Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei by Global Transcriptional Analysis

Abstract

We defined the role of the transcriptional factor-XYR1-in the filamentous fungus Trichoderma reesei during cellulosic material degradation. In this regard, we performed a global transcriptome analysis using RNA-Seq of the Δxyr1 mutant strain of T. reesei compared with the parental strain QM9414 grown in the presence of cellulose, sophorose, and glucose as sole carbon sources. We found that 5885 genes were expressed differentially under the three tested carbon sources. Of these, 322 genes were upregulated in the presence of cellulose, while 367 and 188 were upregulated in sophorose and glucose, respectively. With respect to genes under the direct regulation of XYR1, 30 and 33 are exclusive to cellulose and sophorose, respectively. The most modulated genes in the Δxyr1 belong to Carbohydrate-Active Enzymes (CAZymes), transcription factors, and transporters families. Moreover, we highlight the downregulation of transporters belonging to the MFS and ABC transporter families. Of these, MFS members were mostly downregulated in the presence of cellulose. In sophorose and glucose, the expression of these transporters was mainly upregulated. Our results revealed that MFS and ABC transporters could be new players in cellulose degradation and their role was shown to be carbon source-dependent. Our findings contribute to a better understanding of the regulatory mechanisms of XYR1 to control cellulase gene expression in T. reesei in the presence of cellulosic material, thereby potentially enhancing its application in several biotechnology fields.

Keywords: RNA-seq; Trichoderma reesei; XYR1 transcription factor; enzymes; gene expression.

Figure 1

RNA-seq coverage plots of the xyr1 gene. Number of sequence reads (x-axis) along the genomic region corresponding to the xyr1 gene (Protein ID 122208, scaffold 11, genomic position 201774–204723).

Figure 2

Gene Regulatory Network (GRN) of 1184 differentially expressed genes in Δxyr1/QM9414 in response to different carbon sources (cellulose, sophorose, and glucose). Genes are represented as nodes (shown as circles), and interactions are represented as edges (red lines: up-regulated interactions; and green lines: down-regulated interactions), connecting the nodes: 1674 interactions.

Figure 3

GO enrichment analysis of up- and down-regulated genes of the Δxyr1 strain compared to parental strain QM9414 grown in cellulose, sophorose, and glucose as sole carbon sources. The enriched GO terms according to molecular function, cellular component, and biological process in T. reesei. Significantly enriched categories (P ≤ 0.05) are shown.

Figure 4

Gene expression profiles of Carbohydrate Active enZymes (CAZy) of the Δxyr1 strain compared to parental strain QM9414. Fragments per kilobase of exon per million fragments mapped (FPKM) for each enzyme class cultured in presence of glucose, cellulose, and sophorose. (A) All CAZymes identified in this study; (B) Only the differentially expressed CAZymes of each carbon source were used in this analysis. AA, Auxiliary Activities—redox enzymes that act in conjunction with CAZymes; CBM, Carbohydrate-Binding Modules—adhesion to carbohydrates; CE, Carbohydrate Esterases—hydrolysis of carbohydrate esters; GH, Glycoside Hydrolases—hydrolysis and/or rearrangement of glycosidic bonds; GT, GlycosylTransferases—formation of glycosidic bonds; PL, Polysaccharide Lyases—non-hydrolytic cleavage of glycosidic bonds.

Figure 5

Maximum-likelihood phylogenetic tree of transporters genes identified exclusively in the conditions studied. The radial tree was generated using iTOL (Letunic and Bork, 2007, 2011).

Figure 6

Scheme involving the direct regulation by the Zn2Cys6 transcription regulator XYR1. This figure shows the processes that are expected to be involved in xyr1 regulation of lignocelluosic enzymes in T. reesei. Endoglucanases (EGs) hydrolyze cellulose bonds internally, while cellobiohydrolases cleave cellobiose units from the ends of the polysaccharide chains. The released cellobiose units (disaccharides) are subsequently hydrolyzed by β-glucosidases, releasing glucose, the main carbon source readily metabolisable by T. reesei. Expansin proteins (Swollenin) rapidly induce extension of plant cell walls by weakening the noncovalent interactions that help to maintain their integrity. Cellobiose dehydrogenase (CDH) is a potential electron donor for polysaccharide monooxygenases (PMOs). EGs and PMOs internally cleave cellulose chains releasing chain ends that are targeted by cellobiohydrolases (CBHs). Dotted arrows indicate potential areas of research for enhancement of enzymes secretion in T. reesei: XYR1 activates or represses (directly) the transcription of genes in cellulose and sophorose. PWM_XYR1 defined in Silva-Rocha et al. (2014).