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In fungi, the cell wall plays a crucial role in morphogenesis and response to stress from the external environment. Chitin is one of the main cell wall components in many filamentous fungi. In Aspergillus nidulans, a class III chitin synthase ChsB plays a pivotal role in hyphal extension and morphogenesis. However, little is known about post-translational modifications of ChsB and their functional impacts. In this study, we showed that ChsB is phosphorylated in vivo. We characterized strains that produce ChsB using stepwise truncations of its N-terminal disordered region or deletions of some residues in that region and demonstrated its involvement in ChsB abundance on the hyphal apical surface and in hyphal tip localization. Furthermore, we showed that some deletions in this region affected the phosphorylation states of ChsB, raising the possibility that these states are important for the localization of ChsB to the hyphal surface and the growth of A. nidulans. Our findings indicate that ChsB transport is regulated by its N-terminal disordered region., (© 2023. The Author(s).)

The biosynthesis of citric acid (CA) using Aspergillus niger as a carrier is influenced by mycelium morphology, which is determined by the expression level of morphology-related genes. As a key component of the fungal cell wall, chitin content has an important effect on morphogenesis, and to investigate the effects of this on fermentation performance, we used RNA interference to knockdown chitin synthase C (CHSC) and chitin synthase activator (CHS3) to obtain the single-gene mutant strains A. niger chs3 and chsC and the double mutant A. niger chs3C. We found that the CA fermentation performance of the two single mutants was significantly better than that of the double mutant. The mutant A. niger chs3-4 exhibited CA production potential compared to that of the parent strain in scale-up fermentation; we determined certain characteristics of CA high-yielding strain fermentation pellets. In addition, when chsC alone was silenced, there was very little change in chs3 mRNA levels, whereas those of chsC were significantly reduced when only chs3 was silenced. As this may be because of a synergistic effect between chsC and chs3, and we speculated that the latent activation target of CHS3 is CHSC, our results confirmed this hypothesis. This study is the first application of a separation and combination silence strategy of chitin synthase and chitin synthase activator in the morphology of A. niger CA fermentation. Furthermore, it provides new insights into the method for the morphological study of A. niger fermentation and the interaction of homologous genes. KEY POINTS: • The function of chitin synthase C (chsC) and chitin synthase activator (chs3) is tightly interrelated. • Mycelial morphology was optimized by knockdown of CHS3, resulting in the overproduction of citric acid. • The separation and combination silence strategies are promising tools for the interaction of homologous housekeeping genes., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants P MET3 -OCH1 and P MET3 -CHS3 and the double mutant P MET3 -OCH1/P MET3 -CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the P MET3 -OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.

Although chitin is a major component of the fungal cell wall, in oomycetes (fungal-like organisms), this compound has only been found in very little amounts, mostly in the cell wall of members of the genera Achlya and Saprolegnia. In the oomycetes Phytophthora infestans and P. sojae the presence of chitin has not been demonstrated; however, the gene putatively encoding chitin synthase (CHS), the enzyme that synthesizes chitin, is present in their genomes. The evolutionary significance of the CHS gene in P. infestans and P. sojae genomes is not fully understood and, therefore, further studies are warranted. We have cloned and characterized the putative CHS genes from two Phytophthora spp. and multiple isolates of P. infestans and P. sojae and analyzed their phylogenetic relationships. We also conducted CHS inhibition assays and measured CHS transcriptional activity in Phytophthora spp. during infection of susceptible plants. Results of our investigations suggest that CHS contains all the motifs that are typical in CHS genes of fungal origin and is expressed, at least at the mRNA level, during in vitro and in planta growth. In infected tissues, the highest levels of expression occurred in the first 12 h post inoculation. In addition, results from our inhibition experiments appear to suggest that CHS activity is important for P. infestans normal vegetative growth. Because of the considerable variation in expression during infection when compared to basal expression observed in in vitro cultures of non-sporulating mycelium, we hypothesize that CHS may have a meaningful role in Phytophthora pathogenicity.

Fungal chitin synthase of classes V and VI (or VII), which contain an additional N-terminal myosin motor domain, have been shown to play important roles in pathogenesis. To study the function of BcChsVI in Botrytis cinerea, BcChs6 gene was disrupted through Agrobacterium tumefaciens-mediated transformation. The Bcchs6 disruption mutant exhibited a 45.5 % increasing in its chitin content when compared with wild strain. The qRT-PCR analysis revealed that in Bcchs6 mutant the expression of BcChs6 was significantly decreased, while the expression of BcChs2 and BcChs3a was increased when compared with wild type. It is probable that the disruption of this gene provoked a compensatory mechanism regulating the cellular response to cell wall damage. Interestingly, the radial growth of Bcchs6 mutant was drastically reduced when 50 % solute was removed from the regular PDA medium, and they were more sensitive to Calcofluor white and other cell wall disturbing chemicals. Pathogenicity assays on tomato leaves indicated that they were significantly reduced in their ability to cause disease. Our results demonstrated that BcChs6 is necessary for proper hyphal growth and pathogenicity of B. cinerea on tomato leaves.

Fusarium verticillioides is both an endophyte and a pathogen of maize and is a health threat in many areas of the world because it can contaminate maize with fumonisins, a toxic secondary metabolite. We identified eight putative chitin synthase (CHS) genes in F. verticillioides genomic sequence, and phylogenetic evidence shows that they group into seven established CHS gene classes. We targeted two CHSs (CHS5 and CHS7) for deletion analysis and found that both are required for normal hyphal growth and maximal disease of maize seedlings and ears. CHS5 and CHS7 encode a putative class V and class VII fungal chitin synthase, respectively; they are located adjacent to each other and are divergently transcribed. Fluorescent microscopy found that both CHS deficient strains produce balloon-shaped hyphae, while growth assays indicated that they were more sensitive to cell wall stressing compounds (e.g., the antifungal compound Nikkomycin Z) than wild type. Pathogenicity assays on maize seedlings and ears indicated that both strains were significantly reduced in their ability to cause disease. Our results demonstrate that both CHS5 and CHS7 are necessary for proper hyphal growth and pathogenicity of F. verticillioides on maize.

Gibberella zeae causes Fusarium head blight of cereal crops, and sexual spores of the fungus play an important role as primary inocula. We isolated a restriction enzyme-mediated integration (REMI) transformant, ZH431, of G. zeae with defects in perithecia formation and virulence. Integration of the REMI vector resulted in disruption of GzCHS7 gene, which encodes a putative class VII chitin synthase with high similarity to Fusarium oxysporum ChsVb. A second chitin synthase, GzCHS5, is adjacently located in a head-to-head configuration with GzCHS7, and its deduced protein sequence showed similarity with a class V chitin synthase in F. oxysporum. Neither DeltaGzChs5 nor DeltaGzChs7 mutants produced perithecia or caused disease on barley heads. Microscopic observation revealed that both mutants formed balloon-shaped hyphae and intrahyphal hyphae and that cell wall rigidity of the mutants was weaker than that of the wild-type strain. Transcription profiles of GzCHS5 and GzCHS7 were not altered in DeltaGzChs7 and DeltaGzChs5, respectively, suggesting that transcription regulations of the genes are independent of each other. Our results demonstrate that GzCHS5 and GzCHS7 are indispensable for perithecia formation and pathogenicity as well as normal septa formation and hyphal growth in G. zeae.

We have isolated and characterised the first set of chitin synthase genes from a rust fungus, a large group of economically highly important, obligately biotrophic plant pathogens. Puccinia graminis was used as a model organism for the rust fungi which are not well investigated on the molecular level today. One of the major structural components of most fungal cell walls is the chitin polymer which is synthesised by a family of enzymes called chitin synthases. In P. graminis, we have isolated five new chitin synthase genes from four different classes, chsII, chsIIIa, chsIIIb, chsIV, and chsV. The genes contain a high number of introns, unusual for other known fungal chitin synthases. The dinucleic stage of the fungus seems to contain two slightly different genes or alleles for four isoforms. One isoform, chsIIIa, seems to be expressed only in the youngest stages of fungal growth. Analysis of the derived proteins shows that together with other basidiomycete CHS, the pgtCHS form separate subgroups in the phylogenetic tree. This set of five rust chitin synthase genes, with some unusual features compared to known fungal chitin synthases, allows new insights into chitin synthase classification, and may help in the development of novel functional fungicides.

The chsA and chsC encode classes II and I chitin synthases, respectively, of the filamentous fungus Aspergillus nidulans. The DeltachsA DeltachsC double mutants (DeltaAC mutants) show defects in asexual development: a striking reduction in the number of conidiophores and aberrant conidiophore morphology. Here, we examined the involvement of regulatory genes for asexual development (brlA, abaA, and medA) in the conidiation defects of the DeltaAC mutants. Spatial expression patterns of brlA, abaA, and medA in conidiophores of the wild-type strains and DeltaAC mutants were examined by in-situ staining using a reporter gene; expression of either gene was detected at abnormal sterigmata in the DeltaAC mutants as well as at normal ones in the wild-type strain. However, abaA expression was not prominent at a subset of conidiophores developing long chains of aberrant sterigmata, suggesting that induction of the abaA expression was retarded in the DeltaAC mutants. Based on these results and those previously presented, possible mechanisms involved in the conidiation defects are discussed.

A straightforward and easy-to-apply semi-quantitative RT-PCR method was developed to study multigenic expression in the phytopathogenic fungus Botrytis cinerea. This procedure is based on the one-step reverse transcription-amplification of a specific transcript within total RNA and product amount determination by densitometric analysis of ethidium bromide fluorescence upon gel electrophoresis. The semi-quantitative analysis is achieved, at a fixed PCR cycle-number, within a range of total RNA concentrations that stays in the exponential phase of the PCR. Co-amplification of the transcript of interest with internal controls allowed comparison between different RNA samples. Using this method, we could demonstrate a differential regulation of chitin synthase genes during fungal growth and an effect of the culture carbon source on the expression of two pectin methylesterase genes in B. cinerea. Finally, the method was shown to be applicable to plant-infected tissue, making it a useful tool to detect pathogenicity genes in B. cinerea.

The filamentous fungus, Aspergillus nidulans, genome contains at least five chitin synthase-encoding genes. chsB is essential for normal hyphal growth. chsA and chsC are likely to be cooperatively required for hyphal wall integrity. In this study, we constructed chsA chsB and chsC chsB double mutants, in which chsB expression was under a repressible promoter [ alcA(p)]. While chsA or chsC single mutants did not show obvious growth defects, the chsA chsB and chsC chsB double mutants showed different phenotypes from the chsB single mutant and from each other under alcA(p)-repressing conditions. The chsA chsB double mutant produced fewer aerial hyphae and the chsC chsB double mutant showed reduced cell mass. These observations support the idea that chsA and chsC each play a different role in hyphal morphogenesis. In addition, the chitin contents of these double mutants were higher than those of the chsB single mutant. When chsA was expressed ectopically under the chsB promoter in the chsB mutant, the growth defects caused by chsB repression were not remedied at all, although an increased level of chsA mRNA was observed. Thus, it is suggested that the gene products of chsA and chsB themselves have different functions in hyphal morphogenesis.

Chitin synthesis is a process maintained across the fungal kingdom that, thanks to the power of genetic manipulation of yeast cells, is now beginning to be understood. Chitin synthesis is based on the regulation of distinct chitin synthase isoenzymes whose number ranges from one in Schizosaccharomyces pombe to seven in some filamentous fungi, such as Aspergillus fumigatus. This high diversity makes it difficult to find a unique model of regulation. However, the results available suggest common themes in regulation. The arrival of the genomic era, together with the development of fungal genetic technology should allow experimental approaches to this process.

We cloned and characterized a novel Aspergillus oryzae chitin synthase gene, chsZ, encoding a polypeptide containing a new myosin motor-like domain in its N-terminal half. Alignment analysis revealed that ChsZ was less homologous to known class V enzymes, except for its probable chitin synthase conserved region in the C-terminal half. We also found a chsY gene and found that ChsY showed higher similarity to the class V enzymes than did ChsZ. Phylogenetic analysis clearly demonstrated that the A. oryzae ChsZ, together with Chs4 of Paracoccidioides brasiliensis and Chs6 of Ustilago maydis, formed a new subclass distinct from A. oryzae ChsY and known class V chitin synthases, including A. nidulans CsmA (ChsD) and A. fumigatus ChsE. In conclusion, we propose a new class, class VI chitin synthases, represented by A. oryzae ChsZ, P. brasiliensis Chs4 and U. maydis Chs6. Expression analysis suggested that the regulation of chsZ expression is distinct from that of chsY expression.

A complete chitin synthase gene and one chitin synthase gene fragment of the zygomycete Mucor circinelloides have been cloned and analyzed. Both genes encode zymogenic Class II chitin synthases. Hybridization analysis showed that there must exist at least another Class II chitin synthase gene in M. circinelloides highly homologous to the cloned Mcchs1 and Mcchs2. The expression of these genes during the dimorphic growing stages was analyzed. Northern hybridizations showed that Mcchs1 transcript accumulates only during the exponentially growing hyphal stage, while no expression could be detected in the yeast form. Expression of Mcchs2 could not be detected at any stage. Accumulation of Mcchs1 transcript was not influenced by visible light. The existence of a multigene chitin synthase family and the observation that Mcchs1 transcription depends upon the dimorphic stage indicate that various chitin synthase activities may have different roles in the dimorphic growth of M. circinelloides.

Nucleotide sequences of chitin synthase 1 (CHS1) gene of dermatophytes, Arthroderma benhamiae, A. simii, A. vanbreuseghemii, Trichophyton mentagrophytes var. interdigitale (T. interdigitale), and T. rubrum were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these five dermatophytes showed more than 90% similarity between the species. The phylogenetic analysis of their sequences revealed that A. benhamiae, A. simii, A. vanbreuseghemii, and T. rubrum were genetically distinct from one another, but T. interdigitale was genetically very close to A. vanbreuseghemii. On the other hand, a specific restriction endonuclease site of HinfI was present in the CHS1 gene fragment of T. rubrum but not in those of A. benhamiae, A. simii, A. vanbreuseghemii and T. interdigitale. The molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution.

Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.

Degenerate PCR primers were used to amplify a 600-bp conserved gene region for chitin synthases from genomic DNA of Sporothrix schenckii, a dimorphic fungal pathogen of humans and animals. Three chitin synthase gene homologs were amplified as shown by DNA sequence analysis and by Southern blotting experiments. Based on differences among the predicted amino acid sequences of these homologs, each was placed within one of three different chitin synthase classes. Phylogenies constructed with the sequences and the PAUP 3.1.1 program showed that S. schenckii consistently clustered most closely with Neurospora crassa in each of the three chitin synthase classes. These findings are significant because the phylogenies support by a new method the grouping of the imperfect fungus S. schenckii with the Pyrenomycetes of the Ascomycota.

Chitin is a component of the yeast cell wall which is localized to the septum between mother and daughter cells. Previous work in Saccharomyces cerevisiae has shown that this organism possesses three chitin synthases, 1, 2, and 3. Disruption experiments have shown that loss of chitin synthase 2 has a more profound effect on cell viability than loss of either of the other two and is lethal in complete media. We report here the finding of an S. cerevisiae strain which does not require the chitin synthase 2 structural gene for viability. We present evidence that there is a gene in this strain which suppresses the lethality of disruption of the chitin synthase 2 structural gene and is genetically distinct from the structural genes for chitin synthase 1 and chitin synthase 2. We show that an S. cerevisiae mutant containing the suppressor and lacking both structural genes for chitin synthase 1 and 2 has normal amounts of chitin in its cell wall. We hypothesize that the suppressor gene encodes or controls the expression of chitin synthase 3.