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Robert H Proctor - One of the best experts on this subject based on the ideXlab platform.
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transformation mediated complementation of a fum gene cluster deletion in fusarium verticillioides restores both Fumonisin production and pathogenicity on maize seedlings
Molecular Plant-microbe Interactions, 2008Co-Authors: Anthony E Glenn, Ronald T. Riley, Nicholas C Zitomer, Anne Marie Zimeri, Lonnie D Williams, Robert H ProctorAbstract:The filamentous ascomycete Fusarium verticillioides is a pathogen of maize and produces the Fumonisin mycotoxins. However, a distinct population of F. verticillioides is pathogenic on banana and does not produce Fumonisins. Fumonisin-producing strains from maize cause leaf lesions, developmental abnormalities, stunting, and sometimes death of maize seedlings, whereas Fumonisin-nonproducing banana strains do not. A Southern analysis of banana strains did not detect genes in the Fumonisin biosynthetic gene (FUM) cluster but did detect genes flanking the cluster. Nucleotide sequence analysis of the genomic region carrying the flanking genes revealed that the FUM cluster was absent in banana strains except for portions of FUM21 and FUM19, which are the terminal genes at each end of the cluster. Polymerase chain reaction analysis confirmed the absence of the cluster in all banana strains examined. Cotransformation of a banana strain with two overlapping cosmids, which together contain the entire FUM cluster, yielded Fumonisin-producing transformants that were pathogenic on maize seedlings. Conversely, maize strains that possess the FUM cluster but do not produce Fumonisins because of mutations in FUM1, a polyketide synthase gene, were not pathogenic on maize seedlings. Together, the data indicate that Fumonisin production may have been lost by deletion of the FUM cluster in the banana population of F. verticillioides but that Fumonisin production could be restored by molecular genetic complementation. The results also indicate that Fumonisin production by F. verticillioides is required for development of foliar disease symptoms on maize seedlings.
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the fusarium verticillioides fum gene cluster encodes a zn ii 2cys6 protein that affects fum gene expression and Fumonisin production
Eukaryotic Cell, 2007Co-Authors: Daren W Brown, Mark Busman, Robert A E Butchko, Robert H ProctorAbstract:The discovery of two notorious fungal toxins, Fumonisins produced by Fusarium and aflatoxins produced by Aspergillus, was precipitated by disease and death that resulted from unintended consumption of contaminated maize or maize products. The surge in research that followed revealed that both families of mycotoxins share a polyketide biosynthetic origin, followed by unique and long biochemical pathways. A major long-term goal of mycotoxin research is to understand the biosynthetic mechanisms in order to develop strategies that reduce the presence of mycotoxins in crops. The maize pathogen Fusarium verticillioides (teleomorph Gibberella moniliformis) can infect maize throughout the world and can cause disease of ears, stalks, and seedlings (29). On some occasions, this fungus can synthesize the Fumonisin family of mycotoxins, which, after ingestion, have been associated with a number of animal diseases, including cancer (15), and have been epidemiologically associated with human esophageal cancer in some regions of the world (23). Of recent concern are findings that Fumonisins can disrupt neural tube development in rodents and, based on human epidemiological surveys, may increase the risks of human neural tube defects (24). Fumonisins are structurally similar to the sphingolipid sphinganine and disrupt sphingolipid metabolism by inhibiting the enzyme ceramide synthase (37 and references therein). Sphingolipids play a critical role in cell membranes and a variety of cell signaling pathways. Thus, disruption of sphingolipid metabolism may account for the multiple diseases associated with Fumonisins. Over the past decade, there have been significant advances in the understanding of the genetics and biochemistry of Fumonisin biosynthesis (4, 9, 10, 13, 43). In general, Fumonisins consist of a 19- or 20-carbon backbone with an amine, one to four hydroxyl, two methyl and two tricarboxylic acid constituents. Eighteen of the carbons that make up the Fumonisin backbone are assembled by a polyketide synthase, an enzyme class required for the biosynthesis of numerous toxins, antibiotics, and other biologically active compounds produced by fungi. The linear polyketide precursor of Fumonisins undergoes up to nine oxygenation, esterification, reduction, and dehydration reactions to form mature, biologically active Fumonisins (9). All Fumonisin biosynthetic (FUM) genes characterized to date are localized within a 42.5-kb region of the F. verticillioides genome (32). The clustering of genes involved in the biosynthesis of secondary metabolites in filamentous ascomycetes is common. For example, biosynthetic gene clusters have also been identified for aflatoxin/sterigmatocystin (8) and lovastatin (17) in Aspergillus and trichothecenes (6) and for gibberellins (41) in Fusarium. In most cases, transcriptional regulation is governed by a pathway-specific DNA-binding protein encoded by a gene located within the cluster (16). Despite significant effort, no gene encoding a Fumonisin specific regulator was found within the FUM cluster nor in the 11 to 22 kb of DNA flanking the cluster (32). Four F. verticillioides genes—FCK1, FCC1, PAC1, and ZFR1—that are not located in the Fumonisin gene cluster can affect Fumonisin biosynthesis (2, 12, 13, 36). FCK1, encoding a C-type cyclin-dependent kinase, and FCC1, encoding a cyclin-like protein, interact and are part of a signal transduction pathway that affects both Fumonisin biosynthesis and morphogenesis (36). PAC1 encodes a transcriptional activator or repressor of pH responsive genes and acts to repress transcription of FUM genes under alkaline conditions (12). ZFR1 encodes a Zn(II)2Cys6 DNA-binding protein and markedly affects Fumonisin production (13). The failure of constitutively expressed ZFR1 in a Δfcc1 mutant to restore Fumonisin biosynthesis suggests that the FCC1 protein, Fcc1p, may activate Zfr1p (13). In collaboration with The Institute of Genomics Research (TIGR), we previously characterized over 87,000 expressed sequence tags (ESTs) from F. verticillioides (5). The ESTs represent as many as 11,000 different genes that may correspond to up to 81% of the genes in the F. verticillioides genome. In this report, we identified a previously undescribed gene from analysis of the ESTs. The new gene is located adjacent to FUM1, has eight introns, and is predicted to encode a protein with two motifs found in fungal DNA transcription factors. Gene disruption and complementation analyses indicate that the gene is a positive activator of FUM gene transcription and plays a critical role in Fumonisin biosynthesis. Microarray analysis of alternative splice form (ASF) transcripts of the gene suggests that some are differentially expressed, a result consistent with the hypothesis that ASFs play a role in Fumonisin biosynthesis (5).
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the fusarium verticillioides fum gene cluster encodes a zn ii 2cys6 protein that affects fum gene expression and Fumonisin production
Eukaryotic Cell, 2007Co-Authors: Daren W Brown, Mark Busman, Robert A E Butchko, Robert H ProctorAbstract:Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of Fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The Fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the Fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Deltafum21) mutants produce little to no Fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 mutant with a wild-type copy of the gene restored Fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate Fumonisin biosynthesis.
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Fumonisin production in the maize pathogen fusarium verticillioides genetic basis of naturally occurring chemical variation
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Robert H Proctor, Ronald D Plattner, Anne E. Desjardins, Mark Busman, Robert A E ButchkoAbstract:Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in Fumonisin C-10 hydroxylation or that do not produce any Fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the Fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the Fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered Fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.
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determining the biosynthetic sequence in the early steps of the Fumonisin pathway by use of three gene disruption mutants of fusarium verticillioides
Journal of Agricultural and Food Chemistry, 2004Co-Authors: Ravi S Bojja, Ronald L Cerny, Robert H ProctorAbstract:Fumonisins are polyketide-derived mycotoxins produced by Fusarium verticillioides, a fungal pathogen of corn plants. Although a gene cluster for the biosynthesis of Fumonisins has been cloned, the biosynthetic pathway is still not clear. We have used three gene-disrupted mutants, designated ΔFUM1, ΔFUM6, and ΔFUM8, to study the early steps of the pathway. Fumonisins were not produced in single-strain cultures of the ΔFUM1, ΔFUM6, and ΔFUM8 mutants. However, Fumonisins were produced by ΔFUM1 or ΔFUM8 mutants when they were cocultured with the ΔFUM6 mutant. No Fumonisins were produced when the ΔFUM1 and ΔFUM8 mutants were cocultured. These results suggest that the ΔFUM6 mutant produces a Fumonisin intermediate that can be further metabolized by Fumonisin biosynthetic enzymes in the ΔFUM1 and ΔFUM8 mutants. To isolate the potential intermediates produced by ΔFUM6, we followed a time course of cocultures of the ΔFUM1 and ΔFUM6 and the ΔFUM8 and ΔFUM6 mutants. Liquid chromatographic−mass spectrometric data sug...
Hans-ulrich Humpf - One of the best experts on this subject based on the ideXlab platform.
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identification of n acyl Fumonisin b1 as new cytotoxic metabolites of Fumonisin mycotoxins
Molecular Nutrition & Food Research, 2013Co-Authors: Henning Harrer, Hans-ulrich Humpf, Elad L Laviad, Anthony H FutermanAbstract:Scope Fumonisins are mycotoxins produced by Fusarium species. The predominant derivative, Fumonisin B1 (FB1), occurs in food and feed and is of health concern due to its hepatotoxic and carcinogenic effects. However, the role of FB1 metabolites on the mechanism of the toxicity, the inhibition of the ceramide synthesis, is unknown. The aim of this study was to identify new Fumonisin metabolites and to evaluate their cytotoxic potential. Methods and results MS, molecular biology, and in vitro enzyme assays were used to investigate Fumonisin metabolism in mammalian cells overexpressing human ceramide synthase (CerS) genes. N-acyl-FB1 derivatives were detected as new metabolites in cultured cells at levels of up to 10 pmol/mg of protein. The N-acylation of FB1 and hydrolyzed FB1 was analyzed in several cell lines, including cells overexpressing CerS. The acyl-chain length of the N-acyl Fumonisins depends on the CerS isoform acylating them. The N-acyl Fumonisins are more cytotoxic than the parent Fumonisin B1. Conclusion The identification of N-acyl Fumonisins with various acyl chain lengths together with the observed cytotoxicity of these compounds is a new aspect of Fumonisin-related toxicity. Therefore, these new metabolites might play an important role in the mode of action of Fumonisins.
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Large scale purification of B-type Fumonisins using centrifugal partition chromatography (CPC)
Mycotoxin Research, 2012Co-Authors: Florian Hübner, Henning Harrer, Andrea Fraske, Susanne Kneifel, Hans-ulrich HumpfAbstract:For toxicological studies of B-type Fumonisin in animals, high amounts of pure Fumonisins are needed. In the past, several methods for the isolation and purification of Fumonisins have been published, stating the problem of high losses of Fumonisins during chromatography on solid phases. In this manuscript we describe a new approach based on liquid-liquid partition techniques using centrifugal partition chromatography in combination with ion exchange chromatography for the large-scale isolation of B-type Fumonisins with good recovery rates, minimizing losses of Fumonisins during the purification. A batch of cultures grown on solid media of 2 kg maize yields approximately 1 g of pure Fumonisins with a purity of >98%.
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effects of thermal food processing on the chemical structure and toxicity of Fumonisin mycotoxins
Molecular Nutrition & Food Research, 2004Co-Authors: Hans-ulrich Humpf, Kenneth A. VossAbstract:Fumonisins are Fusarium mycotoxins that occur in corn and corn-based foods. They are toxic to animals and at least one analogue, Fumonisin B1, is carcinogenic to rodents. Their effect on human health is unclear, however, Fumonisins are considered to be risk factors for cancer and possibly neural tube defects in some heavily exposed populations. It is therefore important to minimize exposures in these populations. Cleaning corn to remove damaged or moldy kernels reduces Fumonisins in foods while milling increases their concentration in some and reduces their concentration in other products. Fumonisins are water-soluble and nixtamalization (cooking in alkaline water) lowers the Fumonisin content of food products if the cooking liquid is discarded. Baking, frying, and extrusion cooking of corn at high temperatures ( > or = 190 degrees C) also reduces Fumonisin concentrations in foods, with the amount of reduction achieved depending on cooking time, temperature, recipe, and other factors. However, the chemical fate of Fumonisins in baked, fried, and extruded foods is not well understood and it is not known if the reduced concentrations result from thermal decomposition of Fumonisins or from their binding to proteins, sugars or other compounds in food matrices. These possibilities might or might not be beneficial depending upon the bioavailability and inherent toxicity of decomposition products or the degree to which bound Fumonisins are released in the gastrointestinal tract. In this review the affects of cooking and processing on the concentration and chemical structure of Fumonisins as well as the toxicological consequences of known and likely Fumonisin reaction products are discussed.
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Analysis of Fumonisin B(1) in Fusarium proliferatum-infected asparagus spears and garlic bulbs from Germany by liquid chromatography-electrospray ionization mass spectrometry.
Journal of Agricultural and Food Chemistry, 2002Co-Authors: W Seefelder, Monika Gossmann, Hans-ulrich HumpfAbstract:Fusarium proliferatum is one of a group of fungal species that produce Fumonisins and is considered to be a pathogen of many economically important plants. The occurrence of Fumonisin B(1) (FB(1)) in F. proliferatum-infected asparagus spears from Germany was investigated using a liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method with isotopically labeled Fumonisin FB(1)-d(6) as internal standard. FB(1) was detected in 9 of the 10 samples in amounts ranging from 36.4 to 4513.7 ng/g (based on dry weight). Furthermore, the capability of producing FB(1) by the fungus in garlic bulbs was investigated. Therefore, garlic was cultured in F. proliferatum-contaminated soil, and the bulbs were screened for infection with F. proliferatum and for the occurrence of Fumonisins by LC-MS. F. proliferatum was detectable in the garlic tissue, and all samples contained FB(1) (26.0-94.6 ng/g). This is the first report of the natural occurrence of FB(1) in German asparagus spears, and these findings suggest a potential for natural contamination of garlic bulbs with Fumonisins.
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toxicity assessment of Fumonisins using the brine shrimp artemia salina bioassay
Food and Chemical Toxicology, 2000Co-Authors: Michaela Hartl, Hans-ulrich HumpfAbstract:Abstract The Fusarium mycotoxins Fumonisin B 1 (FB 1 ) (1) and B 2 (FB 2 ) (2), their hydrolysed analogues HFB 1 (3) and HFB 2 (4) and the recently discovered Fumonisin derivatives N -palmitoyl-HFB 1 (5) and N -carboxymethyl-FB 1 (6) were compared for their toxicity in a short term bioassay using brine shrimp ( Artemia salina ). The brine shrimp were hatched in artificial sea water and exposed to the Fumonisins in microwell plates with a mortality endpoint after 48 hours. LC 50 values were calculated after Probit transformation of the resulting data. Of the substances tested, Fumonisin B 1 emerged to be the most toxic whereas its N -carboxymethyl analogue was 100-fold less effective. The hydrolysed Fumonisins showed a four- to sixfold reduced toxicity compared to FB 1 . N -Palmitoyl-HFB 1 had a higher LC 50 value than its precursor HFB 1 . The brine shrimp assay proved to be a convenient and rapid system for toxicity assessment of this group of mycotoxins.
Ronald D Plattner - One of the best experts on this subject based on the ideXlab platform.
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Fumonisin production in the maize pathogen fusarium verticillioides genetic basis of naturally occurring chemical variation
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Robert H Proctor, Ronald D Plattner, Anne E. Desjardins, Mark Busman, Robert A E ButchkoAbstract:Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in Fumonisin C-10 hydroxylation or that do not produce any Fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the Fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the Fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered Fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.
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Discontinuous distribution of Fumonisin biosynthetic genes in the Gibberella fujikuroi species complex.
Mycological research, 2004Co-Authors: Robert H Proctor, Ronald D Plattner, Daren W Brown, Jeong-ah Seo, Yin-won LeeAbstract:Production of the carcinogenic mycotoxins Fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for Fumonisin production and the presence of Fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, Fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, Fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce Fumonisins.
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co expression of 15 contiguous genes delineates a Fumonisin biosynthetic gene cluster in gibberella moniliformis
Fungal Genetics and Biology, 2003Co-Authors: Robert H Proctor, Ronald D Plattner, Daren W Brown, Anne E. DesjardinsAbstract:Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified Fumonisin biosynthetic (FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with Fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for Fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a Fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on Fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of Fumonisins produced.
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FUM1--a gene required for Fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests.
Molecular plant-microbe interactions : MPMI, 2002Co-Authors: Anne E. Desjardins, Ronald D Plattner, Gary P. Munkvold, Robert H ProctorAbstract:We have analyzed the role of Fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for Fumonisin biosynthesis. Maize ear rot, ear infection, and Fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and Fumonisin production. Two independent Fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective Fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that Fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.
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Fumonisin b1 nonproducing strains of fusarium verticillioides cause maize zea mays ear infection and ear rot
Journal of Agricultural and Food Chemistry, 2000Co-Authors: Anne E. Desjardins, Ronald D PlattnerAbstract:Fumonisins are polyketide mycotoxins produced by Fusarium verticillioides (synonym F. moniliforme), a major pathogen of maize (Zea mays) worldwide. Most field strains produce high levels of Fumonisin B(1) (FB(1)) and low levels of the less-oxygenated homologues FB(2) and FB(3), but Fumonisin B(1)-nonproducing field strains have been obtained by natural variation. To test the role of various Fumonisins in pathogenesis on maize under field conditions, one strain producing FB(1), FB(2), and FB(3), one strain producing only FB(2), one strain producing only FB(3), and one Fumonisin-nonproducing strain were applied to ears via the silk channel and on seeds at planting. Disease severity on the harvested ears was evaluated by visible symptoms and by weight percent symptomatic kernels. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of the applied FB(1)-nonproducing strains in kernels was determined by analysis of recovered strains for Fumonisin production and other traits. All three FB(1)-nonproducing strains were able to infect ears following either silk-channel application or seed application at planting and were as effective as the FB(1)-producing strain in causing ear rot following silk-channel application. These results indicate that production of FB(1), FB(2), or FB(3) is not required for F. verticillioides to cause maize ear infection and ear rot.
Anne E. Desjardins - One of the best experts on this subject based on the ideXlab platform.
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Fumonisin production in the maize pathogen fusarium verticillioides genetic basis of naturally occurring chemical variation
Journal of Agricultural and Food Chemistry, 2006Co-Authors: Robert H Proctor, Ronald D Plattner, Anne E. Desjardins, Mark Busman, Robert A E ButchkoAbstract:Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in Fumonisin C-10 hydroxylation or that do not produce any Fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the Fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the Fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered Fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.
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co expression of 15 contiguous genes delineates a Fumonisin biosynthetic gene cluster in gibberella moniliformis
Fungal Genetics and Biology, 2003Co-Authors: Robert H Proctor, Ronald D Plattner, Daren W Brown, Anne E. DesjardinsAbstract:Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified Fumonisin biosynthetic (FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with Fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for Fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a Fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on Fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of Fumonisins produced.
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FUM1--a gene required for Fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests.
Molecular plant-microbe interactions : MPMI, 2002Co-Authors: Anne E. Desjardins, Ronald D Plattner, Gary P. Munkvold, Robert H ProctorAbstract:We have analyzed the role of Fumonisins in infection of maize (Zea mays) by Gibberella moniliformis (anamorph Fusarium verticillioides) in field tests in Illinois and Iowa, United States. Fumonisin-nonproducing mutants were obtained by disrupting FUM1 (previously FUM5), the gene encoding a polyketide synthase required for Fumonisin biosynthesis. Maize ear rot, ear infection, and Fumonisin contamination were assessed by silk-channel injection in 1999 and 2000 and also by spray application onto maize silks, injection into maize stalks, and application with maize seeds at planting in 1999. Ear rot was evaluated by visual assessment of whole ears and by calculating percentage of symptomatic kernels by weight. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of applied strains in kernels was determined by analysis of recovered isolates for genetic markers and Fumonisin production. Two independent Fumonisin-nonproducing (fum1-3 and fum1-4) mutants were similar to their respective Fumonisin-producing (FUM1-1) progenitor strains in ability to cause ear rot following silk-channel injection and also were similar in ability to infect maize ears following application by all four methods tested. This evidence confirms that Fumonisins are not required for G. moniliformis to cause maize ear rot and ear infection.
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Fumonisin b1 nonproducing strains of fusarium verticillioides cause maize zea mays ear infection and ear rot
Journal of Agricultural and Food Chemistry, 2000Co-Authors: Anne E. Desjardins, Ronald D PlattnerAbstract:Fumonisins are polyketide mycotoxins produced by Fusarium verticillioides (synonym F. moniliforme), a major pathogen of maize (Zea mays) worldwide. Most field strains produce high levels of Fumonisin B(1) (FB(1)) and low levels of the less-oxygenated homologues FB(2) and FB(3), but Fumonisin B(1)-nonproducing field strains have been obtained by natural variation. To test the role of various Fumonisins in pathogenesis on maize under field conditions, one strain producing FB(1), FB(2), and FB(3), one strain producing only FB(2), one strain producing only FB(3), and one Fumonisin-nonproducing strain were applied to ears via the silk channel and on seeds at planting. Disease severity on the harvested ears was evaluated by visible symptoms and by weight percent symptomatic kernels. Fumonisin levels in kernels were determined by high-performance liquid chromatography. The presence of the applied FB(1)-nonproducing strains in kernels was determined by analysis of recovered strains for Fumonisin production and other traits. All three FB(1)-nonproducing strains were able to infect ears following either silk-channel application or seed application at planting and were as effective as the FB(1)-producing strain in causing ear rot following silk-channel application. These results indicate that production of FB(1), FB(2), or FB(3) is not required for F. verticillioides to cause maize ear infection and ear rot.
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biosynthetic and genetic relationships of b series Fumonisins produced by gibberella fujikuroi mating population a
Natural Toxins, 1999Co-Authors: Robert H Proctor, Anne E. Desjardins, Ronald D PlattnerAbstract:Fumonisins are mycotoxins produced by the maize pathogen Gibberella fujikuroi mating population A and frequently contaminate maize. Wild-type G. fujikuroi produces four B-series Fumonisins, FB1, FB2, FB3 and FB4. These toxins are identical in structure except for the number and positions of hydroxyls along their linear carbon backbone. To elucidate the genetic and biosynthetic relationships among these Fumonisins, we conducted meiotic and biochemical analyses of G. fujikuroi mutants with altered Fumonisin production that resulted from defective alleles at three loci, Fum1, Fum2 and Fum3. These mutants produced either no Fumonisins, only FB2 and FB4, or only FB3 and FB4. Genetic analyses revealed the orientation of the Fum loci along linkage group 1 of the fungus. The mutants were grown together in pair-wise combinations to determine if their Fumonisin production phenotypes could be complemented. When FB3- and FB2-producing mutants were grown together, complementation occurred. However, when a nonproducing mutant was grown with a FB2- or FB3-producing mutant, complementation did not occur or was incomplete. When purified FB2, FB3, or FB4 was fed to mutant cultures, FB4 was converted primarily to FB2, FB3 was converted to FB1 and FB2 was not converted. The results from these assays suggest a previously unrecognized branch in the Fumonisin biosynthetic pathway. Published in 1999 by John Wiley & Sons, Ltd.
Daren W Brown - One of the best experts on this subject based on the ideXlab platform.
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lae1 regulates expression of multiple secondary metabolite gene clusters in fusarium verticillioides
Fungal Genetics and Biology, 2012Co-Authors: Robert A E Butchko, Daren W Brown, Mark Busman, Bettina Tudzynski, Philipp WiemannAbstract:The filamentous fungus Fusarium verticillioides can cause disease of maize and is capable of producing Fumonisins, a family of toxic secondary metabolites linked to esophageal cancer and neural tube defects in humans and lung edema in swine and leukoencephalomalacia in equines. The expression of Fumonisin biosynthetic genes is influenced by broad-domain transcription factors (global regulators) and Fum21, a pathway-specific transcription factor. LaeA is a global regulator that in Aspergillus nidulans, affects the expression of multiple secondary metabolite gene clusters by modifying heterochromatin structure. Here, we employed gene deletion analysis to assess the effect of loss of a F. verticillioides laeA orthologue, LAE1, on genome-wide gene expression and secondary metabolite production. Loss of Lae1 resulted in reduced expression of gene clusters responsible for synthesis of the secondary metabolites bikaverin, Fumonisins, fusaric acid and fusarins as well as two clusters for which the corresponding secondary metabolite is unknown. Analysis of secondary metabolites revealed that, in contrast to a previously described Fusarium fujikuroi lae1 mutant, bikaverin production is reduced. Fumonisin production is unchanged in the F. verticillioides lae1 mutant. Complementation of the F. verticillioides mutant resulted in increased Fumonisin production.
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the fusarium verticillioides fum gene cluster encodes a zn ii 2cys6 protein that affects fum gene expression and Fumonisin production
Eukaryotic Cell, 2007Co-Authors: Daren W Brown, Mark Busman, Robert A E Butchko, Robert H ProctorAbstract:Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of Fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The Fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the Fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Deltafum21) mutants produce little to no Fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 mutant with a wild-type copy of the gene restored Fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate Fumonisin biosynthesis.
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the fusarium verticillioides fum gene cluster encodes a zn ii 2cys6 protein that affects fum gene expression and Fumonisin production
Eukaryotic Cell, 2007Co-Authors: Daren W Brown, Mark Busman, Robert A E Butchko, Robert H ProctorAbstract:The discovery of two notorious fungal toxins, Fumonisins produced by Fusarium and aflatoxins produced by Aspergillus, was precipitated by disease and death that resulted from unintended consumption of contaminated maize or maize products. The surge in research that followed revealed that both families of mycotoxins share a polyketide biosynthetic origin, followed by unique and long biochemical pathways. A major long-term goal of mycotoxin research is to understand the biosynthetic mechanisms in order to develop strategies that reduce the presence of mycotoxins in crops. The maize pathogen Fusarium verticillioides (teleomorph Gibberella moniliformis) can infect maize throughout the world and can cause disease of ears, stalks, and seedlings (29). On some occasions, this fungus can synthesize the Fumonisin family of mycotoxins, which, after ingestion, have been associated with a number of animal diseases, including cancer (15), and have been epidemiologically associated with human esophageal cancer in some regions of the world (23). Of recent concern are findings that Fumonisins can disrupt neural tube development in rodents and, based on human epidemiological surveys, may increase the risks of human neural tube defects (24). Fumonisins are structurally similar to the sphingolipid sphinganine and disrupt sphingolipid metabolism by inhibiting the enzyme ceramide synthase (37 and references therein). Sphingolipids play a critical role in cell membranes and a variety of cell signaling pathways. Thus, disruption of sphingolipid metabolism may account for the multiple diseases associated with Fumonisins. Over the past decade, there have been significant advances in the understanding of the genetics and biochemistry of Fumonisin biosynthesis (4, 9, 10, 13, 43). In general, Fumonisins consist of a 19- or 20-carbon backbone with an amine, one to four hydroxyl, two methyl and two tricarboxylic acid constituents. Eighteen of the carbons that make up the Fumonisin backbone are assembled by a polyketide synthase, an enzyme class required for the biosynthesis of numerous toxins, antibiotics, and other biologically active compounds produced by fungi. The linear polyketide precursor of Fumonisins undergoes up to nine oxygenation, esterification, reduction, and dehydration reactions to form mature, biologically active Fumonisins (9). All Fumonisin biosynthetic (FUM) genes characterized to date are localized within a 42.5-kb region of the F. verticillioides genome (32). The clustering of genes involved in the biosynthesis of secondary metabolites in filamentous ascomycetes is common. For example, biosynthetic gene clusters have also been identified for aflatoxin/sterigmatocystin (8) and lovastatin (17) in Aspergillus and trichothecenes (6) and for gibberellins (41) in Fusarium. In most cases, transcriptional regulation is governed by a pathway-specific DNA-binding protein encoded by a gene located within the cluster (16). Despite significant effort, no gene encoding a Fumonisin specific regulator was found within the FUM cluster nor in the 11 to 22 kb of DNA flanking the cluster (32). Four F. verticillioides genes—FCK1, FCC1, PAC1, and ZFR1—that are not located in the Fumonisin gene cluster can affect Fumonisin biosynthesis (2, 12, 13, 36). FCK1, encoding a C-type cyclin-dependent kinase, and FCC1, encoding a cyclin-like protein, interact and are part of a signal transduction pathway that affects both Fumonisin biosynthesis and morphogenesis (36). PAC1 encodes a transcriptional activator or repressor of pH responsive genes and acts to repress transcription of FUM genes under alkaline conditions (12). ZFR1 encodes a Zn(II)2Cys6 DNA-binding protein and markedly affects Fumonisin production (13). The failure of constitutively expressed ZFR1 in a Δfcc1 mutant to restore Fumonisin biosynthesis suggests that the FCC1 protein, Fcc1p, may activate Zfr1p (13). In collaboration with The Institute of Genomics Research (TIGR), we previously characterized over 87,000 expressed sequence tags (ESTs) from F. verticillioides (5). The ESTs represent as many as 11,000 different genes that may correspond to up to 81% of the genes in the F. verticillioides genome. In this report, we identified a previously undescribed gene from analysis of the ESTs. The new gene is located adjacent to FUM1, has eight introns, and is predicted to encode a protein with two motifs found in fungal DNA transcription factors. Gene disruption and complementation analyses indicate that the gene is a positive activator of FUM gene transcription and plays a critical role in Fumonisin biosynthesis. Microarray analysis of alternative splice form (ASF) transcripts of the gene suggests that some are differentially expressed, a result consistent with the hypothesis that ASFs play a role in Fumonisin biosynthesis (5).
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Discontinuous distribution of Fumonisin biosynthetic genes in the Gibberella fujikuroi species complex.
Mycological research, 2004Co-Authors: Robert H Proctor, Ronald D Plattner, Daren W Brown, Jeong-ah Seo, Yin-won LeeAbstract:Production of the carcinogenic mycotoxins Fumonisins has been reported in several Fusarium species, most of which are members of the Gibberella fujikuroi (Gf) complex. In this study, we examined 15 Fusarium species in the Gf complex and 12 other species for Fumonisin production and the presence of Fumonisin biosynthetic genes (FUM). Among the species within the Gf complex, Fumonisin production was detected only in F. fujikuroi, F. globosum, F. proliferatum, F. nygamai, F. oxysporum and F. verticillioides. These five species include members of two of the three major clades delineated in the Gf complex. The FUM genes were detected in these same five species and in F. anthophilum, a member of the third clade. Among the species outside the Gf complex, Fumonisin production and FUM genes were detected only in F. oxysporum. Phylogenetic analyses of nucleotide sequences from two FUM gene fragments inferred relationships similar but not identical to those inferred from previous analyses of other genes. The results indicate the FUM genes are discontinuously distributed in the Gf complex and that this distribution gives rise to the differences in the abilities of closely related Fusarium species to produce Fumonisins.
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co expression of 15 contiguous genes delineates a Fumonisin biosynthetic gene cluster in gibberella moniliformis
Fungal Genetics and Biology, 2003Co-Authors: Robert H Proctor, Ronald D Plattner, Daren W Brown, Anne E. DesjardinsAbstract:Fumonisins are mycotoxins produced by the maize pathogen Gibberella moniliformis and are associated with cancer in rodents. In this study, we determined the nucleotide sequence of a 75-kb region of G. moniliformis DNA and identified 18 heretofore undescribed genes flanking a cluster of five previously identified Fumonisin biosynthetic (FUM) genes. Ten of the newly identified genes downstream of the cluster were coregulated with FUM genes and exhibited patterns of expression that were correlated with Fumonisin production. BLASTX analyses indicated that the predicted functions of proteins encoded by the 10 genes were consistent with activities expected for Fumonisin biosynthesis or self-protection. These data indicate that the 10 newly identified genes and the previously identified FUM genes constitute a Fumonisin biosynthetic gene cluster. Disruption of two of the new genes, encoding longevity assurance factors, had no apparent effect on Fumonisin production, but disruption of a third, encoding an ABC transporter, had a subtle effect on ratios of Fumonisins produced.
