Identification of the CRE-1 cellulolytic regulon in Neurospora crassa

Abstract

Background: In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and β-galactosidase.

Methodology/principal findings: Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Δcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Δcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 5'-SYGGRG-3' motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Δcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion.

Conclusions/significance: Our data provide comprehensive information on the CRE-1 regulon in N. crassa and contribute to deciphering the global role of carbon catabolite repression in filamentous ascomycete fungi during plant cell wall deconstruction.

Figure 1. Phenotype of WT and Δcre-1 strains.

Wild type (FGSC 2489) (A) and Δcre-1 (FGSC 10372) (B) strains were grown on different carbon sources at 30°C for 24 hrs. The carbon source in the media is indicated above each plate and all plates contained Vogel's salts and 2% of various carbon sources. C) SDS-PAGE of secreted proteins in culture filtrates from WT and Δcre-1 strains grown on Avicel for 7 days. Protein bands representing β-glucosidase (NCU04952), cellobiohydrolase 1 (NCU07340) and 2 (NCU09680), and endoglucanase 2 (NCU00762) are marked. D) Comparison of endoglucanase activity on azo-CMC, protein concentration, glucose and cellobiose concentration from Avicelase assays of 7-day culture supernatants from WT and Δcre-1 strains.

Figure 2. Complementation and gene expression of cre-1.

A) Pn-cre-1 (his-3::Pnative-cre-1-gfp; Δcre-1) and B) Pc-cre-1 (his-3::Pccg-1-cre-1-gfp; Δcre-1) strains were grown on different carbon sources at 30°C for 24 hrs. The carbon source in the media is indicated above each plate and all plates contained 1× Vogel's salts and 2% of different carbon sources. C) Comparison of the azo-CMC activity of culture supernatants from WT, Δcre-1, Pc-cre-1 and Pn-cre-1 strains grown on Avicel for 7 days. D) Quantitative RT-PCR of cre-1 expression levels in WT, Δcre-1, Pc-cre-1 and Pn-cre-1 strains. All strains were grown in MM for 16 hrs, and then transferred into sucrose or Avicel for an additional 4 hrs.

Figure 3. Subcellular localization of CRE-1-GFP.

Strains with cre-1 under control of the native promoter (Pn-cre-1) or the ccg-1 promoter (Pc-cre-1) were grown on plates or in liquid MM for 16 hrs then transferred onto plates with Vogel's salts and either 2% sucrose (MM) or 2% Avicel a sole carbon source and allowed to grow for an additional 5–6 hrs (25°C). Fluorescence was evaluated using a Deltavision Spectris DV4 deconvolution microscope. Nuclei were also stained by DAPI. Scale bar = 10 µm.

Figure 4. Gene expression patterns in wild type and Δcre-1 strains.

A) Gene expression levels of cbh-1 (NCU07340), cbh-2 (NCU09680), gh5-1 (NCU00762) and β-glucosidase (NCU00130) in WT (FGSC 2489) and Δcre-1 (FGSC 10372) strains. Expression levels for all genes were normalized to 1 in WT. Strains were grown in MM for 16 hrs followed by 4 hrs growth on Avicel. B) Gene expression levels of cbh-1 (NCU07340) and putative CRE-1 target, NCU03181, in WT and Δcre-1 strains. Cultures were inoculated with conidia and harvested at time points shown post-inoculation. C) Gene expression levels of cre-1 under identical conditions to that shown in (B). actin (NCU04173) gene expression levels were used as an endogenous control in all samples. Each reaction was done by triplicate. *P = 0.05.

Figure 5. Venn diagram of the transcriptome of wild type and Δcre-1 strains.

A) Overlap among genes that exhibit statistically significant increased expression level in Δcre-1 strain relative to the WT strain (see Table 1 for details). There are a total of 271 genes that showed increased expression in the Δcre-1 strain (for FunCat analysis, see Figure 7). Data sets marked as c, d, e, f and g were under Avicel growth conditions, with an additional 75 genes that showed increased expression levels in the Δcre-1 strain in MM compared to WT (Table 1). B) Overlap among genes that showed a statistically significant decrease in expression level in the Δcre-1 strain relative to WT. This set includes 381 genes identified from Avicel cultures marked as C, D, E, F and G, plus 80 genes that showed decreased relative expression levels in a Δcre-1 compared to WT when grown on MM (Table 1; for FunCat analysis, see Figure 7).

Figure 6. Relative expression levels of N. crassa genes predicted to be regulated by CRE-1.

A) Genes with increased expression level in the Δcre-1 mutant as compared to WT under MM culture conditions; expression levels of these genes are shown for cultures transferred to MM or Avicel for 4 hours. NCU00721 (proline permease) and NCU09805 (α-amylase A) have been identified as direct targets of CreA in Aspergilli , . B) Expression level of 16 of the 23 predicted cellulases in WT or the Δcre-1 mutant from cultures transferred to MM or Avicel for 4 hours. NCU07340 (cbh-1) is a direct target of CRE1 in H. jecorina . C) Expression levels of 7 of the 19 predicted hemicellulases in WT or the Δcre-1 mutant from cultures transferred to MM or Avicel for 4 hrs. NCU02855 is a known target of CRE-1 in other systems . D) Expression levels of genes with functions associated with plant cell wall degradation that showed increased expression in the Δcre-1 mutant versus WT following transfer to either MM or Avicel for 4 hrs.

Figure 7. Functional category (FunCat) analysis of potential CRE-1 target genes.

A) Functional category (FunCat) analysis of the 271 genes that showed increased expression levels in the Δcre-1 mutant relative to WT (CRE-1 repressed genes). There were 77 genes classified into the category of C-compound and Carbohydrate metabolism, which included most of the predicted cellulase and hemicellulase genes. B) FunCat analysis of 381 genes that showed a decrease in relative expression level in the Δcre-1 mutant as compared to WT (CRE-1 activated genes). Of these, 65 genes were in the C-compound and Carbohydrate metabolism functional category.

Figure 8. Schematic representation of 5′-SYGGRG-3′ motifs in promoter regions and regions enriched by ChIP-PCR.

Vertical black bars indicate the location 5′-SYGGRG-3′ motifs in 1 kbp promoter regions of 9 putative CRE-1 target genes (NCU number on left and predicted function on right). Shaded box indicates region of promoters that showed enrichment via chromatin-immunoprecipitation-PCR experiments. See Tables S4, S5 and Figure S4 for further details.

Figure 9. Enzyme activity and protein profile of culture supernatants from strains containing deletions of genes in the CRE-1 cellulolytic regulon.

A) Total secreted protein, endoglucanase activity on azo-CMC, glucose and cellobiose concentration from 5-hr Avicelase assays of 7-day old culture supernatants from WT and deletion strains grown in Avicel as a sole carbon source. B) SDS-PAGE of proteins from unconcentrated culture supernatants of WT and deletion strains using same samples as from (A). Bars represent standard deviation.