scholarly journals The Plasma Membrane H+-ATPase from the Biotrophic Rust Fungus Uromyces fabae: Molecular Characterization of the Gene (PMA1) and Functional Expression of the Enzyme in Yeast

1998 ◽  
Vol 11 (6) ◽  
pp. 458-465 ◽  
Author(s):  
Christine Struck ◽  
Claudia Siebels ◽  
Oliver Rommel ◽  
Marcus Wernitz ◽  
Matthias Hahn
Keyword(s):  
Plasma Membrane ◽  
Rust Fungus ◽  
Single Gene ◽  
Functional Expression ◽  
Wild Type ◽  
Ph Optimum ◽  
Uromyces Fabae ◽  
Mutant Derivative ◽  
Regulatory Properties

To study the molecular basis of biotrophic nutrient uptake by plant parasitic rust fungi, the gene (Uf-PMA1) encoding the plasma membrane H+-ATPase from Uromyces fabae was isolated. Uf-PMA1 exists probably as a single gene. However, two nearly identical sequences were identified; the similarity apparently is due to two Uf-PMA1 alleles in the dikaryotic hyphae. Multiple Uf-PMA1 transcripts were observed during early rust development, and reduced amounts of a single Uf-PMA1 mRNA were observed in haustoria and rust-infected leaves. This is in contrast to elevated enzyme activity in haustoria compared to germinated spores (C. Struck, M. Hahn, and K. Mendgen. Fungal Genet. Biol. 20:30–35, 1996). Unexpectedly, the PMA1-encoded rust protein is more similar to H+-ATPases from plants (55% identity) than from ascomycetous fungi (36% identity). When the rust PMA1 cDNA was expressed in Saccharomyces cerevisiae, both the wild-type enzyme and a mutant derivative (Δ76) deleted for the 76 C-terminal amino acids were able to support growth of a yeast strain lacking its own H+-ATPases. Compared to the wild-type, the Δ76 mutant enzyme displayed increased affinity to ATP, a higher vanadate sensitivity, and a more alkaline pH optimum. These results indicate that the C-terminal region of the rust enzyme exhibits auto-regulatory properties.

scholarly journals Virulence Characterization of Wheat Stripe Rust Fungus Puccinia striiformis f. sp. tritici in Ethiopia and Evaluation of Ethiopian Wheat Germplasm for Resistance to Races of the Pathogen from Ethiopia and the United States

Plant Disease ◽  
2017 ◽  
Vol 101 (1) ◽  
pp. 73-80 ◽  
Author(s):  
Anmin Wan ◽  
Kebede T. Muleta ◽  
Habtemariam Zegeye ◽  
Bekele Hundie ◽  
Michael O. Pumphrey ◽  
...  
Keyword(s):  
United States ◽  
Stripe Rust ◽  
Puccinia Striiformis ◽  
Rust Fungus ◽  
Single Gene ◽  
The United States ◽  
Wheat Cultivars ◽  
Low Frequencies ◽  
Additional Information

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat in Ethiopia. In total, 97 isolates were recovered from stripe rust samples collected in Ethiopia in 2013 and 2014. These isolates were tested on a set of 18 Yr single-gene differentials for characterization of races and 7 supplementary differentials for additional information of virulence. Of 18 P. striiformis f. sp. tritici races identified, the 5 most predominant races were PSTv-105 (21.7%), PSTv-106 (17.5%), PSTv-107 (11.3%), PSTv-76 (10.3%), and PSTv-41 (6.2%). High frequencies (>40%) were detected for virulence to resistance genes Yr1, Yr2, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, Yr27, Yr28, Yr31, Yr43, Yr44, YrExp2, and YrA. Low frequencies (<40%) were detected for virulence to Yr10, Yr24, Yr32, YrTr1, Hybrid 46, and Vilmorin 23. None of the isolates were virulent to Yr5, Yr15, YrSP, and YrTye. Among the six collection regions, Arsi Robe and Tiyo had the highest virulence diversities, followed by Bekoji, while Bale and Holeta had the lowest. Evaluation of 178 Ethiopian wheat cultivars and landraces with two of the Ethiopian races and three races from the United States indicated that the Ethiopian races were more virulent on the germplasm than the predominant races of the United States. Thirteen wheat cultivars or landraces that were resistant or moderately resistant to all five tested races should be useful for breeding wheat cultivars with resistance to stripe rust in both countries.

scholarly journals Functional Expression and Characterization of Trichome-Specific (-)-Limonene Synthase and (+)-α-Pinene Synthase from Cannabis sativa

2007 ◽  
Vol 2 (3) ◽  
pp. 1934578X0700200 ◽  
Author(s):  
Nils Günnewich ◽  
Jonathan E. Page ◽  
Tobias G. Köllner ◽  
Jörg Degenhardt ◽  
Toni M. Kutchan
Keyword(s):  
Phylogenetic Analysis ◽  
Natural Products ◽  
Cannabis Sativa ◽  
Functional Expression ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Limonene Synthase ◽  
Monoterpene Synthases

Two recombinant, stereospecific monoterpene synthases, a (-)-limonene synthase (CsTPS1) and a (+)-α-pinene synthase (CsTPS2), encoded by Cannabis sativa L. cv. ‘Skunk’ trichome mRNA, have been isolated and characterized. Recombinant CsTPS1 showed a Km value of 6.8 μM, a Vmax of 1.1 × 10−4 μmol/min and Vmax/Km of 0.016; the pH optimum was determined at pH 6.5, and a temperature optimum at 40°C. Recombinant CsTPS2 showed a Km value of 10.5 μM, a Vmax of 2.2 × 10−4 μmol/min and Vmax/Km of 0.021; the pH optimum was determined at pH 7.0, and a temperature optimum at 30°C. Phylogenetic analysis showed that both CsTPSs group within the angiosperms and belong to the Tpsb subgroup of monoterpene synthases. The enzymatic products (-)-limonene and (+)-α-pinene were detected as natural products in C. sativa trichomes.

Control of ammoniagenesis in the kidney of water- and air-breathing osteoglossids: characterization of glutamate dehydrogenase

1978 ◽  
Vol 56 (4) ◽  
pp. 845-851 ◽  
Author(s):  
K. B. Storey ◽  
H. E. Guderley ◽  
M. Guppy ◽  
P. W. Hochachka
Keyword(s):  
Glutamate Dehydrogenase ◽  
Product Inhibition ◽  
Energy Status ◽  
Ph Optimum ◽  
Arapaima Gigas ◽  
Marked Preference ◽  
Regulatory Properties ◽  
Role Activity

Glutamate dehydrogenases (EC 1.4.1.2) from the kidney of Osteoglossum bicirrhosum (called aruana) and Arapaima gigas were kinetically characterized. The two enzymes exhibited several common characteristics including Vmax activity ratio, pH optimum, affinity for cofactors, a marked preference for NAD(H) over NADP(H), and a very low affinity for NH4+. A variety of regulatory metabolites affected both enzymes. GTP and GDP were inhibitory while ADP, ATP, AMP, and leucine activated the enzymes. Both enzymes displayed potent product inhibition which was partially reversed by low levels of ADP. Arapaima kidney glutamate dehydrogenase was tightly regulated by the adenylate and guanylate nucleotides, inhibition by GTP and GDP and deinhibition by ADP and AMP being much stronger for this enzyme than for the aruana enzyme. Aruana glutamate dehydrogenase, however, was more responsive to NAD–NADH control. The enzyme was more sensitive to NAD(H) product inhibition and this inhibition was poorly reversed by ADP. From these data, it was concluded that both fish kidney glutamate dehydrogenases could function in glutamate oxidation in vivo. However, the Arapaima enzyme appeared most clearly adapted to a catabolic role, activity being more tightly linked to the energy status of the mitochondrion. Conversely, the aruana enzyme displayed regulatory properties allowing it the potential to function in NADH oxidation during periods of hypoxic stress.

scholarly journals Establishment and characterization of Neu1-knockout zebrafish and its abnormal clinical phenotypes

2020 ◽  
Author(s):  
Keiji Okada ◽  
Ryo Takase ◽  
Yurie Hamaoka ◽  
Akinobu Honda ◽  
Asami Ikeda ◽  
...  
Keyword(s):  
Animal Model ◽  
Plasma Membrane ◽  
Sialic Acid ◽  
Danio Rerio ◽  
Cancer Metastasis ◽  
Expression Patterns ◽  
Wild Type ◽  
Fish Embryo ◽  
Neu1 Sialidase

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both a2-3 and a2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Further, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa,lamp1a, and tfeb were upregulated in adult Neu1-KO muscle as compared to WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

Gene cloning, functional expression and characterization of a novel GH46 chitosanase from Streptomyces avermitilis (SaCsn46A)

2021 ◽  
Author(s):  
Jing Guo ◽  
Yi Wang ◽  
Wenjun Gao ◽  
Xinrou Wang ◽  
Xin Gao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Functional Expression ◽  
Streptomyces Avermitilis ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Glycosidic Bonds ◽  
Sds Page ◽  
Mature Enzyme ◽  
Layer Chromatography

Abstract A novel glycoside hydrolase (GH) family 46 chitosanase (SaCsn46A) from Streptomyces avermitilis was cloned and functionally expressed in Escherichia coli Rosetta (DE3) strains. SaCsn46A consists of 271 amino acids, which includes a 34-amino acids signal peptide. The protein sequence of SaCsn46A shows maximum identity (83.5%) to chitosanase from Streptomyces sp. SirexAA-E. Then the mature enzyme was purified to homogeneity through Ni-chelating affinity chromatography with a recovery yield of 78% and the molecular mass of purified enzyme was estimated to be 29 kDa by SDS-PAGE. The recombinant enzyme possessed a temperature optimum of 45 oC and a pH optimum of 6.2, and it was stable at pH ranging from 4.0 to 9.0 and below 30 oC. The Km and Vmax values of this enzyme were 1.32 mg∙mL− 1, 526.32 µM∙mg− 1∙min− 1, respectively (chitosan as substrate). The enzyme activity can be enhanced by Mg2+ and especially Mn2+, which could enhance the activity about 3.62-fold at a 3 mM concentration. The enzyme can hydrolyze a variety of polysaccharides which linked by β-1,4-glycosidic bonds such as chitin, xylan and cellulose, but it could not hydrolyze polysaccharides linked by α-1,4-glycosidic bonds. The results of thin layer chromatography and HPLC showed that the enzyme exhibited an endo-type cleavage pattern and could hydrolyze chitosan to glucosamine (GlcN) and (GlcN)2. This study demonstrated that SaCsn46A is a promising enzyme to produce glucosamine and chitooligosaccharides (COS) from chitosan.

scholarly journals Cloning and Characterization of a Novel Invertase from the Obligate Biotroph Uromyces fabae and Analysis of Expression Patterns of Host and Pathogen Invertases in the Course of Infection

2006 ◽  
Vol 19 (6) ◽  
pp. 625-634 ◽  
Author(s):  
Ralf T. Voegele ◽  
Stefan Wirsel ◽  
Ulla Möll ◽  
Melanie Lechner ◽  
Kurt Mendgen
Keyword(s):  
Cell Wall ◽  
Expression Analysis ◽  
Biochemical Characterization ◽  
Higher Plants ◽  
Rust Fungus ◽  
Expression Patterns ◽  
Invertase Activity ◽  
Pathogen Infection ◽  
Uromyces Fabae

Invertases are key enzymes in carbon partitioning in higher plants. They gain additional importance in the distribution of carbohydrates in the event of wounding or pathogen attack. Although many researchers have found an increase in invertase activity upon infection, only a few studies were able to determine whether the source of this activity was host or parasite. This article analyzes the role of invertases involved in the biotrophic interaction of the rust fungus Uromyces fabae and its host plant, Vicia faba. We have identified a fungal gene, Uf-INV1, with homology to invertases and assessed its contribution to pathogenesis. Expression analysis indicated that transcription began upon penetration of the fungus into the leaf, with high expression levels in haustoria. Heterologous expression of Uf-INV1 in Saccharomyces cerevisiae and Pichia pastoris allowed a biochemical characterization of the enzymatic activity associated with the secreted gene product INV1p. Expression analysis of the known vacuolar and cell-wall-bound invertase isoforms of V. faba indicated a decrease in the expression of a vacuolar invertase, whereas one cell-wall-associated invertase exhibited increased expression. These changes were not confined to the infected tissue, and effects also were observed in remote plant organs, such as roots. These findings hint at systemic effects of pathogen infection. Our results support the hypothesis that pathogen infection establishes new sinks which compete with physiological sink organs.

Functional Expression and Characterization of the Wild-Type Mammalian Renal Cortex Sodium/Phosphate Cotransporter and an215R Mutant inSaccharomyces cerevisiae†

Biochemistry ◽  
1999 ◽  
Vol 38 (41) ◽  
pp. 13551-13559 ◽  
Author(s):  
Frank Bernhardt ◽  
Wilhelm Schoner ◽  
Bernd Schroeder ◽  
Gerhard Breves ◽  
Georgios Scheiner-Bobis
Keyword(s):  
Sodium Phosphate ◽  
Renal Cortex ◽  
Functional Expression ◽  
Wild Type

scholarly journals Characterization of a novel NADP+-dependent D-arabitol dehydrogenase from the plant pathogen Uromyces fabae

2005 ◽  
Vol 389 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Tobias Link ◽  
Gertrud Lohaus ◽  
Ingrid Heiser ◽  
Kurt Mendgen ◽  
Matthias Hahn ◽  
...  
Keyword(s):  
Plant Pathogen ◽  
Broad Bean ◽  
Rust Fungus ◽  
Plant Defence ◽  
In Vitro System ◽  
Uromyces Fabae ◽  
Factor Specificity ◽  
Defence Reactions

We have identified and characterized a novel NADP+-dependent D-arabitol dehydrogenase and the corresponding gene from the rust fungus Uromyces fabae, a biotrophic plant pathogen on broad bean (Vicia faba). The new enzyme was termed ARD1p (D-arabitol dehydrogenase 1). It recognizes D-arabitol and mannitol as substrates in the forward reaction, and D-xylulose, D-ribulose and D-fructose as substrates in the reverse reaction. Co-factor specificity was restricted to NADP(H). Kinetic data for the major substrates and co-factors are presented. A detailed analysis of the organization and expression pattern of the ARD1 gene are also given. Immunocytological data indicate a localization of the gene product predominantly in haustoria, the feeding structures of these fungi. Analyses of metabolite levels during pathogenesis indicate that the D-arabitol concentration rises dramatically as infection progresses, and D-arabitol was shown in an in vitro system to be capable of quenching reactive oxygen species involved in host plant defence reactions. ARD1p may therefore play an important role in carbohydrate metabolism and in establishing and/or maintaining the biotrophic interaction in U. fabae.

scholarly journals Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins

1999 ◽  
Vol 19 (11) ◽  
pp. 7870-7885 ◽  
Author(s):  
Nieves Movilla ◽  
Xosé R. Bustelo
Keyword(s):  
Tyrosine Kinases ◽  
Protein Tyrosine Kinases ◽  
Nucleotide Exchange ◽  
Loss Of Function ◽  
Wild Type ◽  
Gtp Binding ◽  
Regulatory Properties ◽  
Identification And Characterization

ABSTRACT We report here the identification and characterization of a novel Vav family member, Vav-3. Signaling experiments demonstrate that Vav-3 participates in pathways activated by protein tyrosine kinases. Vav-3 promotes the exchange of nucleotides on RhoA, on RhoG and, to a lesser extent, on Rac-1. During this reaction, Vav-3 binds physically to the nucleotide-free states of those GTPases. These functions are stimulated by tyrosine phosphorylation in wild-type Vav-3 and become constitutively activated upon deletion of the entire calponin-homology region. Expression of truncated versions of Vav-3 leads to drastic actin relocalization and to the induction of stress fibers, lamellipodia, and membrane ruffles. Moreover, expression of Vav-3 alters cytokinesis, resulting in the formation of binucleated cells. All of these responses need only the expression of the central region of Vav-3 encompassing the Dbl homology (DH), pleckstrin homology (PH), and zinc finger (ZF) domains but do not require the presence of the C-terminal SH3-SH2-SH3 regions. Studies conducted with Vav-3 proteins containing loss-of-function mutations in the DH, PH, and ZF regions indicate that only the DH and ZF regions are essential for Vav-3 biological activity. Finally, we show that one of the functions of the Vav-3 ZF region is to work coordinately with the catalytic DH region to promote both the binding to GTP-hydrolases and their GDP-GTP nucleotide exchange. These results highlight the role of Vav-3 in signaling and cytoskeletal pathways and identify a novel functional cross-talk between the DH and ZF domains of Vav proteins that is imperative for the binding to, and activation of, Rho GTP-binding proteins.

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