Identification and characterization of the glucose dual-affinity transport system in Neurospora crassa: pleiotropic roles in nutrient transport, signaling, and carbon catabolite repression

Abstract

BackgroundThe glucose dual-affinity transport system (low- and high-affinity) is a conserved strategy used by microorganisms to cope with natural fluctuations in nutrient availability in the environment. The glucose-sensing and uptake processes are believed to be tightly associated with cellulase 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 systematically identified and characterized the components of the glucose dual-affinity transport system in the model fungus Neurospora crassa.ResultsUsing RNA sequencing coupled with functional transport analyses, we assigned GLT-1 (K m = 18.42 ± 3.38 mM) and HGT-1/-2 (K m = 16.13 ± 0.95 and 98.97 ± 22.02 µM) to the low- and high-affinity glucose transport systems, respectively. The high-affinity transporters hgt-1/-2 complemented 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 in cells grown on cellulose. The 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 in hexose transporters resulted in a loss of glucose-transporting function, whereas CCR signal transduction was retained, indicating dual functions for HGT-1/-2 as “transceptors.”ConclusionsIn this study, GLT-1 and HGT-1/-2 were identified as the key components of the glucose dual-affinity transport system, which plays diverse roles in glucose transport and carbon metabolism. Given the wide conservation of the glucose dual-affinity transport system across fungal species, the identification of its components and their pleiotropic roles in this study shed important new light on the molecular basis of nutrient transport, signaling, and plant cell wall degradation in fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0705-4) contains supplementary material, which is available to authorized users.

Broad substrate-specific phosphorylation events are associated with the initial stage of plant cell wall recognition inNeurospora crassa

“…Differential phosphorylation of key components in substrate perception was demonstrated for the cellulolytic transcription factors CLR-1, a major cellulase regulator in N. crassa (Coradetti et al, 2012, 2013) and XLR-1 (a cellulase/hemicellulase regulator conserved in fungi (Samal et al, 2017) as well as a cellobionic acid transporter, CBT-1, after exposure to cellulose (Xiong et al, 2014). Carbon catabolite repression (CCR) is another central regulatory mechanism involved in substrate perception and metabolic regulation found in a wide range of microbial organisms that ensures the preferential utilization of glucose over less favorable carbon sources (Aro et al, 2005; Vinuselvi et al, 2012) and therefore affects the fungal response to nutrient availability (Fernandez et al, 2012; Nguyen et al, 2016; Bang Wang et al, 2017). Differential phosphorylation of CCR-related proteins after addition of different carbon sources was previously observed in a proteome analysis of Trichoderma reesei (Nguyen et al, 2016).…”

Enhancing peptaibols production in the biocontrol fungus Trichoderma longibrachiatum SMF2 by elimination of a putative glucose sensor

“…A total of 20 putative hexose (glucose) transporters showed changed expression pattern in Δ Tlstp1 compared to the WT strain (8 upregulated, 12 downregulated). Particularly, the gene SMF2FGGW_104114 encoding a putative hexose transporter that shares high sequence identity (75.9%) with the high‐affinity glucose transporter Hgt‐1 in N. crassa (Wang et al, ) is significantly downregulated during both the 36‐hr and 60‐hr cultivation period. Two relatively close homologs (SMF2FGGW_104773 and SMF2FGGW_101765) of the low‐affinity glucose transporters, Glt‐1 in N. crassa and HxtB in A. nidulans (Dos Reis et al, ; Wang et al, ), also exhibit downregulated expression in the Δ Tlstp1 mutant.…”

“…Tlstp1 is located in the membrane, which is consistent with the sequence analyses that Tlstp1 encodes a putative membrane transporter. Further glucose-mediated analyses showed that, in contrast with the yeast cells expressing the glucose transporter hgt-1 (Wang et al, 2017) which showed significant growth, cells expressing Tlstp1 exhibited hardly any growth when glucose was used as the sole carbon source (Figure 2b), suggesting that TlSTP1…”

“…Particularly, the gene SMF2FGGW_104114 encoding a putative hexose transporter that shares high sequence identity (75.9%) with the high-affinity glucose transporter Hgt-1 in N. crassa(Wang et al, 2017) is significantly downregulated during both the 36-hr and 60-hr cultivation period. Two relatively close homologs (SMF2FGGW_104773 and SMF2FGGW_101765) of the low-affinity glucose transporters, Glt-1 in N. crassa and HxtB in A. nidulans (DosReis et al, 2017;Wang et al, 2017), also exhibit downregulated expression in the ΔTlstp1 mutant.The two NRPS encoding genes, tlx1 and tlx2 responsible for synthesis of peptaibols, are upregulated in the transcriptome, which is in agreement with the quantitative RT-PCR results. We also found that several genes involved in the synthesis of secondary metabolites from NRPS, PKS, and terpenes, are differentially expressed in the…”

Efficient Human Genome Editing Using SaCas9 Ribonucleoprotein Complexes

“…Fresh cells were harvested and RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA extraction was performed as described in a previous study …”