Dataset Information

Genome-Wide Identification and Analysis of Chitinase GH18 Gene Family in Mycogone perniciosa.

ABSTRACT: Mycogone perniciosa causes wet bubble disease in Agaricus bisporus and various Agaricomycetes species. In a previous work, we identified 41 GH18 chitinase genes and other pathogenicity-related genes in the genome of M. perniciosa Hp10. Chitinases are enzymes that degrade chitin, and they have diverse functions in nutrition, morphogenesis, and pathogenesis. However, these important genes in M. perniciosa have not been fully characterized, and their functions remain unclear. Here, we performed a genome-wide analysis of M. perniciosa GH18 genes and analyzed the transcriptome profiles and GH18 expression patterns in M. perniciosa during the time course of infection in A. bisporus. Phylogenetic analysis of the 41 GH18 genes with those of 15 other species showed that the genes were clustered into three groups and eight subgroups based on their conserved domains. The GH18 genes clustered in the same group shared different gene structures but had the same protein motifs. All GH18 genes were localized in different organelles, were unevenly distributed on 11 contigs, and had orthologs in the other 13 species. Twelve duplication events were identified, and these had undergone both positive and purifying selection. The transcriptome analyses revealed that numerous genes, including transporters, cell wall degrading enzymes (CWDEs), cytochrome P450, pathogenicity-related genes, secondary metabolites, and transcription factors, were significantly upregulated at different stages of M. perniciosa Hp10 infection of A. bisporus. Twenty-three out of the 41 GH18 genes were differentially expressed. The expression patterns of the 23 GH18 genes were different and were significantly expressed from 3 days post-inoculation of M. perniciosa Hp10 in A. bisporus. Five differentially expressed GH18 genes were selected for RT-PCR and gene cloning to verify RNA-seq data accuracy. The results showed that those genes were successively expressed in different infection stages, consistent with the previous sequencing results. Our study provides a comprehensive analysis of pathogenicity-related and GH18 chitinase genes' influence on M. perniciosa mycoparasitism of A. bisporus. Our findings may serve as a basis for further studies of M. perniciosa mycoparasitism, and the results have potential value for improving resistance in A. bisporus and developing efficient disease-management strategies to mitigate wet bubble disease.

SUBMITTER: Yang Y

PROVIDER: S-EPMC7829358 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Genome-Wide Identification and Analysis of Chitinase GH18 Gene Family in Mycogone perniciosa.

Yang Yang Y Sossah Frederick Leo FL Li Zhuang Z Hyde Kevin D KD Li Dan D Xiao Shijun S Fu Yongping Y Yuan Xiaohui X Li Yu Y

Frontiers in microbiology 20210111

Mycogone perniciosa causes wet bubble disease in Agaricus bisporus and various Agaricomycetes species. In a previous work, we identified 41 GH18 chitinase genes and other pathogenicity-related genes in the genome of M. perniciosa Hp10. Chitinases are enzymes that degrade chitin, and they have diverse functions in nutrition, morphogenesis, and pathogenesis. However, these important genes in M. perniciosa have not been fully characterized, and their functi ...[more]

PMID: 33505368

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Project description:BackgroundChitinases (EC.3.2.1.14) hydrolyze the beta-1,4-linkages in chitin, an abundant N-acetyl-beta-D-glucosamine polysaccharide that is a structural component of protective biological matrices such as insect exoskeletons and fungal cell walls. The glycoside hydrolase 18 (GH18) family of chitinases is an ancient gene family widely expressed in archea, prokaryotes and eukaryotes. Mammals are not known to synthesize chitin or metabolize it as a nutrient, yet the human genome encodes eight GH18 family members. Some GH18 proteins lack an essential catalytic glutamic acid and are likely to act as lectins rather than as enzymes. This study used comparative genomic analysis to address the evolutionary history of the GH18 multiprotein family, from early eukaryotes to mammals, in an effort to understand the forces that shaped the human genome content of chitinase related proteins.ResultsGene duplication and loss according to a birth-and-death model of evolution is a feature of the evolutionary history of the GH18 family. The current human family likely originated from ancient genes present at the time of the bilaterian expansion (approx. 550 mya). The family expanded in the chitinous protostomes C. elegans and D. melanogaster, declined in early deuterostomes as chitin synthesis disappeared, and expanded again in late deuterostomes with a significant increase in gene number after the avian/mammalian split.ConclusionThis comprehensive genomic study of animal GH18 proteins reveals three major phylogenetic groups in the family: chitobiases, chitinases/chitolectins, and stabilin-1 interacting chitolectins. Only the chitinase/chitolectin group is associated with expansion in late deuterostomes. Finding that the human GH18 gene family is closely linked to the human major histocompatibility complex paralogon on chromosome 1, together with the recent association of GH18 chitinase activity with Th2 cell inflammation, suggests that its late expansion could be related to an emerging interface of innate and adaptive immunity during early vertebrate history.

Project description:The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.Two novel constitutively expressed members of the glycosyl hydrolase (GH18) gene family of chitinases were isolated from the A. astaci strain Gb04. The primary amino acid sequence of these chitinase genes, termed CHI2 and CHI3, is composed of an N-terminal signal peptide directing the post-translational transport of the protein into the extracellular space, the catalytic GH18 domain, a proline-, serine-, and threonine-rich domain and a C-terminal cysteine-rich putative chitin-binding site. The A. astaci mycelium grown in a pepton-glucose medium showed significant temporal changes in steady-state CHI2 and CHI3 mRNA amounts indicating functional constraint. Their different temporal occurrence with maxima at 48 and 24 hours of incubation for CHI2 and CHI3, respectively, is in accordance with the multifunctionality of GH18 family members. To identify A. astaci-specific primer target sites in these novel genes, we determined the partial sequence homologs in the related oomycetes A. frigidophilus, A. invadans, A. helicoides, A. laevis, A. repetans, Achlya racemosa, Leptolegnia caudata, and Saprolegnia parasitica, as well as in the relevant fungi Fusarium solani and Trichosporon cutaneum. An A. astaci-specific primer pair targeting the novel genes CHI2 and CHI3 as well as CHI1 - a third GH18 family member - was multiplexed with primers targeting the 5.8S rRNA used as an endogenous control. A species was typed unambiguously as A. astaci if two peaks were concomitantly detected by melting curve analysis (MCA). For sensitive detection of the pathogen, but also for quantification of agent levels in susceptible crayfish and carrier crayfish, a TaqMan-probe based real-time PCR (qPCR) assay was developed. It targets the same chitinase genes and allows quantification down to 25 target sequences.The simultaneous qualitative detection of multiple sequences by qPCR/MCA represents a promising approach to detect species with elevated levels of genetic variation and/or limited available sequence information. The homogenous closed-tube format, reduced detection time, higher specificity, and the considerably reduced chance of false negative detection achieved by targeting multiple genes (CHI1, CHI2, CHI3, and the endogenous control) at least two of which are subject to high functional constraint, are the major advantages of this multiplex assay compared to other diagnostic methods. Sensitive quantification achieved with TaqMan qPCR facilitates to monitor infection status and pathogen distribution in different tissues and can help prevent disease transmission.

Dataset Information

Genome-Wide Identification and Analysis of Chitinase GH18 Gene Family in Mycogone perniciosa.

Publications

Genome-Wide Identification and Analysis of Chitinase GH18 Gene Family in <i>Mycogone perniciosa</i>.

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