PMC

Schematic outline showing the functional coordination of hemicellulose degradation enzymes (mainly xylan) (). The numbers of related enzymes were retrieved by searching the genome annotation of N. crassa.

Venn diagrams of transcriptomes from wild-type (FGSC 2489) and Δxlr-1 strains when exposed to different carbon sources. Overlap among genes that exhibit a statistically significant 2-fold increase in expression levels in Δxlr-1 strains compared to FGSC 2489 when transferred to either xylan (A), xylose (B), or Avicel (C) for 4 h from 16-h sucrose cultures (MM).

Venn diagram of comparison of transcriptomes and secretomes of the wild-type strain on different carbon sources. (A) Overlap among genes that exhibit a statistically significant 2-fold increase in expression levels when a wild-type strain (FGSC 2489) was transferred to xylan or xylose from a 16-h sucrose culture (MM). (B) Overlap among genes that exhibit a statistically significant 2-fold increase in expression levels in FGSC 2489 when transferred to xylan or Avicel from 16-h sucrose culture (MM). (C) Overlap of secretomes identified from FGSC 2489 growing on beechwood xylan for 4 days versus that growing on Avicel for 7 days (Avicel secretome data are from reference ).

Phylogeny and domain structure of XLR-1. (A) XLR-1 is highly conserved across the genomes of most sequenced filamentous ascomycete species. The neighbor-joining tree shows phylogenetic relationships of fungal XlnR/XYR1 proteins and their relationship to N. crassa XLR-1. Bootstrap values are shown, and the scale bar indicates 0.2 substitutions per amino acid residue. (B) The domain structure of N. crassa XLR-1 contains an N-terminal GAL4-like Zn₂Cys₆ binuclear cluster domain and a C-terminal fungus-specific transcription factor domain.

Phenotype and complementation of Δxlr-1 strain. (A) SDS-PAGE gel of wild-type (FGSC 2489), Δxlr-1, and complemented Δxlr-1 (Δxlr-1-xlr-1) strains grown at 25°C for 4 days on beechwood xylan or 7 days on Avicel. Twenty microliters of supernatant was loaded onto a Criterion 4%-15% gradient SDS-PAGE gel. CBH-1 and CBH-2 were present in the secretome of the Δxlr-1 mutant on Avicel. However, there were two bands (NCU05924, gh10-1, and NCU02855, gh11-1) (see Fig. S3 in the supplemental material) () missing in the secretome of the Δxlr-1 mutant. Numbers at left are molecular masses in kilodaltons. (B) Endoxylanase (Azo-xylan) and endoglucanase (Azo-CMC) activity of culture filtrates from wild-type (FGSC 2489), Δxlr-1, or Δxlr-1-xlr-1 strains grown at 25°C for 4 days on beechwood xylan (black bars) or for 7 days on Avicel (gray bars).

Enzyme activity of culture supernatants from strains containing deletions of genes related to hemicellulose degradation. Total secreted protein, endoxylanase activity on azoxylan, and reducing sugar concentration in assays with culture supernatants from WT (FGSC 2489) and selected isogenic deletion mutants (missing gene denoted as the NCU gene number in figure) grown on beechwood xylan as a sole carbon source for 4 days. Data from FGSC 2489 were set to 100% (WT levels) (see Table S5 in the supplemental material). Bars represent standard deviations.

Regulation of genes encoding xylanolytic enzymes in N. crassa. Metabolites released from hemicellulose degradation by basal levels of secreted hemicellulases trigger the transcription and activation of xlr-1 and other unknown transcription factors (TFs). XLR-1 activates the transcription of certain hemicellulase genes (group A) and also functions with other unknown transcription factors to modulate expression of other hemicellulase genes (group B). An unknown TF(s) also induces the expression of separate XLR-1-independent hemicellulase genes (group C). Action of these TFs results in production of hydrolytic enzymes, which are secreted, thereby deconstructing hemicellulose, producing metabolites and additional signaling molecules. Another unknown transcription factor(s) also coordinates with XLR-1 to modulate the regulation of genes associated with xylose metabolism.

Gene expression levels of the wild-type strain (FGSC 2489) and a xyr-1 (NCU08384; xylose reductase) deletion strain under different growth conditions. Strains were pregrown in MM-sucrose for 18 h, washed, and transferred into minimal medium without any carbon source or with 2% sucrose (MM), 2% xylose, or 2% xylan as a sole carbon source for an additional 4 h. NCU00891 encodes xylitol dehydrogenase, NCU11353 encodes xylulose kinase, and NCU07225 encodes an endoxylanase. RNA was extracted from these samples, and qRT-PCR for these genes and xlr-1 was performed as indicated in Materials and Methods. Error bars indicate errors for 3 replicates.

Hemicellulase gene expression levels of the wild-type strain when exposed to xylose or xylan. (A) The wild-type strain (FGSC 2489) was pregrown in MM for 18 h, washed, and then transferred into MM without any carbon source or MM with 1 mM glucose, 1 mM xylose, 5 mM xylose, or 66 mM xylose as the sole carbon source. Gene expression levels of NCU05924 (endoxylanase, gh10-1) and NCU08189 (endoxylanase, gh10-2) were determined by quantitative RT-PCR (see Materials and Methods). (B) FGSC 2489 was grown in MM-2% sucrose for 18 h or 2 days or grown in MM-2% xylan or MM-2% xylose medium for 2 or 4 days. RNA was extracted from samples, and qRT-PCR was performed as indicated in Materials and Methods. Expression levels of NCU05924 (gh10-1), NCU08189 (gh10-2), NCU02343 (arabinofuranosidase, gh510-1), NCU02855 (endoxylanase, gh11-1), and NCU09652 (β-xylosidase, gh43-5) were determined. Expression of the actin (NCU04173) gene was used as an endogenous control for all experiments. Error bars indicate errors of 3 replicates.

xlr-1 expression levels under xylose, xylan, and Avicel growth conditions. (A) xlr-1 expression was monitored by qRT-PCR in the WT strain (FGSC 2489) (see Materials and Methods). The WT strain was grown in MM-sucrose for 18 h, washed, and then transferred into medium with 2% sucrose (MM), 2% xylose, or 2% xylan as a sole carbon source for an additional 4 h prior to RNA extraction. (B) Expression levels of xlr-1 were monitored by qRT-PCR in the WT when grown continuously in 2% sucrose-MM (18 h and 2 days), 2% xylose medium (2 and 4 days), and 2% xylan medium (2 and 4 days) prior to RNA extraction. (C) xlr-1 expression was monitored by qRT-PCR in WT and Δcre-1 strains when grown in medium containing 2% Avicel as a sole carbon source for 18, 25, and 30 h and 2, 3, and 5 days. RNA was extracted from samples, and qRT-PCR was performed as indicated in Materials and Methods. Error bars indicate errors for 3 replicates.