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 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.

scholarly journals Distribution and Characterization of AKT Homologs in the Tangerine Pathotype of Alternaria alternata

2000 ◽  
Vol 90 (7) ◽  
pp. 762-768 ◽  
Author(s):  
A. Masunaka ◽  
A. Tanaka ◽  
T. Tsuge ◽  
T. L. Peever ◽  
L. W. Timmer ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
Toxin Production ◽  
Brown Spot ◽  
Japanese Pear ◽  
Black Rot ◽  
Acid Moiety ◽  
High Sequence Similarity ◽  
Rough Lemon

The tangerine pathotype of Alternaria alternata produces a host-selective toxin (HST), known as ACT-toxin, and causes Alternaria brown spot disease of citrus. The structure of ACT-toxin is closely related to AK- and AF-toxins, which are HSTs produced by the Japanese pear and strawberry pathotypes of A. alternata, respectively. AC-, AK-, and AF-toxins are chemically similar and share a 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Two genes controlling AK-toxin biosynthesis (AKT1 and AKT2) were recently cloned from the Japanese pear pathotype of A. alternata. Portions of these genes were used as heterologous probes in Southern blots, that detected homologs in 13 isolates of A. alternata tangerine pathotype from Minneola tangelo in Florida. Partial sequencing of the homologs in one of these isolates demonstrated high sequence similarity to AKT1 (89.8%) and to AKT2 (90.7%). AKT homologs were not detected in nine isolates of A. alternata from rough lemon, six isolates of nonpathogenic A. alternata, and one isolate of A. citri that causes citrus black rot. The presence of homologs in the Minneola isolates and not in the rough lemon isolates, nonpathogens or black rot isolates, correlates perfectly to pathogenicity on Iyo tangerine and ACT-toxin production. Functionality of the homologs was demonstrated by detection of transcripts using reverse transcription-polymerase chain reaction (RT-PCR) in total RNA of the tangerine pathotype of A. alternata. The high sequence similarity of AKT and AKT homologs in the tangerine patho-type, combined with the structural similarity of AK-toxin and ACT-toxin, may indicate that these homologs are involved in the biosynthesis of the decatrienoic acid moiety of ACT-toxin.

scholarly journals Contribution of Peroxisomes to Secondary Metabolism and Pathogenicity in the Fungal Plant Pathogen Alternaria alternata

2010 ◽  
Vol 9 (5) ◽  
pp. 682-694 ◽  
Author(s):  
Ai Imazaki ◽  
Aiko Tanaka ◽  
Yoshiaki Harimoto ◽  
Mikihiro Yamamoto ◽  
Kazuya Akimitsu ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Alternaria Alternata ◽  
Marked Reduction ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Defense Responses ◽  
Japanese Pear ◽  
Acid Media ◽  
Content Type ◽  
Mutant Strains

ABSTRACT The filamentous fungus Alternaria alternata includes seven pathogenic variants (pathotypes) which produce different host-selective toxins and cause diseases on different plants. The Japanese pear pathotype produces the host-selective AK-toxin, an epoxy-decatrienoic acid ester, and causes black spot of Japanese pear. Previously, we identified four genes, AKT1, AKT2, AKT3, and AKTR, involved in AK toxin biosynthesis. AKT1, AKT2, and AKT3 encode enzyme proteins with peroxisomal targeting signal type 1 (PTS1)-like tripeptides, SKI, SKL, and PKL, respectively, at the C-terminal ends. In this study, we verified the peroxisome localization of Akt1, Akt2, and Akt3 by using strains expressing N-terminal green fluorescent protein (GFP)-tagged versions of the proteins. To assess the role of peroxisome function in AK-toxin production, we isolated AaPEX6, which encodes a peroxin protein essential for peroxisome biogenesis, from the Japanese pear pathotype and made AaPEX6 disruption-containing transformants from a GFP-Akt1-expressing strain. The ΔAaPEX6 mutant strains did not grow on fatty acid media because of a defect in fatty acid β oxidation. The import of GFP-Akt1 into peroxisomes was impaired in the ΔAaPEX6 mutant strains. These strains completely lost AK toxin production and pathogenicity on susceptible pear leaves. These data show that peroxisomes are essential for AK-toxin biosynthesis. The ΔAaPEX6 mutant strains showed a marked reduction in the ability to cause lesions on leaves of a resistant pear cultivar with defense responses compromised by heat shock. This result suggests that peroxisome function is also required for plant invasion and tissue colonization in A. alternata. We also observed that mutation of AaPEX6 caused a marked reduction of conidiation.

Electron Microscopic Observation of the Longitudinal Organization of Poly (p-Phenylene Terephthalamide) Fibers

1982 ◽  
Vol 40 ◽  
pp. 680-681
Author(s):  
Li Li-Sheng ◽  
L.F. Allard ◽  
W.C. Bigelow
Keyword(s):  
Electron Microscopy ◽  
Mechanical Properties ◽  
Scanning Electron Microscopy ◽  
Microscopic Observation ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Aromatic Polyamides ◽  
Radial Arrangement ◽  
Scanning Electron ◽  
Better Than

The aromatic polyamides form a class of fibers having mechanical properties which are much better than those of aliphatic polyamides. Currently, the accepted morphology of these fibers as proposed by M.G. Dobb, et al. is a radial arrangement of pleated sheets, with the plane of the pleats parallel to the axis of the fiber. We have recently obtained evidence which supports a different morphology of this type of fiber, using ultramicrotomy and ion-thinning techniques to prepare specimens for transmission and scanning electron microscopy.

Observation of Concanavalin-A receptors on hydrated planar erythrocyte membrane film by in situ electron microscopy

1983 ◽  
Vol 41 ◽  
pp. 608-609
Author(s):  
Neng-Bo He ◽  
S.W. Hui
Keyword(s):  
Lipid Bilayers ◽  
Erythrocyte Membrane ◽  
Lipid Membranes ◽  
Surface Film ◽  
Biological Membranes ◽  
Electron Microscopic Observation ◽  
Electron Microscopic ◽  
Black Lipid Membranes ◽  
Fine Mesh ◽  
Planar Films

Monolayers and planar "black" lipid membranes have been widely used as models for studying the structure and properties of biological membranes. Because of the lack of a suitable method to prepare these membranes for electron microscopic observation, their ultrastructure is so far not well understood. A method of forming molecular bilayers over the holes of fine mesh grids was developed by Hui et al. to study hydrated and unsupported lipid bilayers by electron diffraction, and to image phase separated domains by diffraction contrast. We now adapted the method of Pattus et al. of spreading biological membranes vesicles on the air-water interfaces to reconstitute biological membranes into unsupported planar films for electron microscopic study. hemoglobin-free human erythrocyte membrane stroma was prepared by hemolysis. The membranes were spreaded at 20°C on balanced salt solution in a Langmuir trough until a surface pressure of 20 dyne/cm was reached. The surface film was repeatedly washed by passing to adjacent troughs over shallow partitions (fig. 1).

The Scanning Electron Microscopic Observation of the Vestibular Sensory Epithelia

1969 ◽  
Vol 27 ◽  
pp. 40-41
Author(s):  
D.J. Lim ◽  
W.C. Lane
Keyword(s):  
Semicircular Canals ◽  
Electron Microscopic Observation ◽  
Gravitational Acceleration ◽  
Electron Microscopic ◽  
Sensory Epithelia ◽  
Scanning Electron Microscopic ◽  
Vestibular Sensory Epithelia ◽  
And Function ◽  
Sand Piles ◽  
Active Investigation

The morphology and function of the vestibular sensory organs has been extensively studied during the last decade with the advent of electron microscopy and electrophysiology. The opening of the space age also accelerated active investigation in this area, since this organ is responsible for the sensation of balance and of linear, angular and gravitational acceleration.The vestibular sense organs are formed by the saccule, utricle and three ampullae of the semicircular canals. The maculae (sacculi and utriculi) have otolithic membranes on the top of the sensory epithelia. The otolithic membrane is formed by a layer of thick gelatin and sand-piles of calcium carbonate crystals (Fig.l).

Plastic deformation of θ ' in Al-4%Cu alloy

1978 ◽  
Vol 36 (1) ◽  
pp. 576-577
Author(s):  
Shrikant P. Bhat
Keyword(s):  
Deformation Behavior ◽  
Room Temperature ◽  
Electron Microscopic Observation ◽  
Bulk Alloy ◽  
Electron Microscopic ◽  
Cu Alloy ◽  
Cu Alloys ◽  
Orowan Mechanism ◽  
The Subject ◽  
Cyclic Straining

deformation behavior of Al-Cu alloys aged to contain θ ' has been the subject of many investigations (e.g., Ref. 1-5). Since θ ' is strong and hard, dislocations bypass θ ' plates (Orowan mechanism) at low strains. However, at high strains the partially coherent θ ' plates are probably sheared, although the mechanism is complex, depending on the form of deformation. Particularly, the cyclic straining of the bulk alloy is known to produce gross bends and twists of θ '. However, no detailed investigation of the deformation of θ ' has yet been reported; moreover, Calabrese and Laird interpreted the deformation of θ ' as largely being elastic.During an investigation of high temperature cyclic deformation, the detailed electron-microscopic observation revealed that, under reversed straining conditions, θ ' particles are severely distorted--bent and twisted depending on the local matrix constraint. A typical electronmicrograph, showing the twist is shown in Fig. 1. In order to establish whether the deformation is elastic or plastic, a sample from a specimen cycled at room temperature was heated inside the microscope and the results are presented in a series of micrographs (Fig. 2a-e).

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