Identification and Characterization of the Glucose Dual-transporter System: Pleiotropic Roles in Nutrient Transport, Signaling, and Carbon Catabolite Repression in Neurospora crassa. Neurospora crassa OR74A

The glucose dual-affinity transport system (low- and high-affinity) is a conserved strategy exerted by microorganisms to cope with the naturally fluctuating availability of nutrients in the environment. The glucose sensing and uptaking process were believed to be tightly involved in cellulases expression regulation in cellulolytic fungi. However, both the identities and functions of the major molecular components of this evolutionarily conserved system in filamentous fungi remain elusive. Here, we conducted a systematic identification and characterization of the glucose dual-affinity transport system in the model fungus Neurospora crassa. Using RNA sequencing coupled with functional transport analyses, we were able to assign GLT-1 (Km = 18.42 ± 3.38 mM) and HGT-1/-2 (Km = 16.13 ± 0.95 µM and 98.97 ± 22.02 µM) to low- and high-affinity glucose transport systems, respectively. The high-affinity transporters hgt-1/-2 were able to complement a moderate growth defect under high glucose when glt-1 was deleted. Simultaneous deletion of hgt-1/-2 led to extensive derepression of genes for plant cell wall deconstruction on cellulose. This suppression by HGT-1/-2 was connected to both carbon catabolite repression (CCR) and the cyclic adenosine monophosphate-protein kinase A pathway. Alteration of a residue conserved across taxa for hexose-transporters was found to result in a loss of glucose-transporting function, whereas CCR signal transduction was retained, indicating a dual function for HGT-1/-2 as “transceptors”. In this study, GLT-1 and HGT-1/-2 are identified as the key components of the glucose dual-affinity transport system, which play diverse roles in glucose transport and carbon metabolism. Given their wide conservation across fungal species, the glucose dual-affinity transport components and their pleiotropic roles revealed in this study would shed extensive new light on the molecular basis of nutrient transport, signaling, and plant cell wall degradation in fungi. Overall design: Conidia of Neurospora crass wild type were inoculated with 10e6 conidia/mL into 100 mL 1×Vogel’s salts with 2% (w/v) sucrose pregrown for 16 h and then transferred into an indicated glucose gradient Vogel's salts for 1 or 2 hours. For Avicel growth, Conidia of WT and indicated mutants were inoculated with 10e6 conidia/mL into 100 mL 1×Vogel’s salts with 2% (w/v) Avicel grown for indicated hours. Two or three biological replicates were designed for each treatment. Mycelia were harvested through filtration and immediately frozen in liquid nitrogen.Total RNA from frozen sample was isolated with TRIzol reagent (Invitrogen) and further treated with DNase I (RNeasy Mini Kit, QIAGEN). The qualified RNA was prepared with standard protocol from Shenzhen BGI (China) and sequenced on the Illumina HiSeqTM 2000/2500 platform.