The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

 We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion

Although chitin is an essential component of the fungal cell wall (CW), its biosynthesis and role in virulence is poorly understood. In Aspergillus fumigatus, there are eight chitin synthase (CHS) genes belonging to two families CHSA‐C, CHSG in family 1 and CHSF, CHSD, CSMA, CSMB in family 2). To understand the function of these CHS genes, their single and multiple deletions were performed using β‐rec/six system to be able to delete all genes within each family (up to a quadruple ΔchsA/C/B/G mutant in family 1 and a quadruple ΔcsmA/csmB/F/D mutant in family 2). Radial growth, conidiation, mycelial/conidial morphology, CW polysaccharide content, Chs‐activity, susceptibility to antifungal molecules and pathogenicity in experimental animal aspergillosis were analysed for all the mutants. Among the family 1 CHS, ΔchsA, ΔchsB and ΔchsC mutants showed limited impact on chitin synthesis. In contrast, there was reduced conidiation, altered mycelial morphotype and reduced growth and Chs‐activity in the ΔchsG and ΔchsA/C/B/G mutants. In spite of this altered phenotype, these two mutants were as virulent as the parental strain in the experimental aspergillosis models. Among family 2 CHS, phenotypic defects mainly resulted from the CSMA deletion. Despite significant morphological mycelial and conidial growth phenotypes in the quadruple ΔcsmA/csmB/F/D mutant, the chitin content was poorly affected by gene deletions in this family. However, the entire mycelial cell wall structure was disorganized in the family 2 mutants that may be related to the reduced pathogenicity of the quadruple ΔcsmA/csmB/F/D mutant strain compared to the parental strain, in vivo. Deletion of the genes encompassing the two families (ΔcsmA/csmB/F/G) showed that in spite of being originated from an ancient divergence of fungi, these two families work cooperatively to synthesize chitin in A. fumigatus and demonstrate the essentiality of chitin biosynthesis for vegetative growth, resistance to antifungal drugs, and virulence of this filamentous fungus.     

Energy Value Evaluation of Hydrogenated Resistant Maltodextrin

Two chitin synthase genes, designated chsA and chsB, were isolated from Aspergillus nidulans with the Saccharomyces cerevisiae CHS2 gene as the hybridization probe. Nucleotide sequencing showed that chsA and chsB encoded polypeptides consisting of 1013 and 916 amino acid residues, respectively; the hydropathy profiles of the enzymes were similar to those of other fungal chitin synthases. Northern analysis indicated that both genes were transcribed, suggesting that cellular chitin in A. nidulans is synthesized by at least two chitin synthases. For examination of the roles of the chitin synthase genes in cell growth, gene disruption experiments were done. The chsA disruptant grew as well as the wild-type strain, but the chsB disruptant had severe growth defects that could not be overcome by the addition of 1.2 m sorbitol as an osmotic stabilizer. These findings suggested that chsB but not chsA is essential for hyphal growth.     

The Aspergillus nidulans genes chsA and chsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, and chsC) from Aspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, named chsD, from A. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 of Saccharomyces cerevisiae and Chs3 of Candida albicans. Disruption of chsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption of chsA and chsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption of chsC and chsD caused no defect. Thus it appears that chsA and chsD serve redundant functions in conidia formation.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

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.     

A class Vb chitin synthase in Colletotrichum graminicola is localized in the growing tips of multiple cell types, in nascent septa, and during septum conversion to an end wall after hyphal breakage

Summary. Previous complementation of a chitin synthase class Vb null mutant (Colletotrichum graminicola chsA) indicated that the encoded protein is responsible for approximately 30% of the conidial chitin, is essential for conidial wall strength in media with high water potential, and contributes to strength of hyphal tips. We complemented a chsA null mutant with chsA fused to the green-fluorescent protein (sgfp) gene driven by a heterologous constitutively expressed promoter. Comparisons of the strain with the ectopic chsA-sgfp to the wild type indicated that ChsA-sGFP serves the same biological functions as ChsA in that like the wild type, the chsAΔ chsA::sgfp (EC) had conidia that did not explode and hyphal tips that did not swell. Confocal microscopy of ChsA-sGFP (EC) cells stained with the membrane stain FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide) indicated that ChsA is localized in the plasma membrane of the following: growing apices of hyphal branches, conidiophores, and falcate and oval conidia; in nascent septa; and in septa that are being converted to an end wall after hyphal breakage. The data support the hypothesis that chsA either directly or indirectly encodes the information for its localization, that ChsA is localized in the plasma membrane, and that the class Vb enzyme produces chitin synthase in multiple cells and after wall breakage. Correspondence and reprints: Department of Plant Pathology, University of California, Davis, CA 95616-8680, USA.     

Deletion of BGL2 results in an increased chitin level in the cell wall of Saccharomyces cerevisiae

It is shown that the deletion of BGL2 gene leads to increase in chitin content in the cell wall of Saccharomyces cerevisiae. A part of the additional chitin can be removed from the bgl2Δ cell wall by alkali or trypsin treatment. Chitin synthase 1 (Chs1) activity was increased by 60 % in bgl2Δ mutant. No increase in chitin synthase 3 (Chs3) activity in bgl2Δ cells was observed, while they became more sensitive to Nikkomycin Z. The chitin level in the cell walls of a strain lacking both BGL2 and CHS3 genes was higher than that in chs3Δ and lower than that in bgl2Δ strains. Together these data indicate that the deletion of BGL2 results in the accumulation and abnormal incorporation of chitin into the cell wall of S. cerevisiae, and both Chs1 and Chs3 take part in a response to BGL2 deletion in S. cerevisiae cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

几丁质合酶在人类致病真菌中的研究进展

抑制真菌细胞壁的合成常作为防治真菌感染的安全有效手段。几丁质是真菌细胞壁及隔膜的重要结构成分，几丁质合酶是催化几丁质合成的关键酶。真菌细胞中几丁质合酶家族的不同成员在调控几丁质的合成中存在着差异，因此产生不同的生物学效应。本文通过综述几丁质合酶在人体三大条件致病真菌白色念珠菌、烟曲霉、新生隐球菌中的研究进展，分析了几丁质合酶对真菌致病性影响的机制，总结了几丁质合酶调控真菌细胞增殖、形态转换、病原菌与宿主的相互作用和细胞壁损伤诱导的补偿效应，展望了抗真菌感染的新策略及关于真菌几丁质合酶的未来研究方向。     

Proper ascospore maturation requires the chs1+ chitin synthase gene in Schizosaccharomyces pombe

We have cloned chs1+, a Schizosaccharomyces pombe gene with similarity to class II chitin synthases, and have shown that it is responsible for chitin synthase activity present in cell extracts from this organism. Analysis of this activity reveals that it behaves like chitin synthases from other fungi, although with specific biochemical characteristics. Deletion or overexpression of this gene does not lead to any apparent defect during vegetative growth. In contrast, chs1+ expression increases significantly during sporulation, and this is accompanied by an increase in chitin synthase activity. In addition, spore formation is severely affected when both parental strains carry a chs1 deletion, as a result of a defect in the synthesis of the ascospore cell wall. Finally, we show that wild-type, but not chs1-/chs1-, ascospore cell walls bind wheatgerm agglutinin. Our results clearly suggest the existence of a relationship between chs1+, chitin synthesis and ascospore maturation in S. pombe.     

AsSlt2基因对茄链格孢细胞壁完整性的调控

【背景】由茄链格孢(Alternaria solani)引起的马铃薯早疫病被普遍认为是马铃薯生产上的第二大叶部病害,在马铃薯各产区普遍发生,给马铃薯生产造成了巨大的经济损失。【目的】明确AsSlt2基因对茄链格孢细胞壁完整性的影响。【方法】在含有刚果红、细胞壁降解酶和十二烷基硫酸钠(sodiumdodecylsulfate,SDS)等细胞壁胁迫的培养基上观察ΔAsSlt2缺失突变株的生长情况,计算相对生长抑制率;通过实时荧光定量PCR (RT-qPCR)方法检测ΔAsSlt2菌株中细胞壁合成相关基因的表达情况;进一步检测ΔAsSlt2细胞壁中几丁质的含量及胞外酶活性。【结果】ΔAsSlt2缺失突变株对SDS、刚果红、细胞壁降解酶等细胞壁胁迫的敏感性增强,在加入细胞壁降解酶后突变株原生质体释放量显著增多;ΔAsSlt2对外源氧胁迫更敏感,突变株胞外过氧化物酶和漆酶活性均显著降低;进一步研究发现,ΔAsSlt2细胞壁中几丁质含量减少,几丁质合成相关基因与漆酶合成相关基因的表达量均明显降低。【结论】AsSlt2基因在茄链格孢细胞壁的完整性及抵御外界胁迫方面发挥重要作用。     

Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A ( chsA )

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression ofchsA gene in transgenicPetunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal ofchsA gene, and transferred the fusion gene intoPetunia hybrida viaAgrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNAin situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.     

Isolation of a Chitin Synthase Gene (chsC) of Aspergillus nidulans

We isolated a class I chitin synthase gene (chsC) from Aspergillus nidulans. Expression of this gene was confirmed by Northern analysis and by sequencing of the PCR-amplified DNA fragments from cDNA. chsC disruptants showed no difference of morphology in the asexual cycle and no difference of growth rate compared to a wild-type strain.     

Umchs5,a Gene Coding for a Class IV Chitin Synthase inUstilago maydis

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungusUstilago maydiswas amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product ofUmchs5has highest similarity with class IV chitin synthases encoded by theCHS3genes fromSaccharomyces cerevisiaeandCandida albicans, chs-4fromNeurospora crassa,andchsEfromAspergillus nidulans. Umchs5null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase,Umchs6.It is suggested that multigenic control of chitin synthesis inU. maydisoperates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes.     

Chitin synthetase mutants of Phycomyces blakesleeanus

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 M nikkomycin, chsA mutants grew reasonably well in the presence of 50 M nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased K_a for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.     

金针菇几丁质合成酶基因家族鉴定及其表达分析

【背景】几丁质是真菌细胞壁的重要成分,由几丁质合成酶（chitin synthase,CS）催化合成。几丁质合成酶编码基因在大型食用真菌金针菇中的数量及表达规律尚不明确。【目的】探究几丁质合成酶基因在金针菇中存在的数量及其在子实体不同发育时期的表达规律,为其在大型真菌子实体生长发育过程中的功能研究提供基础。【方法】基于已有的金针菇菌株L11基因组数据,结合NCBI其他真菌CS序列鉴定金针菇中几丁质合成酶编码基因的数量,并对其进行生物信息学分析。进一步根据金针菇F19转录组数据以及实时荧光定量PCR （RT-qPCR）技术分析金针菇CS基因家族的表达规律。【结果】在金针菇单核体菌株L11的基因组中鉴定到9个几丁质合成酶基因,系统发育分析表明它们在子实体发育过程中的表达模式可分为4类（皮尔森相关系数=0.85）。【结论】金针菇CS基因家族表达模式在金针菇不同生长发育时期均存在差异,可能参与了子实体发育不同时期和组织的形态建成。     

Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs

Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.