scholarly journals Structural and Functional Complexity of the Genomic Region Controlling AK-Toxin Biosynthesis and Pathogenicity in the Japanese Pear Pathotype of Alternaria alternata

2000 ◽  
Vol 13 (9) ◽  
pp. 975-986 ◽  
Author(s):  
Aiko Tanaka ◽  
Takashi Tsuge
Keyword(s):  
Blot Analysis ◽  
Alternaria Alternata ◽  
Black Spot ◽  
Genomic Region ◽  
Cosmid Clone ◽  
Japanese Pear ◽  
Wild Type ◽  
Single Chromosome ◽  
In The Wild ◽  
Functional Complexity

The Japanese pear pathotype of Alternaria alternata produces host-specific AK-toxin and causes black spot of Japanese pear. Previously, a cosmid clone, pcAKT-1, was isolated that contains two genes, AKT1 and AKT2, within a 5.0-kb region required for AK-toxin biosynthesis. The wild-type strain has multiple, nonfunctional copies of these genes. In the present study, two additional genes, AKTR-1 and AKT3-1, downstream of AKT2 were identified. Transformation of the wild type with AKTR-1- and AKT3-1-targeting vectors produced toxin-deficient (Tox¯), nonpathogenic mutants. DNA gel blot analysis, however, demonstrated that the fragments targeted in Tox¯ mutants were different from those containing AKTR-1 and AKT3-1 on the transforming vectors. A cosmid clone, pcAKT-2, containing the targeted DNA was isolated and shown to carry two genes, AKTR-2 and AKT3-2, with high similarity to AKTR-1 and AKT3-1, respectively. Transcripts from not only AKTR-2 and AKT3-2 but also AKTR-1 and AKT3-1 were found in the wild type. DNA gel blot analysis with pulsed-field gel electrophoresis showed that AKT1, AKT2, AKT3, and AKTR and their homologues are on a single chromosome. These results indicate the structural and functional complexity of the genomic region controlling AK-toxin biosynthesis.

Circular DNA Plasmid in the Phytopathogenic Fungus Alternaria alternata: Its Temperature-Dependent Curing and Association with Pathogenicity

Genetics ◽  
1997 ◽  
Vol 146 (1) ◽  
pp. 111-120
Author(s):  
Satoshi Katsuya ◽  
Isao Kaneko ◽  
Makiko Owaki ◽  
Kouichi Ishikawa ◽  
Takashi Tsujimoto ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Alternaria Alternata ◽  
Black Spot ◽  
Electron Microscopic Observation ◽  
Toxin Production ◽  
Japanese Pear ◽  
Phytopathogenic Fungus ◽  
Temperature Dependent ◽  
Electron Microscopic ◽  
Endonuclease Mapping

We found the presence of plasmid DNA in strain T88-56 of the Japanese pear pathotype of Alternaria alternata, which causes black spot of certain cultivars of Japanese pear by producing host-specific AK-toxin. The plasmid, designated pAAT56, was identified to be an ∼5.4-kilobase (kb) circular molecule by electron microscopic observation and restriction endonuclease mapping. Southern blot analysis showed that pAAT56 DNA had no homology with either nuclear or mitochondrial DNA. Cultures of strain T88-56 grown at 26° showed markedly reduced plasmid levels relative to those grown at lower temperatures. The strain was completely cured of pAAT56 during growth at 29°. Temperature-dependent curing of pAAT56 was confirmed by using single-protoplast isolates from mycelia grown at 23°, most of which maintained the plasmid, and from mycelia grown at 29°, most of which had lost the plasmid. Northern blot analysis detected the presence of three RNA species (∼1.7, 2.7 and 5.4 kb) transcribed from pAAT56. The biological function of pAAT56 was observed using single-protoplast isolates from mycelia that either contained or had been cured of pAAT56. The plasmid-containing isolates tended to be reduced in AK-toxin production and pathogenicity compared with the plasmid-cured isolates.

scholarly journals Insertional Mutagenesis and Cloning of the Genes Required for Biosynthesis of the Host-Specific AK-Toxin in the Japanese Pear Pathotype of Alternaria alternata

1999 ◽  
Vol 12 (8) ◽  
pp. 691-702 ◽  
Author(s):  
Aiko Tanaka ◽  
Hiroshi Shiotani ◽  
Mikihiro Yamamoto ◽  
Takashi Tsuge
Keyword(s):  
Restriction Enzyme ◽  
Alternaria Alternata ◽  
Type Strain ◽  
Insertional Mutagenesis ◽  
Wild Type Strain ◽  
Black Spot ◽  
Toxin Production ◽  
Japanese Pear ◽  
Wild Type ◽  
Functional Analyses

The Japanese pear pathotype of Alternaria alternata causes black spot of Japanese pear by producing a host-specific toxin known as AK-toxin. Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for toxin biosynthesis. Protoplasts of a wild-type strain were treated with a linearized plasmid along with the restriction enzyme used to linearize the plasmid. Of 984 REMI transformants recovered, three produced no detectable AK-toxin and lost pathogenicity on pear leaves. Genomic DNA flanking the integrated plasmid was recovered from one of the mutants. With the recovered DNA used as a probe, a cosmid clone of the wild-type strain was isolated. Structural and functional analyses of an 8.0-kb region corresponding to the tagged site indicated the presence of two genes. One, designated AKT1, encodes a member of the class of carboxyl-activating enzymes. The other, AKT2, encodes a protein of unknown function. The essential roles of these two genes in both AK-toxin production and pathogenicity were confirmed by transformation-mediated gene disruption experiments. DNA gel blot analysis detected AKT1 and AKT2 homologues not only in the Japanese pear pathotype strains but also in strains from the tangerine and strawberry pathotypes. The host-specific toxins of these two pathotypes are similar in structure to AK-toxin. Homologues were not detected in other pathotypes or in non-pathogenic strains of A. alternata, suggesting acquisition of AKT1 and AKT2 by horizontal transfer.

scholarly journals Identification and molecular characterization of a Chlamydomonas reinhardtii mutant that shows a light intensity dependent progressive chlorophyll deficiency

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 142
Author(s):  
Phillip B Grovenstein ◽  
Darryel A Wilson ◽  
Kathryn D Lankford ◽  
Kelsey A Gaston ◽  
Surangi Perera ◽  
...  
Keyword(s):  
Light Intensity ◽  
Insertional Mutagenesis ◽  
Protoporphyrin Ix ◽  
Model Organism ◽  
Genomic Region ◽  
Oxygenic Photosynthesis ◽  
Autotrophic Growth ◽  
Wild Type ◽  
Light Intensities ◽  
In The Wild

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study all aspects of oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in energy metabolism in photosynthetic organisms. These tetrapyrroles are synthesized via a common branched pathway that involves mainly nuclear encoded enzymes. One of the enzymes in the pathway is Mg chelatase (MgChel) which inserts Mg2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

Functional overlap of the dictyostelium RasG, RasD and RasB proteins

2000 ◽  
Vol 113 (8) ◽  
pp. 1427-1434 ◽  
Author(s):  
M. Khosla ◽  
G.B. Spiegelman ◽  
R. Insall ◽  
G. Weeks
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ras Proteins ◽  
Wild Type ◽  
Vegetative Cells ◽  
Protein Levels ◽  
Ras Genes ◽  
Downstream Effector ◽  
In The Wild

Disruption of the rasG gene in Dictyostelium discoideum results in several distinct phenotypes: a defect in cytokinesis, reduced motility and reduced growth. Reintroduction of the rasG gene restores all of the properties of the rasG(-) cells to those of the wild type. To determine whether the defects are due to impaired interactions with a single or multiple downstream effectors, we tested the ability of the highly related but non identical Dictyostelium ras genes, rasD and rasB, to rescue the defects. Introduction of the rasD gene under the control of the rasG promoter into rasG null (rasG(-)) cells corrected all phenotypes except the motility defect, suggesting that motility is regulated by a RasG mediated pathway that is different to those regulating growth or cytokinesis. Western blot analysis of RasD protein levels revealed that vegetative rasG(-)cells contained considerably more protein than the parental AX-3 cells, suggesting that RasD protein levels are negatively regulated in vegetative cells by RasG. The level of RasD was enhanced when the rasD gene was introduced under the control of the rasG promoter, and this increase in protein is presumably responsible for the reversal of the growth and cytokinesis defects of the rasG(-)cells. Thus, RasD protein levels are controlled by the level of RasG, but not by the level of RasD. Introduction of the rasB gene under the control of the rasG promoter into rasG(-) cells produced a complex phenotype. The transformants were extremely small and mononucleate and exhibited enhanced motility. However, the growth of these cells was considerably slower than the growth of the rasG(-) cells, suggesting the possibility that high levels of RasB inhibit an essential process. This was confirmed by expressing rasB in wild-type cells; the resulting transformants exhibited severely impaired growth. When RasB protein levels were determined by western blot analysis, it was found that levels were higher in the rasG(-)cells than they were in the wild-type parental, suggesting that RasG also negatively regulates rasB expression in vegetative cells. Overexpression of rasB in the rasG(-)cells also reduced the level of RasD protein. In view of the fact that alternate Ras proteins correct some, but not all, of the defects exhibited by the rasG(-) cells, we propose that RasG interacts with more than one downstream effector. In addition, it is clear that the levels of the various Ras proteins are tightly regulated in vegetative cells and that overexpression can be deleterious.

scholarly journals The mechanisms of fructose 1,6 bisphosphatase degradation in methylotrophic yeasts Pichia pastoris

2018 ◽  
Vol 22 ◽  
pp. 235-239
Author(s):  
O. V. Dmytruk ◽  
N. V. Bulbotka ◽  
A. A. Sibirny
Keyword(s):  
Pichia Pastoris ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Specific Activity ◽  
Wild Type ◽  
Methylotrophic Yeasts ◽  
Methanol Induction ◽  
Fructose 1,6 Bisphosphatase ◽  
In The Wild

Aim. The study of the mechanisms of fructose-1,6-bisphosphatase degradation in methylotrophic yeasts Pichia pastoris. Methods. Methods of determination the specific activity of fructose-1,6-bisphosphatase in the wild type and mutant strains of methylotrophic yeast P. pastoris after shifting cells from the medium with methanol into the medium with glucose were used. The study of fructose-1,6-bisphosphatase protein degradetion was performed by Western blot analysis. Results. The changes of the specific activity of fructose-1,6-bisphosphatase in the wild type strain GS200, the strain with the deletion of the GSS1 hexose sensor gene and strain defected in autophagy pathway SMD1163 of P. pastoris in short-term and long-term induction with methanol, and with or without the addition of the MG132 (proteasome degradation inhibitor) was investigated. Degradation of fructose‑1,6‑bisphosphatase by the Western blot analysis in GS200, SMD1163 and Δgss1 strains was studied. Conclusions. It was shown that the duration of cell incubation on methanol has no particular effect on the inactivation of the enzyme. The effect of the proteasome inhibitor MG132 was insignificant. Catabolic inactivation of cytosolic and peroxisomal enzymes is damaged in the Δgss1 mutant as glucose signaling is impaired. Fructose-1,6-bisphosphatase degrades by a vacuolar pathway, regardless of the duration of methanol induction, which correlates with the activity data of this enzyme. Keywords: fructose-1,6-bisphosphatase, yeasts, Pichia pastoris, methanol, autophagy.

scholarly journals Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail

2021 ◽  
Author(s):  
Satoshi Ishishita ◽  
Shumpei Kitahara ◽  
Mayuko Takahashi ◽  
Sakura Iwasaki ◽  
Shoji Tatsumoto ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Japanese Quail ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Genomic Region ◽  
Pigment Production ◽  
Wild Type ◽  
Its Gene ◽  
Uterine Mucosa ◽  
In The Wild

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uterus of birds, such as Japanese quail, Coturnix japonica, resulting in brown-speckled eggshells. The molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. We showed that 11 genes, including the 5-aminolevulinic acid synthase 1 (ALAS1) and ferroxidase hephaestin-like 1 (HEPHL1) genes, were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, and their expression was downregulated in these cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail.

scholarly journals Identification and molecular characterization of the second Chlamydomonas gun4 mutant, gun4-II

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 142
Author(s):  
Phillip B Grovenstein ◽  
Darryel A Wilson ◽  
Kathryn D Lankford ◽  
Kelsey A Gaston ◽  
Surangi Perera ◽  
...  
Keyword(s):  
Light Intensity ◽  
Insertional Mutagenesis ◽  
Protoporphyrin Ix ◽  
Model Organism ◽  
Genomic Region ◽  
Oxygenic Photosynthesis ◽  
Autotrophic Growth ◽  
Wild Type ◽  
Light Intensities ◽  
In The Wild

The green micro-alga Chlamydomonas reinhardtii is an elegant model organism to study oxygenic photosynthesis. Chlorophyll (Chl) and heme are major tetrapyrroles that play an essential role in photosynthesis and respiration. These tetrapyrroles are synthesized via a common branched pathway that involves mainly enzymes, encoded by nuclear genes. One of the enzymes in the pathway is Mg chelatase (MgChel). MgChel catalyzes insertion of Mg2+ into protoporphyrin IX (PPIX, proto) to form Magnesium-protoporphyrin IX (MgPPIX, Mgproto), the first biosynthetic intermediate in the Chl branch. The GUN4 (genomes uncoupled 4) protein is not essential for the MgChel activity but has been shown to significantly stimulate its activity. We have isolated a light sensitive mutant, 6F14, by random DNA insertional mutagenesis. 6F14 cannot tolerate light intensities higher than 90-100 μmol photons m-2 s-1. It shows a light intensity dependent progressive photo-bleaching. 6F14 is incapable of photo-autotrophic growth under light intensity higher than 100 μmol photons m-2 s-1. PCR based analyses show that in 6F14 the insertion of the plasmid outside the GUN4 locus has resulted in a genetic rearrangement of the GUN4 gene and possible deletions in the genomic region flanking the GUN4 gene. Our gun4 mutant has a Chl content very similar to that in the wild type in the dark and is very sensitive to fluctuations in the light intensity in the environment unlike the earlier identified Chlamydomonas gun4 mutant. Complementation with a functional copy of the GUN4 gene restored light tolerance, Chl biosynthesis and photo-autotrophic growth under high light intensities in 6F14. 6F14 is the second gun4 mutant to be identified in C. reinhardtii. Additionally, we show that our two gun4 complements over-express the GUN4 protein and show a higher Chl content per cell compared to that in the wild type strain.

scholarly journals Targeted Disruption of a Melanin Biosynthesis Gene Affects Conidial Development and UV Tolerance in the Japanese Pear Pathotype of Alternaria alternata

1999 ◽  
Vol 12 (1) ◽  
pp. 59-63 ◽  
Author(s):  
Chie Kawamura ◽  
Takashi Tsujimoto ◽  
Takashi Tsuge
Keyword(s):  
Structural Analysis ◽  
Alternaria Alternata ◽  
Uv Light ◽  
Japanese Pear ◽  
Wild Type ◽  
Targeted Disruption ◽  
Melanin Biosynthesis ◽  
Uv Tolerance ◽  
Biosynthesis Gene ◽  
Conidial Development

Structural analysis of the BRM2 gene involved in melanin biosynthesis of the Japanese pear pathotype of Alternaria alternata suggested that this gene encodes 1,3,8-trihydroxynaphthalene reductase. Targeted disruption of the BRM2 gene did not affect pathogenicity, vegetative growth, or the number of conidia produced. Targeted disruption, however, did reduce conidial size and septal number, suggesting that melanin is associated with conidial development. The conidia of brm2 mutant transformants were more sensitive to UV light than those of the wild type, demonstrating that melanin confers UV tolerance.

Molecular analysis of high CO2 requiring mutants: involvement of genes in the region of rbc, including rbcS, in the ability of cyanobacteria to grow under low CO2

1991 ◽  
Vol 69 (5) ◽  
pp. 945-950 ◽  
Author(s):  
Judy Lieman-Hurwitz ◽  
Rakefet Schwarz ◽  
Flor Martinez ◽  
Zeev Maor ◽  
Leonora Reinhold ◽  
...  
Keyword(s):  
Small Subunit ◽  
Kanamycin Resistance ◽  
Genomic Region ◽  
Open Reading Frames ◽  
Chemical Mutagenesis ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Low Co2 ◽  
In The Wild ◽  
Type Mutant

Modifications of the genomic region near (and including) rbc, the operon that codes for the large and small subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), resulted in cyanobacterial mutants that demand high CO2 for growth. Mutant EK6 originated from the fusion of the 3' end of rbcS (which codes for the small subunit of Rubisco) with 84 nucleotides from the 5′ flanking region of nptII (kanamycin-resistance gene), leading to a 17-kDa small subunit, as compared to 14 kDa in the wild type. Mutant D4 originated from substitution of the 1.4-kb PstI fragment, downstream of rbc, with nptII, inactivating several open reading frames in this region. Mutant O105 was obtained by chemical mutagenesis and the mutation was mapped approximately 9 kb upstream of rbc. Mutants EK6 and O105 exhibited a very low apparent photosynthetic affinity for inorganic carbon, whereas D4 had an affinity similar to that observed in wild-type cells grown under high CO2. These mutants, and the constructs used to raise them, can be used to study the role of the small subunit of Rubisco and the genomic region near rbc in cyanobacterial photosynthesis. We propose that this region contains a cluster of genes involved in the ability of cyanobacteria to grow under low ambient CO2..

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