Nucleo‐cytoplasmic shuttling dynamics of the transcriptional regulators XYR1 and CRE1 under conditions of cellulase and xylanase gene expression in Trichoderma reesei

Abstract

Trichoderma reesei is a model for investigating the regulation of (hemi-)cellulase gene expression. Cellulases are formed adaptively, and the transcriptional activator XYR1 and the carbon catabolite repressor CRE1 are main regulators of their expression. We quantified the nucleo-cytoplasmic shuttling dynamics of GFP-fusion proteins of both transcription factors under cellulase and xylanase inducing conditions, and correlated their nuclear presence/absence with transcriptional changes. We also compared their subcellular localization in conidial germlings and mature hyphae. We show that cellulase gene expression requires de novo biosynthesis of XYR1 and its simultaneous nuclear import, whereas carbon catabolite repression is regulated through preformed CRE1 imported from the cytoplasmic pool. Termination of induction immediately stopped cellulase gene transcription and was accompanied by rapid nuclear degradation of XYR1. In contrast, nuclear CRE1 rapidly decreased upon glucose depletion, and became recycled into the cytoplasm. In mature hyphae, nuclei containing activated XYR1 were concentrated in the colony center, indicating that this is the main region of XYR1 synthesis and cellulase transcription. CRE1 was found to be evenly distributed throughout the entire mycelium. Taken together, our data revealed novel aspects of the dynamic shuttling and spatial bias of the major regulator of (hemi-)cellulase gene expression, XYR1, in T. reesei.

Diverse Regulation of the CreA Carbon Catabolite Repressor in Aspergillus nidulans

“…Recently, it was observed that CRE1 in T. reesei does not require de novo biosynthesis and is imported into the nucleus from a preformed cytoplasmic pool (Lichius et al 2014). Microscopy was carried out on A. nidulans CreA::GFP germlings, where the creA (AN6195 ) wild-type allele was replaced with the creA::gfp allele (strain TN02a3) (Table S1) (Brown et al 2013), grown for 16 hr in glucose (CreA target genes repressed; CreA localizes to the nucleus), and then transferred to Xylan for 6 hr (CreA target genes derepressed; CreA leaves the nucleus) before glucose was added to the Xylan cultures for 30 min (Table 1).…”

Regulation of the lignocellulolytic response in filamentous fungi

“…The regulation of CreA/CRE1/CRE-1 appears to be at least partially dependent on intracellular localization. Under repressing conditions, CreA/CRE1/CRE-1 is found in the nucleus where it can regulate the expression of target genes, and when under de-repressing conditions, CreA/CRE1/CRE-1 exits the nucleus (Brown et al, 2013;Cupertino et al, 2015;Lichius et al, 2014). However, the genetic regulation of CreA/CRE1/CRE-1 appears to be fairly divergent amongst the filamentous fungi.…”

Transcriptional Regulation and Responses in Filamentous Fungi Exposed to Lignocellulose

“…In filamentous fungi, CCR is mediated by the CreA/CRE1 protein [28], which is a functional homologue of Mig1p. CreA/CRE1 is localized in the cytoplasm under non-repressing conditions and is shuttled to the nucleus under repressing conditions [37,38]. Its localization is regulated by its phosphorylation state [37], similar to Mig1p.…”

“…Section I described how some of the inducers of CAZymes are different in T. reesei compared to A. niger, namely the effects of lactose and sophorose, but these different inducers can still signal through the XYR1 transcription factor in T. reesei. Measurements of the shuttling of XYR1 in T. reesei provide insights into the functioning of this activator [38]. The XYR1 protein was synthesised in the cytoplasm as part of the induction process and when induction ceased the XYR1 protein in the nucleus was rapidly degraded [38].…”

“…Measurements of the shuttling of XYR1 in T. reesei provide insights into the functioning of this activator [38]. The XYR1 protein was synthesised in the cytoplasm as part of the induction process and when induction ceased the XYR1 protein in the nucleus was rapidly degraded [38]. Finally, a recent analysis in five ascomycetes emphasised some of the functional diversity of XlnR/XYR1; there were substantial differences in the CAZyme-encoding genes regulated by the XlnR/XYR1 orthologues in response to induction by xylan [83].…”