An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei)

Steiger, Matthias G.; Mach, Robert L.; Mach-Aigner, Astrid R.

doi:10.1016/j.jbiotec.2009.10.012

Cited by 103 publications

(77 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…All PCR efficiencies were above 90%. Data analysis using sar1 and act as reference genes and calculations using REST 2009 software were performed as published previously (18).…”

Section: Methodsmentioning

confidence: 99%

L -Arabitol Is the Actual Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)

Mach-Aigner

Gudynaite‐Savitch

Mach

2011

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

The saprophytic fungus Hypocrea jecorina (anamorph, Trichoderma reesei) is an important native producer of hydrolytic enzymes, including xylanases. Regarding principles of sustainability, cheap and renewable raw materials, such as D-xylose (the backbone monomer of xylan), have been receiving increasing attention from industries. Recently, it was demonstrated that small (0.5 to 1 mM) amounts of D-xylose induce the highest expression of xylanase in H. jecorina. However, it was also reported that active metabolism of D-xylose is necessary for induction. In this report, we demonstrate that xylitol, the next intermediate in the pentose pathway after D-xylose, does not trigger transcription of xylanase-encoding genes in H. jecorina QM9414. The highest level of transcription of xylanolytic enzyme-encoding genes occurred in an xdh1 (encoding a xylitol dehydrogenase) deletion strain cultured in the presence of 0.5 mM D-xylose, suggesting that a metabolite upstream of xylitol is the inducer. The expression levels of xylanases in an xdh1-lad1 double-deletion strain were lower than that of an xdh1 deletion strain. This observation suggested that L-xylulose is not an inducer and led to the hypothesis that L-arabitol is the actual inducer of xylanase expression. A direct comparison of transcript levels following the incubation of the H. jecorina parental strain with various metabolites of the pentose pathway confirmed this hypothesis. In addition, we demonstrate that xyr1, the activator gene, is not induced in the presence of pentose sugars and polyols, regardless of the concentration used; instead, we observed low constitutive expression of xyr1.

show abstract

“…All PCR efficiencies were above 90%. Data analysis using sar1 and act as reference genes and calculations using REST 2009 software were performed as published previously (18).…”

Section: Methodsmentioning

confidence: 99%

L -Arabitol Is the Actual Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)

Mach-Aigner

Gudynaite‐Savitch

Mach

2011

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Primer sequences are provided in Table S3. Cycling conditions and control reactions were performed as previously described (52). Calculations using sar1 and act as reference genes were performed as previously published (52).…”

Section: Methodsmentioning

confidence: 99%

Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism

Derntl

Kluger

Bueschl

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Fungi can produce a wide range of chemical compounds via secondary metabolism. These compounds are of major interest because of their (potential) application in medicine and biotechnology and as a potential source for new therapeutic agents and drug leads. However, under laboratory conditions, most secondary metabolism genes remain silent. This circumstance is an obstacle for the production of known metabolites and the discovery of new secondary metabolites. In this study, we describe the dual role of the transcription factor Xylanase promoter binding protein 1 (Xpp1) in the regulation of both primary and secondary metabolism of Trichoderma reesei. Xpp1 was previously described as a repressor of xylanases. Here, we provide data from an RNAsequencing analysis suggesting that Xpp1 is an activator of primary metabolism. This finding is supported by our results from a Biolog assay determining the carbon source assimilation behavior of an xpp1 deletion strain. Furthermore, the role of Xpp1 as a repressor of secondary metabolism is shown by gene expression analyses of polyketide synthases and the determination of the secondary metabolites of xpp1 deletion and overexpression strains using an untargeted metabolomics approach. The deletion of Xpp1 resulted in the enhanced secretion of secondary metabolites in terms of diversity and quantity. Homologs of Xpp1 are found among a broad range of fungi, including the biocontrol agent Trichoderma atroviride, the plant pathogens Fusarium graminearum and Colletotrichum graminicola, the model organism Neurospora crassa, the human pathogen Sporothrix schenckii, and the ergot fungus Claviceps purpurea.transcription factor | secondary metabolism | low molecular compounds | fungi | gene regulation F ungi are prominent producers of a broad variety of so-called secondary metabolites (1, 2). They are highly variable in structure and effects but share the following common feature: they are produced via the secondary metabolism (3, 4). Whereas primary metabolites are shared between all living cells, secondary metabolites are highly diverse and frequently produced by a limited number of species or cell types. Fungi use secondary metabolites for different purposes [e.g., protection against predation (5) or harsh environments (6), communication (7), competition and toxicity against bacteria (8) and other fungi (9), and pathogenicity (10)]. Some fungal secondary metabolites have toxic characteristics, such as aflatoxins, fumonisins, trichothecenes, fusarins, zearalenone, and ergot alkaloids (11-16), whereas other secondary metabolites are of major interest because of their potential application in the treatment of infectious diseases [e.g., antibiotics (17)] or cancer [e.g., immunosuppressants (18)] and as a potential source for novel therapeutic agents and drug leads (19). Interestingly, some of these natural products have already been used by ancient human populations (20). Numerous new compounds have been identified within the last decade that are now applied by the biotechnology and pharma...

show abstract

“…We selected 13 genes of different functional classes as candidates ( Table 2). Our gene panel contained (i) housekeeping genes, such as tubulin (tub), actin (actin1), or cytochrome c (cyt-c); (ii) genes chosen on the basis of stable expression in several RNA-seq and qPCR experiments, such as lipase (lip) or methylthioadenosine phosphorylase (phos) (26,27); and (iii) the sar1 gene, described to be the ideal reference gene in the ascomycetes Aspergillus niger (28) and Trichoderma reesei (29). The 13 candidates were classified according to their suitability as internal standards using the geNorm (18), NormFinder (16), and BestKeeper (21) algorithms.…”

Section: Methodsmentioning

confidence: 99%

Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Castanera

López-Varas

Pisabarro

et al. 2015

Appl Environ Microbiol

View full text Add to dashboard Cite

Recently, the lignin-degrading basidiomycete Pleurotus ostreatus has become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes in P. ostreatus time course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10 and mnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.T he basidiomycete Pleurotus ostreatus is an efficient producer of extracellular enzymes, such as laccases (Lacs; EC 1.10.3.2) and manganese peroxidases (MnPs; EC 1.11.1.13), which are notable for their application in multiple industrial and biotechnological processes (1, 2). It has a special relevance in agroindustry due to its worldwide cultivation for mushroom production, and it has also been studied for its nutritional and medicinal value (3). Currently, two genomic sequences of P. ostreatus are publicly available, and it has become a very popular model organism for the study of fungal genetics and comparative genomics (4-6). Additionally, many studies on the production of ligninolytic enzymes, with a focus on culture optimization for enzyme production, have recently been published. Most of these enzymes are encoded by genes organized in gene families, which are occasionally arranged into clusters as a result of gene duplications.Despite the information uncovered by sequencing efforts, substantial work on the functional characterization of gene family members remains to be performed. In this sense, transcriptomic analyses are powerful tools for providing an understanding of gene regulation and the presumptive function of genes. Despite the high productivity and low cost of transcriptome sequencing (RNA-seq), reverse transcription (RT) followed by real-time PCR (RT-quantitative PCR [RT-qPCR]) is likely the best option for analyzing the expression patterns of certain genes under multiple conditions. Additionally, it is the most reliable technique for validating RNA-seq and microarray data because of its specificity, reproducibility, and capacity...

show abstract

An accurate normalization strategy for RT-qPCR in Hypocrea jecorina (Trichoderma reesei)

Cited by 103 publications

References 31 publications

L -Arabitol Is the Actual Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)

L -Arabitol Is the Actual Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)

Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism

Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

Contact Info

Product

Resources

About