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. 2017 Oct 31;83(22):e01578-17.
doi: 10.1128/AEM.01578-17. Print 2017 Nov 15.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels

Doris Tisch  1 , Kyle R Pomraning  2   3 , James R Collett  2 , Michael Freitag  3 , Scott E Baker  2 , Chia-Ling Chen  4 , Paul Wei-Che Hsu  4 , Yu Chien Chuang  4 , Andre Schuster  1 , Christoph Dattenböck  5 , Eva Stappler  5 , Michael Sulyok  6 , Stefan Böhmdorfer  7 , Josua Oberlerchner  7 , Ting-Fang Wang  4 , Monika Schmoll  8   5
Affiliations

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels

Doris Tisch et al. Appl Environ Microbiol. .

Abstract

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.IMPORTANCETrichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

Keywords: Hypocrea jecorina; Trichoderma reesei; carbon source; cellulase; female fertility; mating type; secondary metabolism; sexual development; strain improvement.

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Figures

FIG 1
FIG 1
Strains and their metabolic and regulatory characteristics. (A) CBS999.97 and QM6a are original isolates (15, 55). As the CBS999.97 isolate consisted of a mixture of two strains of different mating types, they were separated to obtain compatible strains of both mating types, designated CBS999.97 MAT1-1 and CBS999.97 MAT1-2 (12). Female fertile CBS999.97 MAT1-1 was used for backcrossing with female sterile QM6a in order to obtain female fertile strains showing the physiological characteristics of QM6a (FF1a, FF1b, and FF1c). FF1a, FF1b, and FF1c were constructed in the same backcrossing procedure, with 10 backcrossings in total, but are progeny of individual separate crossing lines of CBS999.97 MAT1-1 (17). (B) Hierarchical cluster analysis of growth on diverse carbon sources. Growth of QM6a (MAT1-2), FF1a, -b, and -c (female fertile sister strains of mating type MAT1-1, treated as replicates), and FF2a, -b, and -c (female fertile sister strains of mating type MAT1-2, treated as replicates) was analyzed on 95 carbon sources after 72 h. Hierarchical clustering was done for both strain background and carbon source using HCE3.5. Carbon sources with similar expression patterns across strain backgrounds are shown close to each other. (C) Regulation patterns of CAZyme-encoding genes in different genetic backgrounds. Transcript levels of CAZyme genes from CBS999.97 of both mating types and from strains QM6a, FF1, and FF2 were analyzed by hierarchical clustering. CAZyme gene families of which all members present in the QM6a genome are upregulated in strains with the QM6a background (QM6a, FF1, and FF2) are shown as cluster 3, and those with all members downregulated in QM6a background strains compared to their expression in CBS999.97 of both mating types are shown as cluster 1 (see also Data Set S3 in the supplemental material).
FIG 2
FIG 2
Functional categories of genes upregulated in the QM6a background. Genes upregulated in the QM6a genetic background (strains QM6a, FF1, and FF2) compared to their expression in CBS999.97 of both mating types were subjected to functional category analysis (Data Set S5). Significantly enriched categories are highlighted.
FIG 3
FIG 3
Analysis of secreted metabolites of QM6a and CBS999.97. Secreted metabolites were analyzed in minimal medium with glucose as the carbon source as follows: QM6a (A) buffered to pH 5.0 using solid (lanes 1 and 2) and liquid (lanes 3 and 4) medium or unbuffered solid (lanes 5 and 6) and liquid (lanes 7 and 8) minimal medium, CBS999.97 MAT1-1 (B) at pH 5.0 using solid (lane 1) or liquid (lane 2) medium and unbuffered solid (lane 3) or liquid (lane 4) medium, and CBS999.97 MAT1-2 1 at pH 5.0 using solid (lane 5) or liquid (lane 6) medium and unbuffered solid (lane 7) or liquid (lane 8) medium. (C) The same media without fungi were used as controls: buffered solid (lane 1) and liquid (lane 2) media and unbuffered solid (lane 3) and liquid (lane 4) media. Two biological replicates were considered in each case. High-performance thin-layer chromatography (HPTLC) plates were documented at 366 nm without derivatization. (D to F) Quantification of selected metabolites in QM6a and CBS999.97. Strains were grown on minimal medium with cellulose as the carbon source in constant light (LL) and constant darkness (DD). The abundance of each compound was related to the biomass produced and is shown relative to the result for CBS999.97 MAT1-2 in darkness. Error bars indicate standard deviations.
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