Network Analysis Reveals Different Cellulose Degradation Strategies Across Trichoderma harzianum Strains Associated With XYR1 and CRE1

Abstract

Trichoderma harzianum, whose gene expression is tightly controlled by the transcription factors (TFs) XYR1 and CRE1, is a potential candidate for hydrolytic enzyme production. Here, we performed a network analysis of T. harzianum IOC-3844 and T. harzianum CBMAI-0179 to explore how the regulation of these TFs varies between these strains. In addition, we explored the evolutionary relationships of XYR1 and CRE1 protein sequences among Trichoderma spp. The results of the T. harzianum strains were compared with those of Trichoderma atroviride CBMAI-0020, a mycoparasitic species. Although transcripts encoding carbohydrate-active enzymes (CAZymes), TFs, transporters, and proteins with unknown functions were coexpressed with cre1 or xyr1, other proteins indirectly related to cellulose degradation were identified. The enriched GO terms describing the transcripts of these groups differed across all strains, and several metabolic pathways with high similarity between both regulators but strain-specific differences were identified. In addition, the CRE1 and XYR1 subnetworks presented different topology profiles in each strain, likely indicating differences in the influences of these regulators according to the fungi. The hubs of the cre1 and xyr1 groups included transcripts not yet characterized or described as being related to cellulose degradation. The first-neighbor analyses confirmed the results of the profile of the coexpressed transcripts in cre1 and xyr1. The analyses of the shortest paths revealed that CAZymes upregulated under cellulose degradation conditions are most closely related to both regulators, and new targets between such signaling pathways were discovered. Although the evaluated T. harzianum strains are phylogenetically close and their amino acid sequences related to XYR1 and CRE1 are very similar, the set of transcripts related to xyr1 and cre1 differed, suggesting that each T. harzianum strain used a specific regulation strategy for cellulose degradation. More interestingly, our findings may suggest that XYR1 and CRE1 indirectly regulate genes encoding proteins related to cellulose degradation in the evaluated T. harzianum strains. An improved understanding of the basic biology of fungi during the cellulose degradation process can contribute to the use of their enzymes in several biotechnological applications and pave the way for further studies on the differences across strains of the same species.

Whole-Genome Sequencing and Comparative Genomic Analysis of Potential Biotechnological Strains of Trichoderma harzianum, Trichoderma atroviride, andTrichoderma reesei

“…In this study, we detected SVs by aligning our four genomes against T. reesei QM6a (Martinez et al, 2008); although the genomes were assembled at a scaffolding level, unlike the T. reesei QM6a reference genome, which was assembled at the chromosome level, we opted to proceed with this dataset because its annotation file was available . As expected, due to phylogenetic proximity to the reference genome (Rosolen et al, 2022), Tr0711 exhibited a lower number of SVs than the other strains. However, although T. atroviride is phylogenetically distant from T. reesei (Rosolen et al, 2022), Ta0020 exhibited fewer SVs than both T. harzianum strains, and this result might be explained by the uncertain phylogenetic position of fungi in these species (Druzhinina et al, 2010).…”

“…As expected, due to phylogenetic proximity to the reference genome (Rosolen et al, 2022), Tr0711 exhibited a lower number of SVs than the other strains. However, although T. atroviride is phylogenetically distant from T. reesei (Rosolen et al, 2022), Ta0020 exhibited fewer SVs than both T. harzianum strains, and this result might be explained by the uncertain phylogenetic position of fungi in these species (Druzhinina et al, 2010). Although the T. harzianum strains are phylogenetically close (Rosolen et al, 2022), comparison of the SVs identified from the mapping of both genomes against T. reesei QM6a reveals genetic variability across the strains (Martinez et al, 2008).…”

“…These observations might be explained by the complex speciation process within the T. harzianum species group (Druzhinina et al, 2010); therefore, the phylogenetic position is uncertain for fungi from these species. However, the molecular identification of Th3844 and Th0179 based on the ITS and tef1 sequences has already been reported (Rosolen et al, 2022), which confirmed that both strains were phylogenetically close to other T. harzianum strains. Tr0711 was grouped with T. pararessei CBS 125925, and both were phylogenetically in proximity to other T. reesei strains.…”

“…The identity of Trichoderma isolates was authenticated by CBMAI based on phylogenetic studies of their internal transcribed spacer (ITS) region, translational elongation factor 1 ( tef1 ), and RNA polymerase II ( rpb2 ) marker gene. The phylogenetic tree based on the ITS region was previously published (Rosolen et al, 2022); moreover, the methodology used to model the phylogenetic trees based on the tef1 and rpb2 marker genes is described in Supplementary Material 1. Briefly, Th3844, Th0179, Ta0020, and Tr0711 strains were cultivated on potato dextrose agar (PDA) solid medium (ampicillin 100 µg/ml and chloramphenicol 34 µg/ml) for 3 days at 28 °C.…”

“…For the molecular identification of the fungi used in this study, we previously investigated the phylogenetic relationships among Th3844, Th0179, Ta0020, and Tr0711 based on the ITS sequences (Rosolen et al, 2022). Here, we modeled phylogenetic trees based on the rpb2 and tef1 sequences of 16 and 26 Trichoderma spp., respectively, including those of our study strains (Supplementary Material 1: Supplementary Fig.…”