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 Function of Genes Encoding Acyl-CoA Synthetase and Enoyl-CoA Hydratase for Host-Selective ACT-Toxin Biosynthesis in the Tangerine Pathotype of Alternaria alternata

2009 ◽  
Vol 99 (4) ◽  
pp. 369-377 ◽  
Author(s):  
Y. Miyamoto ◽  
Y. Ishii ◽  
A. Honda ◽  
A. Masunaka ◽  
T. Tsuge ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Gene Disruption ◽  
Toxin Production ◽  
Brown Spot ◽  
Acid Moiety ◽  
Targeted Gene Disruption ◽  
Brown Spot Disease ◽  
Alternaria Brown Spot ◽  
Genes Encoding ◽  
Enoyl Coa Hydratase

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease. Sequence analysis of a genomic cosmid clone identified a part of the ACTT gene cluster and implicated two genes, ACTT5 encoding an acyl-CoA synthetase and ACTT6 encoding an enoyl-CoA hydratase, in the biosynthesis of ACT-toxin. Genomic Southern blots demonstrated that both genes were present in tangerine pathotype isolates producing ACT-toxin and also in Japanese pear pathotype isolates producing AK-toxin and strawberry pathotype isolates producing AF-toxin. ACT-, AK-, and AF-toxins from these three pathotypes share a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety. Targeted gene disruption of two copies of ACTT5 significantly reduced ACT-toxin production and virulence. Targeted gene disruption of two copies of ACTT6 led to complete loss of ACT-toxin production and pathogenicity and a putative decatrienoic acid intermediate in ACT-toxin biosynthesis accumulated in mycelial mats. These results indicate that ACTT5 and ACTT6 are essential genes in ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and both are required for full virulence of this fungus.

scholarly journals Citrus Black Rot is Caused by Phylogenetically Distinct Lineages of Alternaria alternata

2005 ◽  
Vol 95 (5) ◽  
pp. 512-518 ◽  
Author(s):  
T. L. Peever ◽  
L. Carpenter-Boggs ◽  
L. W. Timmer ◽  
L. M. Carris ◽  
A. Bhatia
Keyword(s):  
Phylogenetic Analysis ◽  
Alternaria Alternata ◽  
Sequence Data ◽  
Brown Spot ◽  
Ecological Niches ◽  
Black Rot ◽  
Data Set ◽  
Dna Sequence Data ◽  
Rough Lemon ◽  
Combined Data

Phylogenetic analysis revealed that isolates of Alternaria alternata causing black rot of citrus were associated with six well-supported evolutionary lineages. Isolates recovered from brown spot lesions on Minneola tangelo, leaf spot lesions on rough lemon, and healthy citrus tissue and noncitrus hosts were related closely to isolates from black-rotted fruit. Phylogenies estimated independently from DNA sequence data from an endopolygalacturonase gene (endoPG) and two anonymous regions of the genome (OPA1-3 and OPA2-1) had similar topologies, and phylogenetic analysis was performed on the combined data set. In the combined phylogeny, isolates from diverse ecological niches on citrus and noncitrus hosts were distributed in eight clades. Isolates from all lineages, regardless of ecological or host association, caused black rot in fruit inoculation assays, demonstrating that small-spored Alternaria isolates associated with different ecological niches on citrus and other plant hosts are potential black rot pathogens. These data also indicated that the fungi associated with black-rotted fruit do not form a natural evolutionary group distinct from other Alternaria pathogens and saprophytes associated with citrus. The use of the name A. citri to describe fungi associated with citrus black rot is not justified and it is proposed that citrus black rot fungi be referred to as A. alternata.

scholarly journals Limited Microcystin, Anatoxin and Cylindrospermopsin Production by Cyanobacteria from Microbial Mats in Cold Deserts

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 244 ◽  
Author(s):  
Nataliia Khomutovska ◽  
Małgorzata Sandzewicz ◽  
Łukasz Łach ◽  
Małgorzata Suska-Malawska ◽  
Monika Chmielewska ◽  
...  
Keyword(s):  
Microbial Mats ◽  
Sequence Similarity ◽  
Extreme Environment ◽  
Toxin Production ◽  
High Mountain ◽  
Rrna Gene ◽  
High Sequence Similarity ◽  
Small Water ◽  
Two Samples ◽  
Liquid Chromatography Tandem Mass

Toxic metabolites are produced by many cyanobacterial species. There are limited data on toxigenic benthic, mat-forming cyanobacteria, and information on toxic cyanobacteria from Central Asia is even more scarce. In the present study, we examined cyanobacterial diversity and community structure, the presence of genes involved in toxin production and the occurrence of cyanotoxins in cyanobacterial mats from small water bodies in a cold high-mountain desert of Eastern Pamir. Diversity was explored using amplicon-based sequencing targeting the V3-V4 region of the 16S rRNA gene, toxin potential using PCR-based methods (mcy, nda, ana, sxt), and toxins by enzyme-linked immunosorbent assays (ELISAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Molecular identification of cyanobacteria showed a high similarity of abundant taxa to Nostoc PCC-73102, Nostoc PCC-7524, Nodularia PCC-935 and Leptolyngbya CYN68. The PCRs revealed the presence of mcyE and/or ndaF genes in 11 samples and mcyD in six. The partial sequences of the mcyE gene showed high sequence similarity to Nostoc, Planktothrix and uncultured cyanobacteria. LC-MS/MS analysis identified six microcystin congeners in two samples and unknown peptides in one. These results suggest that, in this extreme environment, cyanobacteria do not commonly produce microcystins, anatoxins and cylindrospermopsins, despite the high diversity and widespread occurrence of potentially toxic taxa.

scholarly journals Role of the Host-Selective ACT-Toxin Synthesis Gene ACTTS2 Encoding an Enoyl-Reductase in Pathogenicity of the Tangerine Pathotype of Alternaria alternata

2010 ◽  
Vol 100 (2) ◽  
pp. 120-126 ◽  
Author(s):  
Naoya Ajiro ◽  
Yoko Miyamoto ◽  
Akira Masunaka ◽  
Takashi Tsuge ◽  
Mikihiro Yamamoto ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Essential Gene ◽  
Toxin Production ◽  
Brown Spot ◽  
Gene Encoding ◽  
Bac Clone ◽  
Biosynthesis Gene Cluster ◽  
Enoyl Reductase ◽  
Alternaria Brown Spot ◽  
Rapid Amplification

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerines and tangerine hybrids. Sequence analysis of a genomic BAC clone identified a previously uncharacterized portion of the ACT-toxin biosynthesis gene cluster (ACTT). A 1,034-bp gene encoding a putative enoyl-reductase was identified by using rapid amplification of cDNA ends and polymerase chain reaction and designated ACTTS2. Genomic Southern blots demonstrated that ACTTS2 is present only in ACT-toxin producers and is carried on a 1.9 Mb conditionally dispensable chromosome by the tangerine pathotype. Targeted gene disruption of ACTTS2 led to a reduction in ACT-toxin production and pathogenicity, and transcriptional knockdown of ACTTS2 using RNA silencing resulted in complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS2 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.

scholarly journals The crystal structure of mycobacterial epoxide hydrolase A

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Eike C. Schulz ◽  
Sara R. Henderson ◽  
Boris Illarionov ◽  
Thomas Crosskey ◽  
Stacey M. Southall ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Specificity ◽  
Drug Targets ◽  
Epoxide Hydrolase ◽  
Sequence Similarity ◽  
Structural Similarity ◽  
High Sequence Similarity ◽  
High Structural Similarity ◽  
Potential Drug Targets ◽  
Insight Into

Abstract The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH’s have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis.

scholarly journals A Polyketide Synthase Gene, ACRTS2, Is Responsible for Biosynthesis of Host-Selective ACR-Toxin in the Rough Lemon Pathotype of Alternaria alternata

2012 ◽  
Vol 25 (11) ◽  
pp. 1419-1429 ◽  
Author(s):  
Y. Izumi ◽  
K. Ohtani ◽  
Y. Miyamoto ◽  
A. Masunaka ◽  
T. Fukumoto ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Polyketide Synthase ◽  
Attachment Site ◽  
Toxin Production ◽  
Leaf Spot Disease ◽  
Predicted Amino Acid Sequence ◽  
Type I ◽  
Methyl Transferase ◽  
Reading Frame ◽  
Rough Lemon

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of rough lemon (Citrus jambhiri). The structure of ACR-toxin I (MW = 496) consists of a polyketide with an α-dihydropyrone ring in a 19-carbon polyalcohol. Genes responsible for toxin production were localized to a 1.5-Mb chromosome in the genome of the rough lemon pathotype. Sequence analysis of this chromosome revealed an 8,338-bp open reading frame, ACRTS2, that was present only in the genomes of ACR-toxin-producing isolates. ACRTS2 is predicted to encode a putative polyketide synthase of 2,513 amino acids and belongs to the fungal reducing type I polyketide synthases. Typical polyketide functional domains were identified in the predicted amino acid sequence, including β-ketoacyl synthase, acyl transferase, methyl transferase, dehydratase, β-ketoreductase, and phosphopantetheine attachment site domains. Combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs in ACR-toxin production. ACRTS2 was found to be essential for ACR-toxin production and pathogenicity of the rough lemon pathotype of A. alternata.

scholarly journals Endopolygalacturonase Is Essential for Citrus Black Rot Caused by Alternaria citri but Not Brown Spot Caused by Alternaria alternata

2001 ◽  
Vol 14 (6) ◽  
pp. 749-757 ◽  
Author(s):  
Atsunori Isshiki ◽  
Kazuya Akimitsu ◽  
Mikihiro Yamamoto ◽  
Hiroyuki Yamamoto
Keyword(s):  
Cell Wall ◽  
Alternaria Alternata ◽  
Biochemical Properties ◽  
Brown Spot ◽  
Black Rot ◽  
Citrus Peel ◽  
Degrading Enzyme ◽  
Cell Wall Degrading Enzyme ◽  
A Cell

Alternaria citri, the cause of Alternaria black rot, and Alternaria alternata rough lemon pathotype, the cause of Alternaria brown spot, are morphologically indistinguishable pathogens of citrus: one causes rot by macerating tissues and the other causes necrotic spots by producing a host-selective toxin. To evaluate the role of endopolygalacturonase (endoPG) in pathogenicity of these two Alternaria spp. pathogens, their genes for endoPG were mutated by gene targeting. The endoPGs produced by these fungi have similar biochemical properties, and the genes are highly similar (99.6% nucleotide identity). The phenotypes of the mutants, however, are completely different. An endoPG mutant of A. citri was significantly reduced in its ability to cause black rot symptoms on citrus as well as in the maceration of potato tissue and could not colonize citrus peel segments. In contrast, an endoPG mutant of A. alternata was unchanged in pathogenicity. The results indicate that a cell wall-degrading enzyme can play different roles in the pathogenicity of fungal pathogens. The role of a cell wall-degrading enzyme depends upon the type of disease but not the taxonomy of the fungus.

scholarly journals Characterization of Alternaria alternata causal agent of brown spot in Citrus spp

2017 ◽  
Vol 1 (2) ◽  
pp. 45
Author(s):  
Aline Vanessa Sauer ◽  
Hugo José Tozze Júnior ◽  
Marcel Bellato Spósito ◽  
Eduardo Feichtenberger Feichtenberger ◽  
Nelson Barros Colauto ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Genetic Similarity ◽  
Mycelial Growth ◽  
Potato Dextrose Agar ◽  
Brown Spot ◽  
Growth Speed ◽  
Phenological Stages ◽  
Rough Lemon ◽  
Citrus Spp

The purpose of this study was to characterize culturally, enzymatically and pathogenically Alternaria alternata isolates obtained of tangerine/tangor (TP) and rough lemon (RLP). Significant differences were observed regarding mycelial growth speed and sporulation of isolates when cultivated in starch-agar (SA), potato-dextrose-agar (PDA) and tomato juice agar (V8) media. SA and PDA media promoted better mycelial growth and sporulation, respectively. Eight genetic similarity groups were defined through isoenzymatic characterization but without correlation between isolates and host or site of origin. All isolates produced amylase, cellulase, polygalacturonase and pectynase; however, no lipolytic or proteolytic activity was observed. Disease incubation period varied between 24 to 48 h for all isolates in all phenological stages of the inoculated fruit. Symptomatic fruit incidence in most tangerine and tangor isolates was higher in stage F3 compared to stages F4 and F5. Alternaria alternata have the ability to perform hyphal anastomosis indicating that this can be a mechanism used by the fungus to increase genetic variability.

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.

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