Genomic and Genetic Insights Into a Cosmopolitan Fungus, Paecilomyces variotii (Eurotiales)

Abstract

Species in the genus Paecilomyces, a member of the fungal order Eurotiales, are ubiquitous in nature and impact a variety of human endeavors. Here, the biology of one common species, Paecilomyces variotii, was explored using genomics and functional genetics. Sequencing the genome of two isolates revealed key genome and gene features in this species. A striking feature of the genome was the two-part nature, featuring large stretches of DNA with normal GC content separated by AT-rich regions, a hallmark of many plant-pathogenic fungal genomes. These AT-rich regions appeared to have been mutated by repeat-induced point (RIP) mutations. We developed methods for genetic transformation of P. variotii, including forward and reverse genetics as well as crossing techniques. Using transformation and crossing, RIP activity was identified, demonstrating for the first time that RIP is an active process within the order Eurotiales. A consequence of RIP is likely reflected by a reduction in numbers of genes within gene families, such as in cell wall degradation, and reflected by growth limitations on P. variotii on diverse carbon sources. Furthermore, using these transformation tools we characterized a conserved protein containing a domain of unknown function (DUF1212) and discovered it is involved in pigmentation.

Keywords: Agrobacterium tumefaciens-mediated transformation; Byssochlamys spectabilis; DUF1212; Eurotiales; genome defense; leuA; mitochondrial membrane carrier.

FIGURE 1

Transformation of P. variotii using Agrobacterium-mediated delivery of the exogenous DNA. The construct encodes an enzyme conferring hygromycin resistance for selection and a hybrid protein with a mitochondrial-targeted sequence fused to GFP. (A) Mitochondrial GFP fluorescence of was observed in 100 out of 100 hygromycin-resistant strains obtained after transformation; one representative strain is shown. (B) Cell walls fluoresce blue from staining with calcofluor white. (C) The overlay of the GFP and calcofluor white signals. Scale bar = 20 μm.

FIGURE 2

Paecilomyces variotii produces a mix of uni- and multinucleate spores. Nucleus copy numbers in strain CBS 101075 conidiospores were visualized through the expression of a CFP-Histone H2B fusion construct. (A) CFP fluorescence, (B) DIC image, and (C) overlay, scale bar = 25 μm. (D) Histogram of the distribution of the number of nuclei per spore (n = 138); two nuclei per spore is the most common, and 32.6% of spores are uninucleate.

FIGURE 3

Targeted gene disruption, through homologous recombination, of the leuA gene in P. variotii. (A) Growth of two representative transformants, 15 that is a putative leuA deletion strain and 11 that is an ectopic insertion of the deletion construct, on minimal medium with (+) or without (–) leucine. (B) PCR amplification of the 5′ and 3′ regions adjacent to leuA into the hph selectable marker gene illustrate correct integration of the knockout construct by amplifying sequences unique to a correct integrant (3,440 and 2,850 bp) in transformants 15 and 20, but not in ectopic insertion strains 4 and 11.

FIGURE 4

(A) Mutation of the prmJ gene, in strain AU1_63, results in a pale phenotype on cleared V8 juice (CV8) agar but not on potato dextrose agar (PDA). Two of the three strains transformed with the complementation construct have a wild type phenotype while one resembles the prmJ mutant. (B) PCR analysis of the genotypes of the AU1_63 mutant, wild type CBS 101075, and the three strains after transformation with the complementation construct. The AU1_63CompB transformant, in which the phenotype was not complemented, has not integrated a wild-type copy of the prmJ gene. (C) Location of the T-DNA insert in the prmJ gene. Green represents sequence of the T-DNA and red represents nucleotides lost when the T-DNA integrated into the genome in the mutant strain AU1_63.

FIGURE 5

Strain AU2_33 has a growth and sporulation defect due to mutation of the dspA gene. (A) Sporulation on CV8 was delayed in the mutant AU2_33 at both 3 and 14 days after growth on clear V8 juice medium compared to the wild type CBS 144490 and two complemented strains. The AU2_33 mutant also had impaired growth on minimal medium (MM). (B) PCR analysis of the genotypes of the AU2_33 mutant, wild type and two complemented isolates. (C) The T-DNA insertion is located in the first exon of the dspA gene. Green represents sequence of the T-DNA and red represents sequence lost from the genome in the mutant. (D) Co-localization of mCherry-tagged DspA protein and mitochondrially localized GFP: (i) red fluorescence from the DspA-mCherry fusion, (ii) green fluorescence of citrate synthase-GFP, (iii) blue fluorescence due to calcofluor white staining of the cell wall, and (iv) the merged image. Scale bar = 10 μm.

FIGURE 6

Cross-species complementation of the P. variotii AU1_63 strain with the Aspergillus niger prmJ homolog restores the colony pigmentation to wild type levels of the AU1_63 mutant of CV8 media. (A) Growth of the wild type (CBS 101075), T-DNA insertion mutant (AU1_63), and insertion mutant transformed with the A. niger prmJ gene (AU1_63+AnComp) on cleared V8 juice medium. (B) A PCR analysis for the P. variotii mutated allele and the introduced A. niger alleles in the three strains, with wild type A. niger as a control.

FIGURE 7

Repeat induced point mutation is active in the Eurotiales. (A) PCR analysis indicates that four progeny (P2, P3, P4, and P10) of a cross between the wild type (WT) and AU2_33 contain the hygromycin phosphotransferase gene, despite (B) these progeny being sensitive to hygromycin (HYG). (C) Nucleotide sequence alignment of 780 bp of the hph coding region of both T-DNAs in progeny number 3 showed a pattern of C to T and A to G mutations, consistent with the RIP process. Dots represent identical nucleotides. (D) RIPCAL analysis of the sequencing information in (C) revealed a basis towards mutation of CpA dinucleotides, also consistent with RIP mutation.

FIGURE 8

(A) Paecilomyces variotii isolates show a bipartite genome structure that is characteristic of a consequence of repeat induced point mutation acting in the organism. (B) The genome assembly of strain CBS 144490 contains a greater proportion of AT-rich regions than does that of strain CBS 101075 (C). A putative transposon was identified on contig 123 of CBS 144490 with similarity to some of the AT-rich regions present in both genomes: RIPCAL analysis revealed that most of the putative mutations were CpA to TpA (TpG to TpA in the reverse strand), which is a feature of DNA that has undergone RIP.

FIGURE 9

Paecilomyces variotii has fewer genes than many other species in the Eurotiales. Phylogenetic relationships between the Eurotiales species, with two yeast species as outgroups, were defined from a comparison of 3,374 single copy gene orthologs. The graph shows total numbers of genes in each species and the distribution of the homologs.

FIGURE 10

Paecilomyces variotii and related Thermoascaceae have a reduced expansion in gene families. (A) A limited number of highly similar gene duplicates are observed in P. variotii compared to other Eurotiales. For each genome, a self BLASTp was conducted to identify orthologs by reciprocal best hit via BLAST, then the fraction of orthologs at various identity levels were plotted. x-axis: percent identity, y-axis: lineage, z-axis: fraction of all orthologs at a given % identity. Lineages are colored at the genus level, green: Paecilomyces, purple: Talaromyces, blue: Penicillium, dark red: Aspergillus, red: Saccharomyces, yellow: Schizosaccharomyces. (B) Three Thermoascaceae species have relatively fewer genes in the 100 most populous gene clusters in the comparative cluster analysis obtained through MycoCosm. Similarly, these species showed a more restricted set of (C) secondary metabolite genes and (D) genes encoding Carbohydrate-Active enZYmes (CAZys).

FIGURE 11

Growth of P. variotii compared to other Eurotiales species on different plant polysaccharides as the sole carbon source, compared to glucose. Petri dishes containing minimal medium and differing carbon sources were inoculated with different species of Eurotiales and growth photographed after 2–5 days depending on the species.