ACTTS3 Encoding a Polyketide Synthase Is Essential for the Biosynthesis of ACT-Toxin and Pathogenicity in the Tangerine Pathotype of Alternaria alternata

The tangerine pathotype of Alternaria alternata produces host-selective ACT-toxin and causes Alternaria brown spot disease of tangerine and tangerine hybrids. Sequence analysis of a genomic BAC clone identified part of the ACT-toxin TOX (ACTT) gene cluster, and knockout experiments have implicated several open reading frames (ORF) contained within the cluster in the biosynthesis of ACT-toxin. One of the ORF, designated ACTTS3, encoding a putative polyketide synthase, was isolated by rapid amplification of cDNA ends and genomic/reverse transcription-polymerase chain reactions using the specific primers designed from the BAC sequences. The 7,374-bp ORF encodes a polyketide synthase with putative β-ketoacyl synthase, acyltransferase, methyltransferase, β-ketoacyl reductase, and phosphopantetheine attachment site domains. Genomic Southern blots demonstrated that ACTTS3 is present on the smallest chromosome in the tangerine pathotype of A. alternata, and the presence of ACTTS3 is highly correlated with ACT-toxin production and pathogenicity. Targeted gene disruption of two copies of ACTTS3 led to a complete loss of ACT-toxin production and pathogenicity. These results indicate that ACTTS3 is an essential gene for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata and is required for pathogenicity of this fungus.

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

Phytopathology ◽

10.1094/phyto-100-2-0120 ◽

2010 ◽

Vol 100 (2) ◽

pp. 120-126 ◽

Cited By ~ 33

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.

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Role of the Pathotype-Specific ACRTS1 Gene Encoding a Hydroxylase Involved in the Biosynthesis of Host-Selective ACR-Toxin in the Rough Lemon Pathotype of Alternaria alternata

Phytopathology ◽

10.1094/phyto-02-12-0021-r ◽

2012 ◽

Vol 102 (8) ◽

pp. 741-748 ◽

Cited By ~ 8

Author(s):

Yuriko Izumi ◽

Eri Kamei ◽

Yoko Miyamoto ◽

Kouhei Ohtani ◽

Akira Masunaka ◽

...

Keyword(s):

Alternaria Alternata ◽

Essential Gene ◽

Toxin Production ◽

Open Reading Frames ◽

Leaf Spot Disease ◽

Bac Clone ◽

Biosynthesis Gene Cluster ◽

Rough Lemon ◽

Reading Frames

The rough lemon pathotype of Alternaria alternata produces host-selective ACR-toxin and causes Alternaria leaf spot disease of the rootstock species rough lemon (Citrus jambhiri) and Rangpur lime (C. limonia). Genes controlling toxin production were localized to a 1.5-Mb chromosome carrying the ACR-toxin biosynthesis gene cluster (ACRT) in the genome of the rough lemon pathotype. A genomic BAC clone containing a portion of the ACRT cluster was sequenced which allowed identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. We studied the functional role of one of these open reading frames, ACRTS1 encoding a putative hydroxylase, in ACR-toxin production by homologous recombination-mediated gene disruption. There are at least three copies of ACRTS1 gene in the genome and disruption of two copies of this gene significantly reduced ACR-toxin production as well as pathogenicity; however, transcription of ACRTS1 and production of ACR-toxin were not completely eliminated due to remaining functional copies of the gene. RNA-silencing was used to knock down the remaining ACRTS1 transcripts to levels undetectable by reverse transcription-polymerase chain reaction. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic. These results indicate that ACRTS1 is an essential gene in ACR-toxin biosynthesis in the rough lemon pathotype of A. alternata and is required for full virulence of this fungus.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-12-0155-r ◽

2012 ◽

Vol 25 (11) ◽

pp. 1419-1429 ◽

Cited By ~ 18

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.

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

Phytopathology ◽

10.1094/phyto.2000.90.7.762 ◽

2000 ◽

Vol 90 (7) ◽

pp. 762-768 ◽

Cited By ~ 63

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.

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

Phytopathology ◽

10.1094/phyto-99-4-0369 ◽

2009 ◽

Vol 99 (4) ◽

pp. 369-377 ◽

Cited By ~ 27

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.

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Distribution of QoI Resistance in Populations of Tangerine-Infecting Alternaria alternata in Florida

Plant Disease ◽

10.1094/pdis-04-13-0449-re ◽

2014 ◽

Vol 98 (1) ◽

pp. 67-76 ◽

Cited By ~ 15

Author(s):

Byron Vega ◽

Megan M. Dewdney

Keyword(s):

Alternaria Alternata ◽

Brown Spot ◽

Cross Resistance ◽

Qoi Fungicides ◽

Quinone Outside Inhibitor ◽

Citrus Orchards ◽

Application Frequency ◽

Polymerase Chain ◽

Highly Correlated ◽

Qoi Resistance

Chemical control, based on copper and quinone outside inhibitor (QoI) fungicides, has been essential for the management of brown spot of citrus, caused by Alternaria alternata. However, QoI control failures were detected recently in Florida. From 2008 to 2012, 817 monoconidial isolates of A. alternata from 46 citrus orchards were examined for sensitivity to azoxystrobin (AZ) and pyraclostrobin (PYR). Of the isolates, 57.6% were resistant to both fungicides, with effective concentration to inhibit 50% growth (EC50) values greater than 5 μg/ml for AZ and 1 μg/ml for PYR. The mean EC50 values for sensitive isolates were 0.139 and 0.020 μg/ml for AZ and PYR, respectively. The EC50 values of both fungicides were highly correlated (P < 0.0001), indicating cross resistance. The proportion of resistant isolates differed significantly (P < 0.0001) among cultivars and with QoI application frequency (P < 0.0001). However, resistance was not significantly related (P = 0.364) to disease severity in the field (low, moderate, and high) or isolate virulence (P = 0.397). The molecular basis for QoI resistance was determined for a subset of 235 isolates using polymerase chain reaction restriction fragment length polymorphism of the cytochrome b gene. All resistant isolates showed the point mutation G143A. Based on the presence of one or two introns, isolates were classified as profile I and profile II, respectively. The resistance frequency was significantly higher (P < 0.0001) in isolate profile II, suggesting a higher selection pressure for resistant population profile II.

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Proper Functions of Peroxisomes Are Vital for Pathogenesis of Citrus Brown Spot Disease Caused by Alternaria alternata

Journal of Fungi ◽

10.3390/jof6040248 ◽

2020 ◽

Vol 6 (4) ◽

pp. 248

Author(s):

Pei-Ching Wu ◽

Chia-Wen Chen ◽

Celine Yen Ling Choo ◽

Yu-Kun Chen ◽

Jonar I. Yago ◽

...

Keyword(s):

Alternaria Alternata ◽

Molecular Mechanisms ◽

Protein Import ◽

Toxin Production ◽

Brown Spot ◽

Wild Type ◽

Targeted Deletion ◽

Brown Spot Disease ◽

Citrus Leaves ◽

Proper Functions

In addition to the production of a host-selective toxin, the tangerine pathotype of Alternaria alternata must conquer toxic reactive oxygen species (ROS) in order to colonize host plants. The roles of a peroxin 6-coding gene (pex6) implicated in protein import into peroxisomes was functionally characterized to gain a better understanding of molecular mechanisms in ROS resistance and fungal pathogenicity. The peroxisome is a vital organelle involved in metabolisms of fatty acids and hydrogen peroxide in eukaryotes. Targeted deletion of pex6 had no impacts on the biogenesis of peroxisomes and cellular resistance to ROS. The pex6 deficient mutant (Δpex6) reduced toxin production by 40% compared to wild type and barely induce necrotic lesions on citrus leaves. Co-inoculation of purified toxin with Δpex6 conidia on citrus leaves, however, failed to fully restore lesion formation, indicating that toxin only partially contributed to the loss of Δpex6 pathogenicity. Δpex6 conidia germinated poorly and formed fewer appressorium-like structures (nonmelanized enlargement of hyphal tips) than wild type. Δpex6 hyphae grew slowly and failed to penetrate beyond the epidermal layers. Moreover, Δpex6 had thinner cell walls and lower viability. All of these defects resulting from deletion of pex6 could also account for the loss of Δpex6 pathogenicity. Overall, our results have demonstrated that proper peroxisome functions are of vital importance to pathogenesis of the tangerine pathotype of A. alternata.

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Functional Analysis of a Multicopy Host-Selective ACT-Toxin Biosynthesis Gene in the Tangerine Pathotype of Alternaria alternata Using RNA Silencing

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-12-1591 ◽

2008 ◽

Vol 21 (12) ◽

pp. 1591-1599 ◽

Cited By ~ 44

Author(s):

Y. Miyamoto ◽

A. Masunaka ◽

T. Tsuge ◽

M. Yamamoto ◽

K. Ohtani ◽

...

Keyword(s):

Alternaria Alternata ◽

Rna Silencing ◽

Toxin Production ◽

Brown Spot ◽

Plasmid Construct ◽

Dispensable Chromosome ◽

Alternaria Brown Spot ◽

Multiple Copies ◽

Commercially Important ◽

Serious Disease

Alternaria brown spot, caused by the tangerine pathotype of Alternaria alternata, is a serious disease of commercially important tangerines and their hybrids. The pathogen produces host-selective ACT toxin, and several genes (named ACTT) responsible for ACT-toxin biosynthesis have been identified. These genes have many paralogs, which are clustered on a small, conditionally dispensable chromosome, making it difficult to disrupt entire functional copies of ACTT genes using homologous recombination-mediated gene disruption. To overcome this problem, we attempted to use RNA silencing, which has never been employed in Alternaria spp., to knock down the functional copies of one ACTT gene with a single silencing event. ACTT2, which encodes a putative hydrolase and is present in multiple copies in the genome, was silenced by transforming the fungus with a plasmid construct expressing hairpin ACTT2 RNAs. The ACTT2 RNA-silenced transformant (S-7-24-2) completely lost ACTT2 transcripts and ACT-toxin production as well as pathogenicity. These results indicated that RNA silencing may be a useful technique for studying the role of ACTT genes responsible for host-selective toxin biosynthesis in A. alternata. Further, this technique may be broadly applicable to the analysis of many genes present in multiple copies in fungal genomes that are difficult to analyze using recombination-mediated knockdowns.

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Draft Genome Sequence of Saccharomonospora sp. Strain LRS4.154, a Moderately Halophilic Actinobacterium with the Biotechnologically Relevant Polyketide Synthase and Nonribosomal Peptide Synthetase Systems

Genome Announcements ◽

10.1128/genomea.00392-17 ◽

2017 ◽

Vol 5 (21) ◽

Author(s):

Scarlett Alonso-Carmona ◽

Blanca Vera-Gargallo ◽

Rafael R. de la Haba ◽

Antonio Ventosa ◽

Horacio Sandoval-Trujillo ◽

...

Keyword(s):

Genome Sequence ◽

Polyketide Synthase ◽

Draft Genome ◽

Nonribosomal Peptide Synthetase ◽

Open Reading Frames ◽

Draft Genome Sequence ◽

Nonribosomal Peptide ◽

Moderately Halophilic ◽

Peptide Synthetase ◽

Reading Frames

ABSTRACT The draft genome sequence of Saccharomonospora sp. strain LRS4.154, a moderately halophilic actinobacterium, has been determined. The genome has 4,860,108 bp, a G+C content of 71.0%, and 4,525 open reading frames (ORFs). The clusters of PKS and NRPS genes, responsible for the biosynthesis of a large number of biomolecules, were identified in the genome.

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