Progress 11/15/00 to 04/30/05
Outputs
Zearalenone is a mycotoxin of worldwide economic and health importance. It is most commonly found as a contaminant in stored grain and has chronic estrogenic effects on mammals. Zearalenone is synthesized by several species of the filamentous fungus, Fusarium. Since the 1970s, the compound zearalenone has been known to be a polyketide, derived from the sequential addition of multiple acetate units by a polyketide synthase (PKS). However, the genetics of zearalenone biosynthesis has not been elucidated. Polyketides are a class of secondary metabolites exhibit a vast diversity of form and function. In fungi, with the recent advent of genomic era, it is possible to clone and characterize the entire suite of PKS genes within a genome, contributing to the overall analysis of the total polyketide potential of an organism. Although PKSs are composed of several highly conserved domains, the extensive diversity of the PK products arise from the number and kind of domains
present on the enzyme and the type of starter and extender units selected by the enzyme. In this study we identified all fifteen PKS genes within the genome of the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum), the causal agent of head blight of wheat. We genetically disrupted each of them to determine their function. Through this process, we succeeded in identifying five genes responsible for producing zearalenone, aurofusarin, fusarin C and the black perithecial pigment by analysis of disrupted mutants of all fifteen genes. A comprehensive expression study revealed several interesting expression patterns. This is the first study to genetically characterize a complete set of PKS genes from a single organism. With the cloning of the PKS genes, we identified two genes, designated ZEA1 and ZEA2 that encode the polyketide synthases responsible for zearalenone production in G. zeae. Disruption of either of these genes results in the loss of zearalenone production by
the resulting mutant. ZEA1 and ZEA2 are transcribed divergently from a common promoter region. Analysis of this region gives clues to the regulation of these genes. Examination of the region of the chromosome involved in zearalenone biosynthesis shows the presence of two possible regulatory proteins. Strikingly, no other genes typically associated with polyketide modification flank the two PKS genes. An oxidoreductase that is regulated similarly may be involved in modifications of zearalenone to form natural derivatives. A putative mechanism for the synthesis of zearalenone by the two PKSs has been determined. A cDNA library from corn kernels colonized with G. zeae mycelia was completed and the sequencing of several hundred clones has been accomplished. Analysis of these clones is still in progress. Finally, analysis of the gene expression during kernel colonization has been accomplished through the use of a newly released Affymetrix Fusarium Genechip. The analysis of that data is in
progress.
Impacts
Zearalenone is a mycotoxin of worldwide economic and health importance and has chronic estrogenic effects on mammals. In the process of identifying the genes for zearalenone biosynthesis, we identified 13 other polyketide synthase genes in G. zeae and have begun the process of functional characterization of these. PKS genes for two other mycotoxins were identified. The identification of the genes responsible for biosynthesis and regulation of these mycotoxins are an important step towards understanding how its accumulation in grain can be controlled.
Publications
- Characterization of two distinct polyketide synthase genes involved in zearalenone biosynthesis in Gibberella zeae. Iffa Gaffoor and Frances Trail. Submitted, 2005.
- Functional analysis of the polyketide synthase genes in the filamentous fungus Gibberella zeae (anamorph Fusarium graminearum). Iffa Gaffoor, Daren Brown, Ron Plattner, Robert Proctor, Weihong Qi, and Frances Trail. Submitted, 2005.
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Progress 01/01/04 to 12/31/04
Outputs
The major objective of this project was to clone the polyketide synthase gene for zearalenone biosynthesis using degenerate primers. We were able to clone 4 PKS genes from G. zeae using the method of degenerate primers. When these were disrupted, they were not responsible for zearalenone biosynthesis. Upon obtaining the genomics sequence last spring, we were able to identify 15 genes that appeared to have strong homology to other fungal PKS genes. We have disrupted all 15 and 2 additional genes which had some PKS-type moieties. All of these mutated transformants have been tested for zearalenone biosynthesis, and are being screened for sexual development, conidiation, deoxynivalenol production and pathogenicity. We have identified one gene which we are currently confirming as the zearalenone PKS. We have identified another pks which produce the pigments of G. zeae, giving the characteristic red color. We have identified this compound as aurofusarin. We have also
identified the pks encoding the black perithecial pigment, and disrupted a second gene in that cluster which will allow us to identify the structure of the pigment monomer. To begin to elucidate a possible role for this mycotoxin in the life cycle of G. zeae, wild type cultures have been supplemented with zearalenone to study the effects of exogenous zearalenone on perithecial development. Preliminary data indicates that cultures grown on defined medium supplemented with zearalenone levels higher than 0.01 ng/ml inhibit perithecial development. We will repeat these studies on the zearalenone minus mutant. To date about 500 random insertional mutants have been screened for loss of zearalenone production. This is in an effort to identify other genes involved in the regulation, biosynthesis and transport of zearalenone. The preliminary screen yielded eight mutants which did not produce detectable levels of zearalenone. These are being screened more rigorously to confirm loss of
zearalenone production. Once confirmed, the affected genes will be identified and characterized.
Impacts
Zearelnone is an important component of the environmental estrogen load encountered by consumers of corn and wheat products. We are developing an understanding of the conditions under which zearalenone is synthesized and how that biosynthsis is triggered in the fungus. Through this understanding, we will identify novel targets for control of this important food contaminant.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
Zearalenone is produced by the filamentous fungus, Gibberella zeae, the pathogen which causes the disease known as head blight on wheat and barley. Zearalenone is a mycotoxin which mimicks estrogen in its effects on mammals and is therefore a concern in foodstuffs. Little is known or understood about the synthesis of zearlenone in G. zeae. It is known to be classified among the chemicals known as polyketides. One of our goals is to isolate the gene for the polyketide synthase which is involved in synthesis of zearalenone. We have identified 15 putative polyketide synthase genes in the genomic sequence generated by us and our collaborators this year. Each of these has been individually disrupted within the G. zeae genome, and we are in the process of determining whether they encode the polyketide synthase involved in zearalonone biosynthesis. In addition, we have begun screened 1000 insertional mutants for absence of zearalenone. Eight of these show absence of
zearalenone production and the genes involved are being examined.
Impacts
Zearelnone is an important component of the environmental estrogen load encountered by consumers of corn and wheat products. We are developing an understanding of the conditions under which zearalenone is synthesized and how that biosynthsis is triggered in the fungus. Through this understanding, we will identify novel targets for control of this important food contaminant.
Publications
- 2003. Publication in preparation on two of the polyketide synthase genes.
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Progress 01/01/02 to 12/31/02
Outputs
Zearalenone is produced by the filamentous fungus, Gibberella zeae, the pathogen which causes the disease known as head blight on wheat and barley. Zearalenone is a mycotoxin which mimicks estrogen in its effects on mammals and is therefore a concern in foodstuffs. Little is known or understood about the synthesis of zearlenone in G. zeae. It is known to be classified among the chemicals known as polyketides. One of our goals is to isolate the gene for the polyketide synthase which is involved in synthesis of zearalenone. Towards this end, we have isolated seven putative polyketide synthase genes by use of degenerate primers. These have been individually disrupted within the G. zeae genome, and we are in the process of determining whether they encode the polyketide synthase involved in zearalonone biosynthesis. In addition, we have begun screening 5000 insertional mutants for absence of zearalenone using a novel yeast assay. We have developed the conditions of growth
for zearalenone biosynthesis in grain and will begin initiation of cDNA libraries in the coming weeks.
Impacts
Zearelnone is an important component of the environmental estrogen load encountered by consumers of corn and wheat products. We are developing an understanding of the conditions under which zearalenone is synthesized and how that biosynthsis is triggered in the fungus. Through this understanding, we will identify novel targets for control of this important food contaminant.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Zearalenone is a polyketide produced by the fungus Gibberella zeae. Zearalenone is considered to be a mycotoxin and of concern to food safety because of its estrogenic effects in mammals. It is hoped through understanding how and why the fungus produces this mycotoxin that novel methods can be identified for its control. Specific Aims: Objective 1. To identify the polyketide synthase gene involved in zearalenone production. Objective 2: To determine the role of zearalenone in the life cycle of G. zeae. Objective 3. To use microarray technology to identify genes involved in zearalenone production and grain colonization. Results: We encountered two unexpected problems in this work, both of which have been resolved this year. Initially, we thought we had observed reasonable levels of zearalenone production in culture and were able to detect these amounts readily. However, detecting the zearalenone in the culture medium that produced the highest levels was problematic
with the ELISA assay, due to precipitation of zearalenone at high pH values. We were able to resolve this problem by changing to a different fungal strain, different culture medium for production and a thin-layer chromatography assay for detection. These procedures have also yielded good cellular mRNA, important for the next steps in isolating the polyketide synthase (PKS) genes. However, it took us several months to sort through this. A total of 9 fragments (approximately 700 bp in length) have been amplified using PCR and 3 pairs of primers designed for isolating fungal PKS genes. These fragments are possibly the PKS genes that are responsible for zearalenone biosynthesis. We have cloned these fragments into pT7Blue3 from Novagen. We have tested 5 of these fragments for hybridization in northern blots of total RNA extracted both from cultures producing zearalenone and those not producing it. All five show hybridization to an approximately 7 kb band on a northern blot. This size is
an appropriate size for PKS genes. The hybridization occurred only to the total RNA from cultures producing zearalenone, but was absent in the total RNA from cultures that were not producing zearalenone. We are currently sequencing these PCR-generated clones, to see if they represent the same gene or several genes. We are testing the clones for expression at additional time-points. We anticipate just one polyketide synthase (PKS) being involved in synthesis of this toxin. We have a genomic library available and will be using this library to pull out full-length PKS genes from those genes that are expressed at the right time. We anticipate this work will begin next month. Once one or several putative PKS genes are identified, we will specifically mutate them one by one to determine which is responsible for zearalenone production. Due to the complications with zearalenone production and detection, we have not begun Objectives 2 and 3. We will be initiating those during the coming
year.
Impacts
This work should result in an understanding of how and when this fungus makes zearalenone, the role the mycotoxin has in the fungal life cycle, and novel means of controling contamination of foodstuffs by the mycotoxin.
Publications
- No publications reported this period
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