scholarly journals Transcription Factor NsdD Regulates the Expression of Genes Involved in Plant Biomass-Degrading Enzymes, Conidiation, and Pigment Biosynthesis inPenicillium oxalicum

2018 ◽  
Vol 84 (18) ◽  
Author(s):  
Qi-Peng He ◽  
Shuai Zhao ◽  
Jiu-Xiang Wang ◽  
Cheng-Xi Li ◽  
Yu-Si Yan ◽  
...  

ABSTRACTSoil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor,Penicillium oxalicumNsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes inP. oxalicum. PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development inAspergillus. In the present study, the regulatory roles of PoxNsdD inP. oxalicumwere further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdDstrain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70parental strain. During cultivation, ΔPoxNsdDcultures changed color, from pale orange to brick red, while the ΔPoxKu70cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed thatPoxNsdDdynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover,in vitrobinding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering ofP.oxalicumfor potential industrial and medical applications.IMPORTANCEMost filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungusPenicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential ofP.oxalicumvia genetic engineering.

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Leandro José de Assis ◽  
Lilian Pereira Silva ◽  
Ozgur Bayram ◽  
Paul Dowling ◽  
Olaf Kniemeyer ◽  
...  

ABSTRACT Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C2H2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects. IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.


mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xiaolong Shao ◽  
Weitong Zhang ◽  
Mubarak Ishaq Umar ◽  
Hei Yuen Wong ◽  
Zijing Seng ◽  
...  

ABSTRACT Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species. IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.


mSystems ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Amanda Cristina Campos Antonieto ◽  
Karoline Maria Vieira Nogueira ◽  
Renato Graciano de Paula ◽  
Luísa Czamanski Nora ◽  
Murilo Henrique Anzolini Cassiano ◽  
...  

ABSTRACT Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans. Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo. Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei. IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.


2010 ◽  
Vol 9 (8) ◽  
pp. 1225-1235 ◽  
Author(s):  
Rasmus J. N. Frandsen ◽  
Klaus Selk Albertsen ◽  
Peter Stougaard ◽  
Jens L. Sørensen ◽  
Kristian F. Nielsen ◽  
...  

ABSTRACT Methylenetetrahydrofolate reductases (MTHFRs) play a key role in biosynthesis of methionine and S-adenosyl-l-methionine (SAM) via the recharging methionine biosynthetic pathway. Analysis of 32 complete fungal genomes showed that fungi were unique among eukaryotes by having two MTHFRs, MET12 and MET13. The MET12 type contained an additional conserved sequence motif compared to the sequences of MET13 and MTHFRs from other eukaryotes and bacteria. Targeted gene replacement of either of the two MTHFR encoding genes in Fusarium graminearum showed that they were essential for survival but could be rescued by exogenous methionine. The F. graminearum strain with a mutation of MET12 (FgΔMET12) displayed a delay in the production of the mycelium pigment aurofusarin and instead accumulated nor-rubrofusarin and rubrofusarin. High methionine concentrations or prolonged incubation eventually led to production of aurofusarin in the MET12 mutant. This suggested that the chemotype was caused by a lack of SAM units for the methylation of nor-rubrofusarin to yield rubrofusarin, thereby imposing a rate-limiting step in aurofusarin biosynthesis. The FgΔMET13 mutant, however, remained aurofusarin deficient at all tested methionine concentrations and instead accumulated nor-rubrofusarin and rubrofusarin. Analysis of MET13 mutants in F. graminearum and Aspergillus nidulans showed that both lacked extracellular reduction potential and were unable to complete mycelium pigment biosynthesis. These results are the first to show that MET13, in addition to its function in methionine biosynthesis, is required for the generation of the extracellular reduction potential necessary for pigment production in filamentous fungi.


mSystems ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Yue-Jin Peng ◽  
Jia-Jia Wang ◽  
Hai-Yan Lin ◽  
Jin-Li Ding ◽  
Ming-Guang Feng ◽  
...  

ABSTRACT In pathogenic filamentous fungi, conidial germination not only is fundamental for propagation in the environment but is also a critical step of infection. In the insect mycopathogen Beauveria bassiana, we genetically characterized the role of the basic leucine zipper (bZIP) transcription factor HapX (BbHapX) in conidial nutrient reserves and pathogen-host interaction. Ablation of BbHapX resulted in an almost complete loss of virulence in the topical inoculation and intrahemocoel injection assays. Comparative transcriptomic analysis revealed that BbHapX is required for fatty acid (FA)/lipid metabolism, and biochemical analyses indicated that BbHapX loss caused a significant reduction in conidial FA contents. Exogenous oleic acid could partially or completely restore the impaired phenotypes of the ΔBbHapX mutant, including germination rate, membrane integrity, vegetative growth, and virulence. BbHapX mediates fungal iron acquisition which is not required for desaturation of stearic acid. Additionally, inactivation of the Δ9-fatty acid desaturase gene (BbOle1) generated defects similar to those of the ΔBbHapX mutant; oleic acid also had significant restorative effects on the defective phenotypes of the ΔBbOle1 mutant. A gel retarding assay revealed that BbHapX directly regulated the expression of BbOle1. Lipidomic analyses indicated that both BbHapX and BbOle1 contributed to the homeostasis of phospholipids with nonpolar tails derived from oleic acid; therefore, exogenous phospholipids could significantly restore membrane integrity. These data reveal that the HapX-Ole1 pathway contributes to conidial fatty acid/lipid reserves and that there are important links between the lipid biology and membrane functionality involved in the early stages of infection caused by B. bassiana. IMPORTANCE Conidial maturation and germination are highly coupled physiological processes in filamentous fungi that are critical for the pathogenicity of mycopathogens. Compared to the mechanisms involved in conidial germination, those of conidial reserves during maturation are less understood. The insect-pathogenic fungus Beauveria bassiana, as a representative species of filamentous fungi, is important for applied and fundamental research. In addition to its conserved roles in fungal adaptation to iron status, the bZIP transcription factor HapX acts as a master regulator involved in conidial virulence and regulates fatty acid/lipid metabolism. Further investigation revealed that the Δ9-fatty acid desaturase gene (Ole1) is a direct downstream target of HapX. This study reveals the HapX-Ole1 pathway involved in the fatty acid/lipid accumulation associated with conidial maturation and provides new insights into the startup mechanism of infection caused by spores from pathogenic fungi.


2013 ◽  
Author(s):  
Remco Stam ◽  
Graham Motion ◽  
Petra C. Boevink ◽  
Edgar Huitema

Phytophthora spp. secrete vast arrays of effector molecules upon infection. A main class of intracellular effectors are the CRNs. They are translocated into the host cell and specifically localise to the nucleus where they are thought to perturb many different cellular processes. Although CRN proteins have been implicated as effectors, direct evidence of CRN mediated perturbation of host processes has been lacking. Here we show that a conserved CRN effector from P. capsici directly binds to tomato transcription factor SlTCP14-2. Previous studies in Arabidopsis thaliana have revealed that transcription factor TCP14 may be key immune signalling protein, targeted by effectors from divergent species. We extend on our understanding of TCP targeting by pathogen effectors by showing that the P. capsici effector CRN12_997 binds to SlTCP14-2 in plants. SlTCP14-2 over-expression enhances immunity to P. capsici, a phenotypic outcome that can be abolished by co-expression of CRN12_997. We show that in the presence of CRN12_997, SlTCP14-2 association with nuclear chromatin is diminished, resulting in altered SlTCP14 subnuclear localisation. These results suggest that CRN12_997 prevents SlTCP14 from positively regulating defence against P. capsici. Our work demonstrates a direct interaction between an oomycete CRN and a host target required for suppression of immunity. Collectively, our results hint at a virulence strategy that is conserved within the oomycetes and may allow engineering of resistance to a wide range of crop pathogens.


2014 ◽  
Vol 14 (4) ◽  
pp. 345-358 ◽  
Author(s):  
Steffen Nordzieke ◽  
Thomas Zobel ◽  
Benjamin Fränzel ◽  
Dirk A. Wolters ◽  
Ulrich Kück ◽  
...  

ABSTRACT S arco l emmal m embrane- a ssociated p rotein (SLMAP) is a tail-anchored protein involved in fundamental cellular processes, such as myoblast fusion, cell cycle progression, and chromosomal inheritance. Further, SLMAP misexpression is associated with endothelial dysfunctions in diabetes and cancer. SLMAP is part of the conserved str iatin- i nteracting p hosphatase a nd k inase (STRIPAK) complex required for specific signaling pathways in yeasts, filamentous fungi, insects, and mammals. In filamentous fungi, STRIPAK was initially discovered in Sordaria macrospora , a model system for fungal differentiation. Here, we functionally characterize the STRIPAK subunit PRO45, a homolog of human SLMAP. We show that PRO45 is required for sexual propagation and cell-to-cell fusion and that its f ork h ead- a ssociated (FHA) domain is essential for these processes. Protein-protein interaction studies revealed that PRO45 binds to STRIPAK subunits PRO11 and SmMOB3, which are also required for sexual propagation. Superresolution s tructured- i llumination m icroscopy (SIM) further established that PRO45 localizes to the nuclear envelope, endoplasmic reticulum, and mitochondria. SIM also showed that localization to the nuclear envelope requires STRIPAK subunits PRO11 and PRO22, whereas for mitochondria it does not. Taken together, our study provides important insights into fundamental roles of the fungal SLMAP homolog PRO45 and suggests STRIPAK-related and STRIPAK-unrelated functions.


2013 ◽  
Vol 58 (3) ◽  
pp. 1434-1442 ◽  
Author(s):  
Xianyun Sun ◽  
Kangji Wang ◽  
Xinxu Yu ◽  
Jie Liu ◽  
Hanxing Zhang ◽  
...  

ABSTRACTAntifungal azoles are widely used for controlling fungal infections. Fungi are able to change the expression of many genes when they adapt to azole stress, and increased expression of some of these genes can elevate resistance to azoles. However, the regulatory mechanisms behind transcriptional adaption to azoles in filamentous fungi are poorly understood. In this study, we found that deletion of the transcription factor geneccg-8, which is known to be a clock-controlled gene, madeNeurospora crassahypersensitive to azoles. A comparative genome-wide analysis of the responses to ketoconazole of the wild type and theccg-8mutant revealed that the transcriptional responses to ketoconazole of 78 of the 488 transcriptionally ketoconazole-upregulated genes and the 427 transcriptionally ketoconazole-downregulated genes in the wild type were regulated by CCG-8. Ketoconazole sensitivity testing of all available knockout mutants for CCG-8-regulated genes revealed that CCG-8 contributed to azole adaption by regulating the ketoconazole responses of many genes, including the target gene (erg11), an azole transporter gene (cdr4), a hexose transporter gene (hxt13), a stress response gene (locus number NCU06317, namedkts-1), two transcription factor genes (NCU01386 [namedkts-2] andfsd-1/ndt80), four enzyme-encoding genes, and six unknown-function genes. CCG-8 also regulated phospholipid synthesis inN. crassain a manner similar to that of its homolog inSaccharomyces cerevisiae, Opi1p. However, there was no cross talk between phospholipid synthesis and azole resistance inN. crassa. CCG-8 homologs are conserved and are common in filamentous fungi. Deletion of the CCG-8 homolog-encoding gene inFusarium verticillioides(Fvccg-8) also made this fungus hypersensitive to antifungal azoles.


mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Lara Hassan ◽  
Liangcai Lin ◽  
Hagit Sorek ◽  
Laura E. Sperl ◽  
Thomas Goudoulas ◽  
...  

ABSTRACTIt is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available forNeurospora crassato investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCEIn fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.


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