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Genome-wide transcriptional response of Trichoderma reesei to lignocellulose using RNA sequencing and comparison with Aspergillus niger.

Ries L, Pullan ST, Delmas S, Malla S, Blythe MJ, Archer DB - BMC Genomics (2013)

Bottom Line: This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi.This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate.The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biology, University of Nottingham, Nottingham NG7 2RD, UK.

ABSTRACT

Background: A major part of second generation biofuel production is the enzymatic saccharification of lignocellulosic biomass into fermentable sugars. Many fungi produce enzymes that can saccarify lignocellulose and cocktails from several fungi, including well-studied species such as Trichoderma reesei and Aspergillus niger, are available commercially for this process. Such commercially-available enzyme cocktails are not necessarily representative of the array of enzymes used by the fungi themselves when faced with a complex lignocellulosic material. The global induction of genes in response to exposure of T. reesei to wheat straw was explored using RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to wheat straw.

Results: In T. reesei, levels of transcript that encode known and predicted cell-wall degrading enzymes were very high after 24h exposure to straw (approximately 13% of the total mRNA) but were less than recorded in A. niger (approximately 19% of the total mRNA). Closer analysis revealed that enzymes from the same glycoside hydrolase families but different carbohydrate esterase and polysaccharide lyase families were up-regulated in both organisms. Accessory proteins which have been hypothesised to possibly have a role in enhancing carbohydrate deconstruction in A. niger were also uncovered in T. reesei and categories of enzymes induced were in general similar to those in A. niger. Similarly to A. niger, antisense transcripts are present in T. reesei and their expression is regulated by the growth condition.

Conclusions: T. reesei uses a similar array of enzymes, for the deconstruction of a solid lignocellulosic substrate, to A. niger. This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi. This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate. The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.

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Sense and antisense transcription of gene with ID 76852. (A) IGV (Integrative Genome Viewer) output of the alignment of RNA-seq reads to the genome region corresponding to gene transcript ID 76852 under each condition. Blue reads represent sense RNAs, red reads represent antisense RNAs. (B) RT-PCR using gene specific primers (indicated by a yellow line) on oligo(dT) primed cDNA. The expected band size from spliced, sense transcripts is 476 bp and the size of the non-spliced antisense transcripts is 670 bp and has the same size as products from reactions run on gDNA. (C) Strand-specific RT-PCR using one of the standard PCR primers to synthesise cDNA from one strand only and then the PCR step was performed by using the same primer together with the opposing gene-specific primer. In 24 h straw and 5 h glucose both antisense (extend differently over both introns) and sense (differently spliced introns) transcripts are present, explaining the presence of multiple PCR products.
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Figure 3: Sense and antisense transcription of gene with ID 76852. (A) IGV (Integrative Genome Viewer) output of the alignment of RNA-seq reads to the genome region corresponding to gene transcript ID 76852 under each condition. Blue reads represent sense RNAs, red reads represent antisense RNAs. (B) RT-PCR using gene specific primers (indicated by a yellow line) on oligo(dT) primed cDNA. The expected band size from spliced, sense transcripts is 476 bp and the size of the non-spliced antisense transcripts is 670 bp and has the same size as products from reactions run on gDNA. (C) Strand-specific RT-PCR using one of the standard PCR primers to synthesise cDNA from one strand only and then the PCR step was performed by using the same primer together with the opposing gene-specific primer. In 24 h straw and 5 h glucose both antisense (extend differently over both introns) and sense (differently spliced introns) transcripts are present, explaining the presence of multiple PCR products.

Mentions: Confirmation of the presence of NATs in T. reesei was achieved by strand-specific PCR analysis for a gene [JGI:76852] with NATs (Figure 3). This gene is predicted to encode a secreted β-glucuronidase, belonging to GH family 2. Strand-specific RT-PCR confirmed the presence of spliced and non-spliced S and AS transcripts of different sizes (Figure 3B and C) in all three conditions as was previously reported for a gene containing NATs in A. niger[3]. Regulation at the post-transcriptional level presents an interesting area for further research.


Genome-wide transcriptional response of Trichoderma reesei to lignocellulose using RNA sequencing and comparison with Aspergillus niger.

Ries L, Pullan ST, Delmas S, Malla S, Blythe MJ, Archer DB - BMC Genomics (2013)

Sense and antisense transcription of gene with ID 76852. (A) IGV (Integrative Genome Viewer) output of the alignment of RNA-seq reads to the genome region corresponding to gene transcript ID 76852 under each condition. Blue reads represent sense RNAs, red reads represent antisense RNAs. (B) RT-PCR using gene specific primers (indicated by a yellow line) on oligo(dT) primed cDNA. The expected band size from spliced, sense transcripts is 476 bp and the size of the non-spliced antisense transcripts is 670 bp and has the same size as products from reactions run on gDNA. (C) Strand-specific RT-PCR using one of the standard PCR primers to synthesise cDNA from one strand only and then the PCR step was performed by using the same primer together with the opposing gene-specific primer. In 24 h straw and 5 h glucose both antisense (extend differently over both introns) and sense (differently spliced introns) transcripts are present, explaining the presence of multiple PCR products.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750697&req=5

Figure 3: Sense and antisense transcription of gene with ID 76852. (A) IGV (Integrative Genome Viewer) output of the alignment of RNA-seq reads to the genome region corresponding to gene transcript ID 76852 under each condition. Blue reads represent sense RNAs, red reads represent antisense RNAs. (B) RT-PCR using gene specific primers (indicated by a yellow line) on oligo(dT) primed cDNA. The expected band size from spliced, sense transcripts is 476 bp and the size of the non-spliced antisense transcripts is 670 bp and has the same size as products from reactions run on gDNA. (C) Strand-specific RT-PCR using one of the standard PCR primers to synthesise cDNA from one strand only and then the PCR step was performed by using the same primer together with the opposing gene-specific primer. In 24 h straw and 5 h glucose both antisense (extend differently over both introns) and sense (differently spliced introns) transcripts are present, explaining the presence of multiple PCR products.
Mentions: Confirmation of the presence of NATs in T. reesei was achieved by strand-specific PCR analysis for a gene [JGI:76852] with NATs (Figure 3). This gene is predicted to encode a secreted β-glucuronidase, belonging to GH family 2. Strand-specific RT-PCR confirmed the presence of spliced and non-spliced S and AS transcripts of different sizes (Figure 3B and C) in all three conditions as was previously reported for a gene containing NATs in A. niger[3]. Regulation at the post-transcriptional level presents an interesting area for further research.

Bottom Line: This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi.This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate.The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biology, University of Nottingham, Nottingham NG7 2RD, UK.

ABSTRACT

Background: A major part of second generation biofuel production is the enzymatic saccharification of lignocellulosic biomass into fermentable sugars. Many fungi produce enzymes that can saccarify lignocellulose and cocktails from several fungi, including well-studied species such as Trichoderma reesei and Aspergillus niger, are available commercially for this process. Such commercially-available enzyme cocktails are not necessarily representative of the array of enzymes used by the fungi themselves when faced with a complex lignocellulosic material. The global induction of genes in response to exposure of T. reesei to wheat straw was explored using RNA-seq and compared to published RNA-seq data and model of how A. niger senses and responds to wheat straw.

Results: In T. reesei, levels of transcript that encode known and predicted cell-wall degrading enzymes were very high after 24h exposure to straw (approximately 13% of the total mRNA) but were less than recorded in A. niger (approximately 19% of the total mRNA). Closer analysis revealed that enzymes from the same glycoside hydrolase families but different carbohydrate esterase and polysaccharide lyase families were up-regulated in both organisms. Accessory proteins which have been hypothesised to possibly have a role in enhancing carbohydrate deconstruction in A. niger were also uncovered in T. reesei and categories of enzymes induced were in general similar to those in A. niger. Similarly to A. niger, antisense transcripts are present in T. reesei and their expression is regulated by the growth condition.

Conclusions: T. reesei uses a similar array of enzymes, for the deconstruction of a solid lignocellulosic substrate, to A. niger. This suggests a conserved strategy towards lignocellulose degradation in both saprobic fungi. This study provides a basis for further analysis and characterisation of genes shown to be highly induced in the presence of a lignocellulosic substrate. The data will help to elucidate the mechanism of solid substrate recognition and subsequent degradation by T. reesei and provide information which could prove useful for efficient production of second generation biofuels.

Show MeSH
Related in: MedlinePlus