs1 allele carries a T-to-G transversion, leading to an amino acid substitution (Y441D) in a domain suggested to be involved in the catalytic function of the protein and/or membrane interaction.     

Disruption of Botrytis cinerea pectin methylesterase gene Bcpme1 reduces virulence on several host plants

The pectinolytic enzyme pectin methylesterase (PME) hydrolyses pectin in methanol and polygalacturonic acid. In the expressed sequence tag library of Botrytis cinerea T4, we identified a 1,041 bp Bcpme1 cDNA potentially encoding a 346-amino acid protein of 37 kDa showing 46.8% identity with Aspergillus sp. PMEs. Bcpme1 is a single copy gene and is similarly expressed in glucose and pectin containing media. To evaluate the role of Bcpme1 in Botrytis cinerea virulence, a mutant in Bcpme1 was generated by gene disruption. The Bcpme1 mutant showed similar growth on rich medium but reduced growth on pectin medium. Two isozymes of pI 7.4 and 7.1 were detected in pectin liquid-culture supernatants of wild-type strain Bd90 analyzed by isoelectric focusing-polyacrylamide gel electrophoresis, while those of Bcpme1 mutant possessed only the pI 7.1 isozyme. BCPME1, the pI 7.4 isozyme, is the major PME activity, as PME activity is 75% reduced in Bcpme1 mutant. Moreover, the Bcpme1 mutant was less virulent on apple fruits, grapevine, and Arabidopsis thaliana leaves. Those phenotypes were complemented by reintroducing a Bcpme1 copy in the Bcpme1 mutant. These results showed that B. cinerea possessed more than one PME-encoding gene and that BCPME1 is an important determinant of B. cinerea virulence.     

Disruption of the Eng18B ENGase gene in the fungal biocontrol agent Trichoderma atroviride affects growth, conidiation and antagonistic ability

The recently identified phylogenetic subgroup B5 of fungal glycoside hydrolase family 18 genes encodes enzymes with mannosyl glycoprotein endo-N-acetyl-β-D-glucosaminidase (ENGase)-type activity. Intracellular ENGase activity is associated with the endoplasmic reticulum associated protein degradation pathway (ERAD) of misfolded glycoproteins, although the biological relevance in filamentous fungi is not known. Trichoderma atroviride is a mycoparasitic fungus that is used for biological control of plant pathogenic fungi. The present work is a functional study of the T. atroviride B5-group gene Eng18B, with emphasis on its role in fungal growth and antagonism. A homology model of T. atroviride Eng18B structure predicts a typical glycoside hydrolase family 18 (αβ)(8) barrel architecture. Gene expression analysis shows that Eng18B is induced in dual cultures with the fungal plant pathogens Botrytis cinerea and Rhizoctonia solani, although a basal expression is observed in all growth conditions tested. Eng18B disruption strains had significantly reduced growth rates but higher conidiation rates compared to the wild-type strain. However, growth rates on abiotic stress media were significantly higher in Eng18B disruption strains compared to the wild-type strain. No difference in spore germination, germ-tube morphology or in hyphal branching was detected. Disruption strains produced less biomass in liquid cultures than the wild-type strain when grown with chitin as the sole carbon source. In addition, we determined that Eng18B is required for the antagonistic ability of T. atroviride against the grey mould fungus B. cinerea in dual cultures and that this reduction in antagonistic ability is partly connected to a secreted factor. The phenotypes were recovered by re-introduction of an intact Eng18B gene fragment in mutant strains. A putative role of Eng18B ENGase activity in the endoplasmic reticulum associated protein degradation pathway of endogenous glycoproteins in T. atroviride is discussed in relation to the observed phenotypes.     

Biosynthesis of endochitinase of Serratia marcescens.

The growth of a mutant strain of Serratia marcescens with high chitinase activity and the biosynthesis of endochitinase by this strain were investigated. The study was carried out using semisynthetic culture medium without inducers and culture medium containing colloidal chitin as a sole nitrogen and carbon source, with and without mitomycin C. The mutant strain, unlike the native one, was shown to produce endochitinase and to secrete the enzyme into the medium during the growth on culture medium without the inducers, chitin and mitomycin C. During growth on the medium with chitin the mutant strain differed from the native one with a short lag-phase of growth, the early appearance of endochitinase in the culture liquid and a high level of endochitinase activity. The difference between the strains disappeared after the addition of mitomycin C, an inducer of the cell SOS-response, to the culture medium containing chitin. Specific endochitinase activity of S. marcescens mutant strain grown on various culture media had two maxima, namely at the beginning and at the end of the stationary phase. Mitomycin C increased the specific activity in a second peak of endochitinase activity during the growth of the mutant strain.     

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.     

Class III Chitin Synthase ChsB of Aspergillus nidulans Localizes at the Sites of Polarized Cell Wall Synthesis and Is Required for Conidial Development

Class III chitin synthases play important roles in tip growth and conidiation in many filamentous fungi. However, little is known about their functions in those processes. To address these issues, we characterized the deletion mutant of a class III chitin synthase-encoding gene of Aspergillus nidulans, chsB, and investigated ChsB localization in the hyphae and conidiophores. Multilayered cell walls and intrahyphal hyphae were observed in the hyphae of the chsB deletion mutant, and wavy septa were also occasionally observed. ChsB tagged with FLAG or enhanced green fluorescent protein (EGFP) localized mainly at the tips of germ tubes, hyphal tips, and forming septa during hyphal growth. EGFP-ChsB predominantly localized at polarized growth sites and between vesicles and metulae, between metulae and phialides, and between phalides and conidia in asexual development. These results strongly suggest that ChsB functions in the formation of normal cell walls of hyphae, as well as in conidiophore and conidia development in A. nidulans.Chitin, a polymer of β-1,4-linked N-acetylglucosmine, is one of the major structural components of the fungal cell wall. Its metabolism, including synthesis, degradation, assembly, and cross-linking to other cell wall components, is thought to be very important for many fungi (5, 22, 24, 36, 45). Fungal chitin synthases have been classified into seven groups, classes I to VII, depending on the structures of their conserved regions (6). The genes encoding the synthases belonging to classes III, V, VI, and VII are only found in fungi with high chitin contents in their cell walls. We have identified six chitin synthase genes from Aspergillus nidulans and designated them chsA, chsB, chsC, chsD, csmA, and csmB; these gene products belong to classes II, III, I, IV, V, and VI, respectively (9, 13, 30, 31, 44, 52). The chsB deletion mutant grew very slowly and formed small colonies with highly branched hyphae, suggesting its important role in hyphal tip growth (3, 52). Repression of chsB expression in the deletion mutant of chsA, chsC, or chsD exaggerated the defects in the formation of aerial hyphae, the production of cell mass, or the growth under high-osmolarity conditions, respectively, compared to each single mutant. These results indicate that chsB functions at various stages of development (15, 16).The deletion of class III chitin synthase-encoding genes leads to severe defects in most of the filamentous fungi thus far investigated. However, their detailed functions are currently unknown. In Neurospora crassa, inactivation of the gene encoding Chs-1, a class III chitin synthase with 63% identity to A. nidulans ChsB, leads to slow growth, aberrant hyphal morphology, and a decrease in chitin synthase activity. The mutant of chs-1 became sensitive to Nikkomycin Z, a chitin synthase inhibitor (53). In Aspergillus fumigatus, two genes encoding class III chitin synthases, chsC and chsG, have been identified. Their gene products showed 66 and 89% identity, respectively, to A. nidulans ChsB. The chsG deletion mutant showed slow growth and defects in conidiation, and its hyphae were highly branched. chsC deletion did not cause any phenotypic change. The chsC chsG double deletion mutant showed almost the same phenotype as the chsG single deletion mutant (28). Class III chitin synthases have been reported to be involved in the virulence of some pathogens. Deletion of Bcchs3a in the phytopathogenic fungus Botrytis cinerea and double deletion of WdCHS3 and class I chitin synthase WdCHS2 in the human pathogen Wangiella dermatitidis both caused a reduction of virulence (40, 48). On the other hand, the deletion mutant of a class III chitin synthase-encoding gene, CgChsIII, of the maize pathogen Colletotrichum graminicola did not exhibit the significant phenotypic difference from the wild-type strain (50). Deletion of a gene, chs1, encoding a class III chitin synthase of the maize pathogenic dimorphic fungi Ustilago maydis caused minor defects in the growth of haploid yeastlike cells and conjugation tube formation (49). These results indicate that the functions of class III chitin synthases has evolutionally diverged.In the present study, we characterized the cytological defects of the A. nidulans chsB deletion mutant and investigated the localization of ChsB using FLAG- or enhanced green fluorescent protein (EGFP)-tagged ChsB. We reveal that the deletion mutant formed hyphae with aberrant cell wall structures and that ChsB tagged with EGFP primarily localized at polarized growth sites during germination, hyphal growth, septation, and conidiation. These findings suggest that ChsB functions at the polarized growth sites and forming septa during the hyphal growth and conidia development.     

Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune

In the presence of MgSO₄ as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (α-1,3-glucan), R-glucan (β-1,3, β-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion to hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 μg/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.     

Transportation of Aspergillus nidulans Class III and V Chitin Synthases to the Hyphal Tips Depends on Conventional Kinesin

Cell wall formation and maintenance are crucial for hyphal morphogenesis. In many filamentous fungi, chitin is one of the main structural components of the cell wall. Aspergillus nidulans ChsB, a chitin synthase, and CsmA, a chitin synthase with a myosin motor-like domain (MMD) at its N-terminus, both localize predominantly at the hyphal tip regions and at forming septa. ChsB and CsmA play crucial roles in polarized hyphal growth in A. nidulans. In this study, we investigated the mechanism by which CsmA and ChsB accumulate at the hyphal tip in living hyphae. Deletion of kinA, a gene encoding conventional kinesin (kinesin-1), impaired the localization of GFP-CsmA and GFP-ChsB at the hyphal tips. The transport frequency of GFP-CsmA and GFP-ChsB in both anterograde and retrograde direction appeared lower in the kinA-deletion strain compared to wild type, although the velocities of the movements were comparable. Co-localization of GFP-ChsB and GFP-CsmA with mRFP1-KinA^rigor, a KinA mutant that binds to microtubules but does not move along them, was observed in the posterior of the hyphal tip regions. KinA co-immunoprecipitated with ChsB and CsmA. Co-localization and association of CsmA with KinA did not depend on the MMD. These findings indicate that ChsB and CsmA are transported along microtubules to the subapical region by KinA.     

Zinc ions alter morphology and chitin deposition in an ericoid fungus

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.     

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (deltacsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine-coated coverslips and AFM force--displacement curves were collected. When grown in complete medium, wild-type hyphae had a cell wall spring constant of 0.29 +/- 0.02 N/m. When wild-type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 +/- 0.01 and 0.18 +/- 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild-type hyphae grown in complete medium was determined to be 110 +/- 10 MPa. This decreased to 64 +/- 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl-supplemented medium were found to have an elastic modulus of 67 +/- 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion-exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild-type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.     

Evidence that the Aspergillus nidulans class I and class II chitin synthase genes, chsC and chsA, share critical roles in hyphal wall integrity and conidiophore development

Although many chitin synthase genes have been identified in a broad range of fungal species, there have been only a few reports about their role in fungal morphogenesis. In most cases, single gene disruption or replacement did not reveal their function, possibly because of functional redundancy among them. We obtained null mutants of Aspergillus nidulans chsA and chsC genes encoding non-essential class II and class I chitin synthases, respectively. The DeltachsA DeltachsC mutant exhibited growth defects on media supplemented with sodium dodecyl sulfate (SDS), high concentration of salts, chitin-binding dyes, or chitin synthase competitive inhibitors, suggesting loss of integrity of hyphal wall. Moreover, remarkable abnormalities of the double mutant were observed microscopically during its asexual development. The conidiophore population was drastically reduced. Interestingly, secondary conidiophores were occasionally produced from vesicles of the primary ones. The morphology of these conidiophores was similar to those of the A. nidulans developmental mutants, medusa (medA), abacus (abaA), and some kinds of bristle (brlA). In situ staining patterns suggested that chsA was mainly expressed in the metulae, phialides, and conidia, whereas chsC was expressed in hyphae as well as conidiophores. These results suggest that ChsA and ChsC share critical functions in hyphal wall integrity and differentiation.     

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.