scholarly journals Arabidopsis Sucrose Transporter AtSuc1 introns act as strong enhancers of expression

2020 ◽  
Vol 61 (6) ◽  
pp. 1054-1063 ◽  
Author(s):  
Praphapan Lasin ◽  
Andreas Weise ◽  
Anke Reinders ◽  
John M Ward
Keyword(s):  
Constitutive Expression ◽  
Sucrose Transporter ◽  
Anthocyanin Accumulation ◽  
Sucrose Uptake ◽  
Loss Of Function ◽  
Defective Allele ◽  
Gus Transgenics

Abstract The expression of AtSUC1 is controlled by the promoter and intragenic sequences. AtSUC1 is expressed in roots, pollen and trichomes. However, AtSUC1 promoter-GUS transgenics only show expression in trichomes and pollen. Here, we show that the root expression of AtSUC1 is controlled by an interaction between the AtSUC1 promoter and two short introns. The deletion of either intron from whole-gene-GUS constructs results in no root expression, showing that both introns are required. The two introns in tandem, fused to GUS, produce high constitutive expression throughout the vegetative parts of the plant. When combined with the promoter, the expression driven by the introns is reduced and localized to the roots. In Arabidopsis seedlings, exogenously applied sucrose induces the expression of AtSUC1 in roots and causes anthocyanin accumulation. atsuc1 loss-of-function mutants are defective in sucrose-induced anthocyanin accumulation. We show that an AtSUC1 whole-gene-GUS construct expressing a nonfunctional AtSUC1 (D152N) mutant, that is transport inactive, is defective in sucrose-induced AtSUC1 expression when expressed in an atsuc1-null background. We also show that the transport-defective allele does not complement the loss of sucrose-induced anthocyanin accumulation in null atsuc1 mutants. The results indicate that sucrose uptake via AtSUC1 is required for sucrose-induced AtSUC1 expression and sucrose-induced anthocyanin accumulation and that the site for sucrose detection is intracellular.

scholarly journals Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

2013 ◽  
Vol 13 (1) ◽  
pp. 53-65 ◽  
Author(s):  
Elodie Bovier ◽  
Carole H. Sellem ◽  
Adeline Humbert ◽  
Annie Sainsard-Chanet
Keyword(s):  
Transcription Factors ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Constitutive Expression ◽  
Podospora Anserina ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Content Type ◽  
Zinc Cluster ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

scholarly journals Seed-specific overexpression of a potato sucrose transporter increases sucrose uptake and growth rates of developing pea cotyledons

2002 ◽  
Vol 30 (2) ◽  
pp. 165-175 ◽  
Author(s):  
Elke Rosche ◽  
Daniel Blackmore ◽  
Mechthild Tegeder ◽  
Terese Richardson ◽  
Hart Schroeder ◽  
...  
Keyword(s):  
Growth Rates ◽  
Sucrose Transporter ◽  
Sucrose Uptake

scholarly journals Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

2015 ◽  
Vol 14 (11) ◽  
pp. 1073-1080 ◽  
Author(s):  
Kevin K. Fuller ◽  
Shan Chen ◽  
Jennifer J. Loros ◽  
Jay C. Dunlap
Keyword(s):  
Aspergillus Fumigatus ◽  
Gene Disruption ◽  
Polyketide Synthase ◽  
High Efficiency ◽  
Constitutive Expression ◽  
Targeted Mutagenesis ◽  
Genomic Alteration ◽  
Loss Of Function ◽  
Content Type ◽  
Targeted Gene Disruption

ABSTRACTLow rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogenAspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use inA. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of theA. fumigatuspolyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious toA. fumigatusgrowth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies inA. fumigatusand has the potential to bolster the genetic toolbox for this important pathogen.

scholarly journals Arabidopsis CALMODULIN-BINDING PROTEIN 60b plays dual roles in plant immunity

2021 ◽  
Author(s):  
Weijie Huang ◽  
Zhongshou Wu ◽  
Hainan Tian ◽  
Xin Li ◽  
Yuelin Zhang
Keyword(s):  
Systemic Acquired Resistance ◽  
Binding Protein ◽  
Plant Immunity ◽  
Interleukin 1 ◽  
Constitutive Expression ◽  
Defense Responses ◽  
Negative Regulator ◽  
Loss Of Function ◽  
Constitutive Defense ◽  
Calmodulin Binding

AbstractArabidopsis SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g) are two master transcription factors that regulate many defense-related genes in plant immunity. They are required for immunity downstream of the receptor-like protein SUPPRESSOR OF NPR1-1, CONSTITUTIVE 2 (SNC2). Constitutive defense responses in the gain-of-function autoimmune snc2-1D mutant are modestly affected by either sard1 or cbp60g single mutants, but completely suppressed by the sard1 cbp60g double mutant. Here we report that CBP60b, another member of the CBP60 family, also functions as a positive regulator of SNC2-mediated immunity. Loss-of-function mutations of CBP60b suppress the constitutive expression of SARD1 and enhanced disease resistance in cbp60g-1 snc2-1D, whereas over-expression of CBP60b leads to elevated SARD1 expression and constitutive defense responses. In addition, transient expression of CBP60b in Nicotiana benthamiana activates the expression of the pSARD1::luciferase reporter gene. Chromatin immunoprecipitation assay further showed that CBP60b is recruited to the promoter region of SARD1, suggesting that it directly regulates SARD1 expression. Interestingly, knocking out CBP60b in the wild type background leads to ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)-dependent autoimmunity, suggesting that CBP60b is required for the expression of a guardee/decoy or a negative regulator in immunity mediated by receptors carrying an N-terminal TIR (Toll-interleukin-1 receptor-like) domain.Significance statementArabidopsis SARD1 serves as a master transcription factor in plant immunity. In this study, we showed that CBP60b positively regulates SARD1 expression, and TIR signaling is activated when CBP60b is inactivated.

scholarly journals Molecular Regulation of Arabinan and l-Arabinose Metabolism in Hypocrea jecorina (Trichoderma reesei)

2009 ◽  
Vol 8 (12) ◽  
pp. 1837-1844 ◽  
Author(s):  
Eda Akel ◽  
Benjamin Metz ◽  
Bernhard Seiboth ◽  
Christian P. Kubicek
Keyword(s):  
Trichoderma Reesei ◽  
Aldose Reductase ◽  
Parent Strain ◽  
Constitutive Expression ◽  
Molecular Regulation ◽  
Transcriptional Analysis ◽  
Loss Of Function ◽  
Hypocrea Jecorina ◽  
Slight Effect ◽  
Encoding Genes

ABSTRACT Hypocrea jecorina (anamorph: Trichoderma reesei) can grow on plant arabinans by the aid of secreted arabinan-degrading enzymes. This growth on arabinan and its degradation product l-arabinose requires the operation of the aldose reductase XYL1 and the l-arabinitol dehydrogenase LAD1. Growth on arabinan and l-arabinose is also severely affected in a strain deficient in the general cellulase and hemicellulase regulator XYR1, but this impairment can be overcome by constitutive expression of the xyl1 encoding the aldose reductase. An inspection of the genome of H. jecorina reveals four genes capable of degrading arabinan, i.e., the α-l-arabinofuranosidase encoding genes abf1, abf2, and abf3 and also bxl1, which encodes a β-xylosidase with a separate α-l-arabinofuranosidase domain and activity but no endo-arabinanase. Transcriptional analysis reveals that in the parent strain QM9414 the expression of all of these genes is induced by l-arabinose and to a lesser extent by l-arabinitol and absent on d-glucose. Induction by l-arabinitol, however, is strongly enhanced in a Δlad1 strain lacking l-arabinitol dehydrogenase activity and severely impaired in an aldose reductase (Δxyl1) strain, suggesting a cross talk between l-arabinitol and the aldose reductase XYL1 in an α-l-arabinofuranosidase gene expression. Strains bearing a knockout in the cellulase regulator xyr1 do not show any induction of abf2 and bxl1, and this phenotype cannot be reverted by constitutive expression of xyl1. The loss of function of xyr1 has also a slight effect on the expression of abf1 and abf3. We conclude that the expression of the four α-l-arabinofuranosidases of H. jecorina for growth on arabinan requires an early pathway intermediate (l-arabinitol or l-arabinose), the first enzyme of the pathway XYL1, and in the case of abf2 and bxl1 also the function of the cellulase regulator XYR1.

scholarly journals Aberrant and constitutive expression of FOXL2 impairs ovarian development and functions in mice

2020 ◽  
Vol 103 (5) ◽  
pp. 966-977
Author(s):  
Barbara Nicol ◽  
Karina Rodriguez ◽  
Humphrey H-C Yao
Keyword(s):  
Ovarian Development ◽  
Constitutive Expression ◽  
Cell Types ◽  
Primary Ovarian Insufficiency ◽  
Fetal Life ◽  
Loss Of Function ◽  
Ovarian Insufficiency ◽  
Ovarian Cell ◽  
Theca Cells ◽  
The Impact

Abstract Development and functions of the ovary rely on appropriate signaling and communication between various ovarian cell types. FOXL2, a transcription factor that plays a key role at different stages of ovarian development, is associated with primary ovarian insufficiency and ovarian cancer as a result of its loss-of-function or mutations. In this study, we investigated the impact of aberrant, constitutive expression of FOXL2 in somatic cells of the ovary. Overexpression of FOXL2 that started during fetal life resulted in defects in nest breakdown and consequent formation of polyovular follicles. Granulosa cell differentiation was impaired and recruitment and differentiation of steroidogenic theca cells was compromised. As a consequence, adult ovaries overexpressing FOXL2 exhibited defects in compartmentalization of granulosa and theca cells, significant decreased steroidogenesis and lack of ovulation. These findings demonstrate that fine-tuned expression of FOXL2 is required for proper folliculogenesis and fertility.

scholarly journals Transformation of Human and Murine Fibroblasts without Viral Oncoproteins

2005 ◽  
Vol 25 (15) ◽  
pp. 6464-6474 ◽  
Author(s):  
Jesse S. Boehm ◽  
Meghan T. Hession ◽  
Sara E. Bulmer ◽  
William C. Hahn
Keyword(s):  
Human Fibroblasts ◽  
Constitutive Expression ◽  
Genetic Alterations ◽  
Human Cells ◽  
Loss Of Function ◽  
Viral Oncoproteins ◽  
Murine Fibroblasts ◽  
Murine Embryo ◽  
Embryo Fibroblasts ◽  
Multiple Strains

ABSTRACT Murine embryo fibroblasts are readily transformed by the introduction of specific combinations of oncogenes; however, the expression of those same oncogenes in human cells fails to convert such cells to tumorigenicity. Using normal human and murine embryonic fibroblasts, we show that the transformation of human cells requires several additional alterations beyond those required to transform comparable murine cells. The introduction of the c-Myc and H-RAS oncogenes in the setting of loss of p53 function efficiently transforms murine embryo fibroblasts but fails to transform human cells constitutively expressing hTERT, the catalytic subunit of telomerase. In contrast, transformation of multiple strains of human fibroblasts requires the constitutive expression of c-Myc, H-RAS, and hTERT, together with loss of function of the p53, RB, and PTEN tumor suppressor genes. These manipulations permit the development of transformed human fibroblasts with genetic alterations similar to those found associated with human cancers and define specific differences in the susceptibility of human and murine fibroblasts to experimental transformation.

scholarly journals Loss of function of the carbon catabolite repressor CreA leads to low but inducer‐independent expression from the feruloyl esterase B promoter in Aspergillus niger

2021 ◽  
Author(s):  
Jos Reijngoud ◽  
Mark Arentshorst ◽  
Claudine Ruijmbeek ◽  
Ian Reid ◽  
Ebru Demirci Alazi ◽  
...  
Keyword(s):  
Catabolite Repression ◽  
Allyl Alcohol ◽  
Carbon Catabolite Repression ◽  
Constitutive Expression ◽  
Genetic Screen ◽  
Feruloyl Esterase ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Targeted Deletion ◽  
Reporter Strains

Abstract Objective With the aim to decipher the mechanisms involved in the transcriptional regulation of feruloyl esterase encoded by faeB, a genetic screen was performed to isolate A. niger mutants displaying inducer-independent expression from the faeB promoter. Result PfaeB-amdS and PfaeB-lux dual reporter strains were constructed and used to isolate trans-acting mutants in which the expression of both reporters was increased, based on the ability to grow on acetamide plates and higher luciferase activity, respectively. The genetic screen on the non-inducing carbon source D-fructose yielded in total 111 trans-acting mutants. The genome of one of the mutants was sequenced and revealed several SNPs, including a point mutation in the creA gene encoding a transcription factor known to be involved in carbon catabolite repression. Subsequently, all mutants were analyzed for defects in carbon catabolite repression by determining sensitivity towards allyl alcohol. All except four of the 111 mutants were sensitive to allyl alcohol, indicating that the vast majority of the mutants are defective in carbon catabolite repression. The creA gene of 32 allyl alcohol sensitive mutants was sequenced and 27 of them indeed contained a mutation in the creA gene. Targeted deletion of creA in the reporter strain confirmed that the loss of CreA results in constitutive expression from the faeB promoter. Conclusion Loss of function of CreA leads to low but inducer-independent expression from the faeB promoter in A. niger.

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