The genome of the xerotolerant mold Wallemia sebi reveals adaptations to osmotic stress and suggests cryptic sexual reproduction

Wallemia (Wallemiales, Wallemiomycetes) is a genus of xerophilic Fungi of uncertain phylogenetic position within Basidiomycota. Most commonly found as food contaminants, species of Wallemia have also been isolated from hypersaline environments. The ability to tolerate environments with reduced water activity is rare in Basidiomycota. We sequenced the genome of W. sebi in order to understand its adaptations for surviving in osmotically challenging environments, and we performed phylogenomic and ultrastructural analyses to address its systematic placement and reproductive biology. W. sebi has a compact genome (9.8 Mb), with few repeats and the largest fraction of genes with functional domains compared with other Basidiomycota. We applied several approaches to searching for osmotic stress-related proteins. In silico analyses identified 93 putative osmotic stress proteins; homology searches showed the HOG (High Osmolarity Glycerol) pathway to be mostly conserved. Despite the seemingly reduced genome, several gene family expansions and a high number of transporters (549) were found that also provide clues to the ability of W. sebi to colonize harsh environments. Phylogenetic analyses of a 71-protein dataset support the position of Wallemia as the earliest diverging lineage of Agaricomycotina, which is confirmed by septal pore ultrastructure that shows the septal pore apparatus as a variant of the Tremella-type. Mating type gene homologs were identified although we found no evidence of meiosis during conidiogenesis, suggesting there may be aspects of the life cycle of W. sebi that remain cryptic.Keywords: Ion homeostasis, Aqua(glycero)porins, Solute accumulation, Electron microscopy, Xerophile, Halophil

RM7 mutant presents a single gene mutation at locus AN8741.2.

<p>A) Diagram showing the genomic insert present in the complementation vector from library pRG3-AMA1-NOT1. The insert contains two ORFs corresponding to AN8741.2 and adjacent AN8740.2. The coding region at AN8741.2 locus encodes a putative C₂H₂ zinc finger domain transcription factor. The revertant mutation in RM7 occurred at AN8741.2, designated as <i>mtfA</i> gene. B) Amino acid alignment of <i>A. nidulans</i> MtfA (Ani) and putative orthologs in <i>A. terreus</i> (Ate), <i>A. flavus</i> (Afl), <i>A. clavatus</i> (Acl) and <i>A. fumigatus</i> (Afu). ClustalW (<a href="http://www.ebi.ac.uk/Tools/clustalw2/index.htm" target="_blank">http://www.ebi.ac.uk/Tools/clustalw2/index.htm</a>) land boxshade (<a href="http://www.ch.embnet.org/software/BOX_form.html" target="_blank">http://www.ch.embnet.org/software/BOX_form.html</a>) multiple sequence alignment software programs were utilized in this analysis. The mutation occurred at the codon corresponding to the first methionine. The two conserved zinc finger domains are indicated in A) and B).</p

Expression profile of <i>sltA</i> during vegetative growth, asexual and sexual-developmental phases in a wild-type strain.

<p>A wild-type <i>velvet</i> strain (<i>veA</i>⁺, strain WIM126) was cultured in supplemented liquid GMM for vegetative mycelial growth. Samples were collected at the times indicated on top of the figure. For induction of asexual or sexual development, vegetative mycelia were collected by filtration after 24 hours of culture in liquid GMM and placed on top of solid GMM. Samples were collected at the time points indicated on top of the figure. Extracted total RNAs were probed with a radioactively labeled <i>sltA</i> coding region. The 26S ribosomal RNA is shown as loading control.</p

SltA is necessary for normal ST biosynthesis particularly in the presence of potassium while calcium sustains toxin production in the absence of SltA.

<p>A) Wild type (FGSC4) and ∆<i>sltA, veA</i>⁺ (RSS1.6P) strains were top-agar inoculated with 5 x 10⁶ conidia per plate on GMM, GMM supplemented with KCl (200 and 400 mM) or CaCl₂ (10 mM), followed by incubation at 37°C for 5 days. ST toxin was extracted and analyzed by thin layer chromatography (TLC) as described in the experimental procedure section. B) Densitometry displaying the intensity of the ST bands. The densitometry was carried out using the Scion Image 4.03 software. Values are the means of two replicates. The error bar represents standard error.</p

Effect of <i>sltA</i> deletion on <i>A. nidulans</i> cleistothecial production.

<p>Micrographs of fruiting bodies (cleistothecia) in ∆<i>sltA, veA</i>⁺ (RSS1.6P) and wild type (FGSC4). Strains were top-inoculated with 5 x10⁶ conidia per plate on GMM, GMM supplemented with KCl (200 mM or 400 mM), or GMM supplemented with 10 mM CaCl₂, and incubated at 37°C for 10 days. Plates were wrapped to promote sexual development. Cultures were sprayed with 70% ethanol to facilitate the visualization of cleistothecia. Arrows indicate cleistothecia in the ∆<i>sltA, veA</i>⁺ culture. Magnification: x50.</p

Calcium remediated the growth defect in the <i>sltA</i> deletion mutant.

<p>Photographs of point-inoculated colonies of <i>A. nidulans</i> ∆<i>sltA, veA</i>⁺ (RSS1.6P) and its control strain (FGSC4). The strains were grown on GMM, GMM supplemented with 200 mM KCl, or GMM supplemented with 10 mM CaCl₂. The plates were incubated for 5 days at 37°C.</p

SltA is required for normal Hülle cell production and regulates <i>nsdD</i> and <i>steA</i> expression.

<p>A) Quantification of Hülle cells produced by wild-type (FGSC4) and ∆<i>sltA, veA</i>⁺ strains (RSS1.6P). The strains were first grown in liquid shaken cultures for 24 hours at 250 rpm. Then equal amounts of mycelia were homogeneously spread onto solid GMM, GMM supplemented with 200 mM KCl, or GMM supplemented with 10 mM CaCl₂ (time point - 0 h) and further incubated at 37°C. B) and C) Expression levels of <i>nsdD</i> and <i>steA</i>, respectively, analyzed by qRT-PCR. Total RNA was extracted using Trizol®, from mycelial samples collected at 24 and 48 hours after the shift. The values were normalized to the wild-type (grown on GMM -24 h) levels considered as 100. The error bar represents standard error. Values are the means of three replicates.</p

Expression of <i>brlA</i> is affected by SltA.

<p>A) <i>brlA</i> expression analysis by qRT-PCR. The values were normalized to the wild-type (grown on GMM -24 h) levels considered as 100. Strains were initially grown in liquid medium in a shaker incubator for 24 hours at 250 rpm. Then equal amounts of mycelia were homogeneously spread onto solid medium GMM, or GMM, supplemented with 200 mM KCl or with 10 mM CaCl₂ (time point - 0 h) and further incubated at 37°C. Total RNA was extracted using Trizol® from mycelial samples collected at 24 and 48 hours after the shift. B) Quantification of conidia produced by ∆<i>sltA, veA</i>⁺ (RSS1.6P) and its wild-type control (FGSC4) under the same experimental conditions. An 8-mm-diameter core was extracted from the cultures at 24 and 48 hours after the shift and homogenized in water. Spores were counted with a hemocytometer. Values are means of three replicates. The error bar indicates standard error. Asterisk indicates not detected.</p

Effects of <i>mtfA</i> deletion on ST production in <i>A</i>. <i>nidulans</i> strains with a <i>veA+</i> allele.

<p>A) TLC analysis showing ST production in GMM cultures. Wild type (WT) <i>veA</i>+ control (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mtfA</i>) and Δ<i>mtfA</i>-com complementation strain (TRVΔ<i>mtfA</i>-com) were spread-inoculated with 5 mL of top agar containing 10⁶ conidia mL⁻¹ and incubated at 37°C in the dark or in the light for 48 h and 72 h. ST was extracted and analyzed by TLC as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074122#s2" target="_blank">Materials and Methods</a> section. White arrows indicate unknown compounds whose synthesis is also affected by the presence or absence of <i>mtfA.</i> B) Effect of the <i>mftA</i> deletion on <i>aflR</i> and <i>stcU</i> expression. Wild type (WT) <i>veA</i>+ control (TRV50.2), Δ<i>mtfA</i> (TRVpΔ<i>mftA</i>) and Δ<i>mtfA</i>-com complementation strain (TRVΔ<i>mtfA</i>-com) were inoculated in liquid GMM. Mycelia were collected 24 h and 48 h after inoculation. Cultures were grown in a shaker incubator at 37°C at 250 rpm. Expression of <i>aflR</i> and <i>stcU</i> was analyzed by Northern blot. 18S rRNA serves as loading control. Asterisk indicates not detected. C) TLC showing accumulation of ST in the cultures described in (B). Densitometries were carried out with the Scion Image Beta 4.03 software.</p

Fungal strains used in the study.

<p>FGSC, Fungal Genetics Stock Center.</p