Genome sequences of 24 Aspergillus niger sensu stricto strains to study strain diversity, heterokaryon compatibility, and sexual reproduction

Abstract

Mating-type distribution within a phylogenetic tree, heterokaryon compatibility, and subsequent diploid formation were studied in 24 Aspergillus niger sensu stricto strains. The genomes of the 24 strains were sequenced and analyzed revealing an average of 6.1 ± 2.0 variants/kb between Aspergillus niger sensu stricto strains. The genome sequences were used together with available genome data to generate a phylogenetic tree revealing 3 distinct clades within Aspergillus niger sensu stricto. The phylogenetic tree revealed that both MAT1-1 and MAT1-2 mating types were present in each of the 3 clades. The phylogenetic differences were used to select for strains to analyze heterokaryon compatibility. Conidial color markers (fwnA and brnA) and auxotrophic markers (pyrG and nicB) were introduced via CRISPR/Cas9-based genome editing in a selection of strains. Twenty-three parasexual crosses using 11 different strains were performed. Only a single parasexual cross between genetically highly similar strains resulted in a successful formation of heterokaryotic mycelium and subsequent diploid formation, indicating widespread heterokaryon incompatibility as well as multiple active heterokaryon incompatibility systems between Aspergillus niger sensu stricto strains. The 2 vegetatively compatible strains were of 2 different mating types and a stable diploid was isolated from this heterokaryon. Sclerotium formation was induced on agar media containing Triton X-100; however, the sclerotia remained sterile and no ascospores were observed. Nevertheless, this is the first report of a diploid Aspergillus niger sensu stricto strain with 2 different mating types, which offers the unique possibility to screen for conditions that might lead to ascospore formation in A. niger.

Keywords: Aspergillus niger; CRISPR/Cas9; heterokaryon incompatibility; heterozygous diploid; mating type; parasexual cycle; sclerotia; sexual cycle; whole genome sequencing.

Fig. 1.

Schematical overview of the MAT1-1 and MAT1-2 loci in A. niger. Conserved genes between the 2 MAT loci have been color coded. White genes are not conserved between the 2 loci and gray genes are positioned outside of the 2 MAT loci. In red, the mating-type genes MAT1-1-1 and MAT1-2-1. These genes are transcription factors, where MAT1-1-1 contains an “alpha1 HMG-box” domain and MAT1-2-1 contains a high mobility group (HMG) domain. In aspergilli, these mating-type genes are normally flanked by the DNA lyase apnB and a cytoskeleton assembly control factor slaB. However, in A. niger the slaB gene is located more than 10-kb downstream of the MAT genes. a) Mating-type locus MAT1-1. This mating-type locus appears to have a flipped orientation when compared to the MAT1-2 locus and when compared to MAT1-1 and MAT1-2 loci of other aspergilli. b) Mating-type locus MAT1-2. Only recently described in the A. niger neotype strain CBS554.65, the orientation of the genes in this locus corresponds with expectations based on the MAT1-1 and MAT1-2 locus organization of other aspergilli. The mating-type loci, the individual genes, and their possible functions in A. niger have been discussed more extensively in a recent study (Ellena et al. 2021).

Fig. 2.

Diagnostic PCR for the presence of the mating-type genes. a) An in silico representation of the diagnostic PCRs performed to investigate presence of the mating-type genes. The primers were designed to be idiomorph-specific based on the haploid parents. If the MAT1-1-1 gene is present, amplification with the MT1_fw and MT1_rv primers will result in a 734 bps band on the gel. If the MAT1-2-1 gene is present, amplification with the MT2_fw and MT2_rv primers will result in a 1009 bps band on the gel. b) This gel shows the diagnostic PCR results confirming the presence of both the MAT1-1-1 gene and the MAT1-2-1 gene in the 6 diploid strains. Columns 1–6 are PCR products resulting from amplification on gDNA from individually obtained diploid strains SJS150.1, SJS150.2, SJS150.3, SJS151.1, SJS151.2, and SJS151.3. In the diploid strains, both PCR products are present confirming the presence of both mating-type genes. Column 7 contains the GeneRuler 1 kb DNA ladder (Thermo Scientific). Columns 8 and 9 are PCR products resulting from amplification on gDNA from MAT1-2 containing CBS147323 parental strains (SJS111 and NS4, respectively). Columns 10 and 11 are PCR products resulting from amplification on gDNA from MAT1-1 containing CBS147347 parental strains (SJS114 and NS8, respectively). In the haploid parental strains, only a single mating-type gene is present. Note that the CBS 147347 parental strains show off-target amplification but do not contain the MAT1-2-1 gene.

Fig. 3.

Phylogenetic tree of Aspergillus section Nigri strains. This phylogenetic tree was based on 3268 single-copy orthologs proteins found in all strains. Bootstrap values of all branches were calculated but not visualized to increase visibility. All branches had bootstrap values of 100, except branches between individual A. niger sensu stricto strains sequenced in this study (see Fig. 4) and between A. tubingensis and A. costaricaensis (bootstrap value = 69). The tree was visualized using iTOL v4 (Letunic and Bork 2019).

Fig. 4.

Phylogenetic tree of A. niger sensu stricto. This phylogenetic tree was based on 7718 single-copy orthologous proteins found in all 33 included strains. Most A. niger sensu stricto strains are part of 3 distinct clades, clade A, clade B, or clade C. Mating types of each strain is visualized with ∗ for the MAT1-1 locus containing genomes and ∗∗ for the MAT1-2 locus containing genomes. Heterokaryon formation via forced protoplast fusion was investigated; successful parasexual crosses are visualized by the green lines and unsuccessful parasexual crosses are visualized by the red lines. A single parasexual crossing resulted in successful heterokaryon formation, between strain CBS147323 and CBS147347 located in clade B, which are of different mating type. Bootstrap values of all branches were calculated, to improve visibility of the figure only bootstrap values indicating uncertainty (smaller than 100) are shown. The tree was visualized using iTOL v4 (Letunic and Bork 2019).

Fig. 5.

Sclerotia induction of A. niger N402 on MEA + Triton X-100 plates. Plates had an inner diameter of 88 mm. Here, the effect of Triton X-100 on sclerotium formation in A. niger N402 was assessed. Plates were inoculated by confluently plating ∼25 conidia per plate. Pictures of plates were taken after 6 days of incubation at 30°C. a) Control plate A. niger colonies growing on MEA for 6 days at 30°C. b) Plate containing MEA + 1% Triton X-100 (v/v). Sclerotia formation is induced in A. niger N402 by the addition of Triton X-100. c) Picture of a single colony from the plate shown at (b), taken after 14 days using a stereo microscope (Leica EZ4 D). The sclerotium formation in A. niger N402 colonies growing on MEA + 1% Triton X-100 is hyper-induced, as all individual colonies formed sclerotia on all sides of the colony.

Fig. 6.

Sclerotia induction in diploid A. niger strains and its parental strains. Pictures were taken after 4 months of growth at 30°C in the dark in all cases. a) Diploid strains SJS150.1 and SJS151.1 were grown on sclerotia inducing media containing 1% Triton X-100. Only a single PDA and a single OA plate containing Triton X-100 showed sclerotia formation and this was limited to a small sector on the plate in both cases. b) Light microscopy was used to assess the cell structures of the obtained sclerotia obtained from the diploid strains. We found sclerotia composed of pseudoparenchymatous cells and these cells appeared to be empty, with no asci or ascospores present. c) Conidia of wild-type strains CBS147323 and CBS147347 were mixed and plated sclerotia inducing media containing 1% Triton X-100. Sclerotia formation visualized with a stereo microscope. Sclerotia formation in these 2 wild-type strains is hyper-induced as seen in N402 (Fig. 5). d) Again, light microscopy was used to assess cell structures of the obtained sclerotia from the mixed parental strains and only pseudoparenchymatous cells (of the sclerotia) without asci or ascospores were observed.