Rab GDP-dissociation inhibitor gdiA is an essential gene required for cell wall chitin deposition in Aspergillus niger

https://doi.org/10.1016/j.fgb.2019.103319Get rights and content
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Highlights

  • Aspergillus niger cell wall chitin mutant characterized by co-segregation analysis.

  • GDP-dissociation inhibitor A (gdiA) was identified as an essential gene.

  • Intronic SNPs in cell wall mutant gdiA cause inefficient splicing.

  • CRISPR/Cas9 genome editing was successfully used to reconstitute mutant gdiA SNPs.

  • The gdiA mutant allele is a candidate for GdiA-related secretion studies in fungi.

Abstract

The cell wall is a distinctive feature of filamentous fungi, providing them with structural integrity and protection from both biotic and abiotic factors. Unlike plant cell walls, fungi rely on structurally strong hydrophobic chitin core for mechanical strength together with alpha- and beta-glucans, galactomannans and glycoproteins. Cell wall stress conditions are known to alter the cell wall through the signaling cascade of the cell wall integrity (CWI) pathway and can result in increased cell wall chitin deposition. A previously isolated set of Aspergillus niger cell wall mutants was screened for increased cell wall chitin deposition. UV-mutant RD15.8#16 was found to contain approximately 60% more cell wall chitin than the wild type. In addition to the chitin phenotype, RD15.8#16 exhibits a compact colony morphology and increased sensitivity towards SDS. RD15.8#16 was subjected to classical genetic approach for identification of the underlying causative mutation, using co-segregation analysis and SNP genotyping. Genome sequencing of RD15.8#16 revealed eight SNPs in open reading frames (ORF) which were individually checked for co-segregation with the associated phenotypes, and showed the potential relevance of two genes located on chromosome IV. In situ re-creation of these ORF-located SNPs in a wild type background, using CRISPR/Cas9 genome editing, showed the importance Rab GTPase dissociation inhibitor A (gdiA) for the phenotypes of RD15.8#16. An alteration in the 5′ donor splice site of gdiA reduced pre-mRNA splicing efficiency, causing aberrant cell wall assembly and increased chitin levels, whereas gene disruption attempts showed that a full gene deletion of gdiA is lethal.

Keywords

Cell wall
Chitin
Rab GTPase family GDI
Intron
Splicing
Alternative splicing
Co-segregation analysis
CRISPR/Cas9
Aspergillus niger

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