Network reconstruction and systems analysis of plant cell wall deconstruction by Neurospora crassa

Abstract

Background: Plant biomass degradation by fungal-derived enzymes is rapidly expanding in economic importance as a clean and efficient source for biofuels. The ability to rationally engineer filamentous fungi would facilitate biotechnological applications for degradation of plant cell wall polysaccharides. However, incomplete knowledge of biomolecular networks responsible for plant cell wall deconstruction impedes experimental efforts in this direction.

Results: To expand this knowledge base, a detailed network of reactions important for deconstruction of plant cell wall polysaccharides into simple sugars was constructed for the filamentous fungus Neurospora crassa. To reconstruct this network, information was integrated from five heterogeneous data types: functional genomics, transcriptomics, proteomics, genetics, and biochemical characterizations. The combined information was encapsulated into a feature matrix and the evidence weighted to assign annotation confidence scores for each gene within the network. Comparative analyses of RNA-seq and ChIP-seq data shed light on the regulation of the plant cell wall degradation network, leading to a novel hypothesis for degradation of the hemicellulose mannan. The transcription factor CLR-2 was subsequently experimentally shown to play a key role in the mannan degradation pathway of N. crassa.

Conclusions: Here we built a network that serves as a scaffold for integration of diverse experimental datasets. This approach led to the elucidation of regulatory design principles for plant cell wall deconstruction by filamentous fungi and a novel function for the transcription factor CLR-2. This expanding network will aid in efforts to rationally engineer industrially relevant hyper-production strains.

Keywords: Biofuels; CLR-2; Mannan; Network reconstruction; Neurospora crassa; Plant cell wall degradation network; Systems biology; Transcriptional regulatory networks.

Fig. 1

Schematic illustration of the structure of different plant cell wall polysaccharides along with the associated reactions and genes in the PCWDN of N. crassa. Cellulose has an unbranched structure composed of linear chains of β-1,4-linked d-glucose residues. Hemicellulose comprises several branched polymers including xylan, xyloglucan (XG), mannan, and mixed-linkage glucan (MLG). Pectin is a family of several polymers including homogalacturonan, xylogalacturonan, and rhamnogalacturonan I. Starch is a polymer composed of amylose and amylopectin. On the right, the number of reactions and genes involved in the degradation of cellulose, hemicelluloses, pectin, and starch are indicated that are captured in our PCWDN

Fig. 2

Schematic illustration of the pipeline for reconstruction and annotation of the PCWDN of N. crassa. An initial scaffold network, PCWDN v0.0, was assembled based on annotation information in several databases. Extensive literature-based manual curation was then performed to fill knowledge gaps in the initial PCWDN v0.0. Gene annotations in the final PCWDN v1.0 were refined based on multi-level supporting evidence from five heterogeneous data types

Fig. 3

Feature matrix and annotation confidence scores for genes encoding AA9 LPMOs in N. crassa. Combined annotation information from the five different data types was captured in a feature matrix, and a method was devised to assign annotation confidence scores to PCWDN genes. A differential weighting system was used to account for the different levels of confidence associated with the information from each data type. The majority of genes encoding AA9 LPMOs of class 1 (3 out of 5 genes) and class 2 (2 out of 3 genes) are well characterized, while only 1 out of 6 genes encoding AA9 LPMOs of class 3 is well characterized. NoC no carbon, CB cellobiose, XG xyloglucan, MLG mixed-linkage glucan, OPP orange peel powder

Fig. 4

Comparative analysis of global transcriptome profiles of N. crassa in nine conditions. a Correlation of the whole transcriptomes across each pair of conditions. Hierarchical clustering of the pairwise correlation matrix led to the identification of four clusters of highly correlated conditions. b Principal component analysis of the whole transcriptomes in all nine conditions. The first and second principal components explain >58% and 20%, respectively, of the total variance. Thus, the first two components together explain more than 79% of the total variance. c Horizontal bar plot showing the sizes of regulons for seven conditions and the overlap of each regulon with the 168 PCWDN genes. Statistically significant overlaps (p < 10⁻⁶) were marked with an asterisk. The vertical bar plot shows the 26 intersection sets among the seven regulons with 5 or more genes and was generated using UpSetR [108]

Fig. 5

Hierarchical clustering of transcriptome profiles within the context of the PCWDN of N. crassa. The heatmap shows the result of two-dimensional clustering of the RNA-seq data for all 168 PCWDN genes in nine conditions corresponding to Avicel, xylan, XG, mannan, MLG, pectin, starch, sucrose, and NoC, which led to the identification of six major clusters. Note that the clustering of conditions was similar to that obtained from correlation and principal component analysis of the global transcriptome profiles (Fig. 4a, b)

Fig. 6

Direct regulation of the PCWDN genes by key transcription factors for cellulose and xylan utilization. The PCWDN genes have been grouped based on their biochemical function and participation in degradation pathways of different plant cell wall polysaccharides. It is observed that CLR-1 directly regulates mostly genes involved in cellulose utilization, XLR-1 directly regulates mostly genes involved in xylan utilization, while CLR-2 has a much broader role in the regulation of genes involved in plant cell wall deconstruction

Fig. 7

a Schematic diagram of the mannan degradation pathway. b Expression of genes encoding enzymes in the mannan degradation pathway and key transcription factors for cellulose and hemicellulose utilization across different conditions. Expression values for genes with more than two fold up-regulation relative to the starvation condition (NoC) are shaded in pink, while those with more than two fold down-regulation are shaded in blue. XG xyloglucan, MLG mixed-linkage glucan

Fig. 8

clr-2 plays a major role in mannan and xyloglucan (XG) degradation. a–c Growth phenotypes of WT, Δgh5-7, Δclr-1, Δclr-2, Δxlr-1, Pc1 (Pccg-1-clr-1), Pc2 (Pccg-1-clr-2), and Px1 (Pccg-1-xlr-1) strains of N. crassa in the medium containing glucomannan as the sole carbon source after growth for 4 days. a Photograph of 3 mL cultures with replicates in 24-well deep-well plates. b Fungal dry weights after 4 days. Bars represent standard deviations. The asterisk indicates a significant difference from WT with an unadjusted p value of <0.003 using one-way ANOVA. c Secreted protein in culture supernatants (SN) relative to WT. Bars represent standard deviations. The concentration of secreted protein is shown relative to WT, which was set to 100%. d Growth phenotypes of WT, Pc1, Pc2, and Px1 strains of N. crassa in the medium containing pure mannan as the sole carbon source after growth for 4 days. e Growth phenotypes of WT, Pc1, Pc2, and Px1 strains of N. crassa in the medium containing XG as the sole carbon source after growth for 4 days

Fig. 9

Schematic diagram summarizing the systems approach undertaken here to reconstruct and analyze the plant cell wall degradation network (PCWDN) of N. crassa. We compiled information from diverse sources to build the network of biochemical reactions responsible for degrading plant cell wall polysaccharides into simple sugars. The combined annotation information was encapsulated in the form of a feature matrix and a simple method was devised to assign an annotation confidence score to each PCWDN gene. To demonstrate the utility of our PCWDN, we performed comparative transcriptomics analysis using RNA-seq data and integrated genome-wide binding data from ChIP-seq experiments for three key transcription factors regulating the plant cell wall degradation response. Integration of next-generation sequencing data within the PCWDN led to the hypothesis that CLR-2 is a key TF for deconstruction of mannan and XG, a hypothesis that was subsequently validated through experimentation