The effect of elevated temperature on gene transcription and aflatoxin biosynthesis.

The molecular regulation of aflatoxin biosynthesis is complex and influenced by several environmental conditions; one of these is temperature. Aflatoxins are produced optimally at 28-30 C, and production decreases as temperatures approach 37 C, the optimum temperature for fungal growth. To better characterize the influence of temperature on aflatoxin biosynthesis, we monitored the accumulation of aflatoxin and the expression of more than 5000 genes in Aspergillus flavus at 28 C and 37 C. A total of 144 genes were expressed differentially (P < 0.001) between the two temperatures. Among the 103 genes more highly expressed at 28 C, approximately 25% were involved in secondary metabolism and about 30% were classified as hypothetical. Genes encoding a catalase and superoxide dismutase were among those more highly expressed at 37 C. As anticipated we also found that all the aflatoxin biosynthetic genes were much more highly expressed at 28 C relative to 37 C. To our surprise expression of the pathway regulatory genes aflR and aflS, as well as aflR antisense, did not differ between the two temperatures. These data indicate that the failure of A. flavus to produce aflatoxin at 37 C is not due to lack of transcription of aflR or aflS. One explanation is that AFLR is nonfunctional at high temperatures. Regardless, the factor(s) sensing the elevated temperatures must be acute. When aflatoxin-producing cultures are transferred to 37 C they immediately stop producing aflatoxin.

Whole genome comparison of Aspergillus flavus and A. oryzae.

Aspergillus flavus is a plant and animal pathogen that also produces the potent carcinogen aflatoxin. Aspergillus oryzae is a closely related species that has been used for centuries in the food fermentation industry and is Generally Regarded As Safe (GRAS). Whole genome sequences for these two fungi are now complete, providing us with the opportunity to examine any genomic differences that may explain the different ecological niches of these two fungi, and perhaps to identify pathogenicity factors in A. flavus. These two fungi are very similar in genome size and number of predicted genes. The estimated genome size (36·8 Mb) and predicted number of genes (12 197) for A. flavus is similar to that of A. oryzae (36·7 Mb and 12 079, respectively). These two fungi have significantly larger genomes than Aspergillus nidulans (30·1) and Aspergillus fumigatus (29·4). The A. flavus and A. oryzae genomes are enriched in genes for secondary metabolism, but do not differ greatly from one another in the predicted number of polyketide synthases, nonribosomal peptide synthases or the number of genes coding for cytochrome P450 enzymes. A micro-scale analysis of the two fungi did show differences in DNA correspondence between the two species and in the number of transposable elements. Each species has approximately 350 unique genes. The high degree of sequence similarity between the two fungi suggests that they may be ecotypes of the same species and that A. oryzae has resulted from the domestication of A. flavus.

Genetics and physiology of aflatoxin biosynthesis.

Aflatoxins are the most thoroughly studied mycotoxins. Elegant early research on the biosynthetic scheme of the pathway has allowed a molecular characterization of aflatoxin biosynthesis and its regulation. Genetic studies on aflatoxin biosynthesis in Aspergillus flavus and A. parasiticus, and sterigmatocystin biosynthesis in A. nidulans, led to the cloning of 17 genes responsible for 12 enzymatic conversions in the AF/ST pathways. Pathway-specific regulation is by a Zn(II)2Cys6 DNA-binding protein that regulates the transcription of all pathway genes. Less is known about the global factors that regulate aflatoxin biosynthesis, but there is a clear link between development and aflatoxin biosynthesis. There is also a large body of information on physiological factors involved in aflatoxin biosynthesis, but it has been difficult to understand their role in the regulation of this pathway. This chapter discusses current knowledge on the molecular biology and genetics of the pathway, and provides a summary of the physiological factors known to influence aflatoxin formation.

Aflatoxin production in peanut lines selected to represent a range of linoleic acid concentrations.

To determine whether concentrations of linoleate in peanut (Arachis hypogaea L.) seed oil could be used to predict an ability to support aflatoxin production, seeds of genotypes representing a range of linoleate content were inoculated with Aspergillus flavus Link ex Fries and assayed for aflatoxin content. Seeds were blanched and quartered, inoculated with conidia of A. flavus, placed on moistened filter paper in petri dishes, and incubated for 8 days at 28 degrees C. Multiple regression analysis was used to account for the variation among lines with the use of fatty acid concentrations as independent variables. In test 1, linoleate accounted for 39 to 44% of the variation among lines for aflatoxin B1 and B2 and total aflatoxin (26 to 27% after log transformation). Oleate accounted for substantial additional variation (27 to 29%) among lines (20 to 23% after log transformation). Other fatty acids accounted for small fractions of among-line variation. In test 2, linoleate accounted for about 35 to 44% of the variation among entries across traits (29 to 37% for log-transformed data); arachidate accounted for 19 to 29% (27 to 33% after log transformation). Eicosenoate accounted for a small part of the total entry variation. In both experiments, residual variation among entries was significant. Low-linoleate lines consistently contained more aflatoxin, whereas normal- to high-linoleate lines contained variable amounts. Although fatty acid concentrations accounted for significant portions of genetic variation, it is not practical to use them as predictors for susceptibility to aflatoxin contamination, especially for lines in the normal range for oleate and linoleate.

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus.

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.

Comparison of the omtA genes encoding O-methyltransferases involved in aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus.

O-methyltransferase (OMT) is one of the key enzymes in aflatoxin (AF) biosynthesis in the fungi, Aspergillus flavus (Af) and A. parasiticus (Ap). Genomic DNA clones containing the omtA genes from Ap strain SRRC 143 and Af strain CRA01-2B were sequenced. Comparison of the genomic DNA sequences with the cDNA of this Ap gene revealed the presence of four introns ranging from 52 to 60 bp in length in both species; the region encoding the putative S-adenosylmethionine-binding motif was located between the third and fourth introns. The coding sequence of omtA from Ap strain SRRC 143 demonstrated a greater than 97% sequence identity with that from Af strain CRA01-2B, within the coding region.

Corn Seed Proteins Inhibitory to Aspergillus flavus and Aflatoxin Biosynthesis.

ABSTRACT This study reports the presence of two fractions from corn seeds inhibitory to aflatoxin formation. Using a sensitive laboratory assay that can measure both inhibition of fungal growth and inhibition of aflatoxin biosynthesis, we examined aqueous extracts from seeds of Tex6, a corn inbred shown to be highly resistant to aflatoxin accumulation in field and laboratory evaluations. In these extracts, we identified two biologically active fractions. One inhibited growth of Aspergillus flavus and, thus, aflatoxin accumulation, and the other inhibited aflatoxin formation with little effect on fungal growth. The compounds responsible for these activities appear to be proteaceous, as they are water soluble, heat labile, and sensitive to proteinase K treatment. The compounds were partially purified by ultrafiltration and chromatography. The estimated molecular mass of the growth inhibitor is approximately 28 kDa, and that of the aflatoxin biosynthesis inhibitor appears to be greater than 100 kDa. Partially purified preparations of the growth inhibitor and aflatoxin biosynthesis inhibitor cause 50% inhibition at 26 and 75 mug of protein/ml, respectively. The presence of these compounds in Tex6 may explain its resistance to aflatoxin accumulation.

Infection and Fumonisin Production by Fusarium verticillioides in Developing Maize Kernels.

ABSTRACT Fusarium ear rot and fumonisin contamination are serious problems for maize growers, particularly in the southeastern United States. The lack of maize genotypes highly resistant to infection by Fusarium verticillioides or to fumonisin contamination emphasizes the need for management strategies to prevent contamination by this mycotoxin. Information on the initial appearance of infection and fumonisin contamination of kernels and their increase over time is needed to determine if early harvest may be an appropriate control strategy. Maize ears from replicated studies at two locations in eastern North Carolina were harvested weekly, starting 2 weeks after pollination and continuing for 14 weeks. The percentage of kernels infected with F. verticillioides and the fumonisin contamination in the harvested samples were determined. Kernel infection by F. verticillioides and fumonisin contamination appeared as kernels neared physiological maturity and increased up to the average harvest date for maize in North Carolina. Beyond this date, the concentrations of fumonisin fluctuated. Under years conducive for fumonisin contamination, early harvest (greater than 25% grain moisture) may help reduce the level of contamination.

Growth of an Aspergillus flavus transformant expressing Escherichia coli beta-glucuronidase in maize kernels resistant to aflatoxin production.

Kernels of a maize inbred that demonstrated resistance to aflatoxin production in previous studies were inoculated with an Aspergillus flavus strain containing the Escherichia coli beta-D-glucuronidase reporter gene linked to a beta-tubulin gene promoter and assessed for both fungal growth and aflatoxin accumulation. Prior to inoculation, kernels were pin-wounded through the pericarp to the endosperm, pin-wounded in the embryo region, or left unwounded. After 7 days incubation with the fungus, beta-glucuronidase activity (fungal growth) in the kernels was quantified using a fluorogenic assay and aflatoxin B content of the same kernels was analyzed. Kernels of a susceptible inbred, similarly treated, served as controls. Results indicate a positive relationship between aflatoxin levels and the amount of fungal growth. However, resistant kernels wounded through the pericarp to the endosperm before inoculation supported an increase in aflatoxin B over levels observed in nonwounded kernels, without an increase in fungal growth. Wounding kernels of the resistant inbred through the embryo resulted in both the greatest fungal growth and the highest levels of aflatoxin B1 for this genotype. Maintenance of resistance to aflatoxin B1 in endosperm-wounded kernels may be due to the action of a mechanism which limits fungal access to the kernel embryo.

Aspergillus niger absorbs copper and zinc from swine wastewater.

Wastewater from swine confined-housing operations contains elevated levels of copper and zinc due to their abundance in feed. These metals may accumulate to phytotoxic levels in some agricultural soils of North Carolina due to land application of treated swine effluent. We evaluated fungi for their ability to remove these metals from wastewater and found Aspergillus niger best suited for this purpose. A. niger was able to grow on plates amended with copper at a level five times that inhibitory to the growth of Saccharomyes cerevisiae. We also found evidence for internal absorption as the mechanism used by A. niger to detoxify its environment of copper, a property of the fungus that has not been previously exploited for metal bioremediation. In this report, we show that A. niger is capable of removing 91% of the copper and 70% of the zinc from treated swine effluent.

Comparison of Aflatoxin Production in Normal- and High-Oleic Backcross-Derived Peanut Lines.

The effect of the high-oleate trait of peanut on aflatoxin production was tested by comparing normal oleic lines with high-oleic backcross-derived lines. Seeds were blanched, quartered, and inoculated with Aspergillus flavus conidia, placed on moistened filter paper in petri dishes, and incubated for 8 days. In one experiment, dishes were stacked in plastic bags in a Latin square design with bags and positions in stacks as blocking variables. High-oleic lines averaged nearly twice as much aflatoxin as normal lines. Background genotype had no significant effect on aflatoxin content, and interaction between background genotype and oleate level was not detected. In a second experiment, dishes were arranged on plastic trays enclosed in plastic bags and stacked with PVC spacers between trays. Fungal growth and aflatoxin production were greater than in the first experiment. Background genotype, oleate level, and their interaction were significant. The mean of high-oleic lines was almost twice that of normal lines, but the magnitude of the difference varied with background genotype. Special care should be taken with high-oleic lines to prevent growth of Aspergillus spp. and concomitant development of aflatoxin contamination.

Function and regulation of aflJ in the accumulation of aflatoxin early pathway intermediate in Aspergillus flavus.

aflJ resides within the aflatoxin biosynthetic gene cluster adjacent to the pathway regulatory gene aflR and is involved in aflatoxin production, but its function is unknown. Over-expression of aflJ in the aflatoxin-producing strain 86-10 resulted in increased aflatoxin. In an effort to study the function and regulation of aflJ, strain 649-1 lacking the entire biosynthetic cluster was transformed with either reporter constructs, expression constructs, or cosmid clones and analysed for gene expression or metabolite accumulation. Over-expression of aflJ did not result in elevated transcription of ver-1, omtA or aflR. To determine if over-expression of aflJ leads to an increase in early pathway intermediates, strain 649-1 was transformed with cosmid 5E6 and either gpdA::aflJ alone, gpdA::aflR alone, or aflJ and aflR together. Cosmid 5E6 contains the genes pksA, nor-1, fas-1, and fas-2, which are required for the biosynthesis of the early pathway intermediate averantin. 649-1 transformants containing 5E6 alone produced no detectable averantin. In contrast, 5E6 transformants with gpdA::aflR produced averantin, but only half as much as those transformants containing both aflR and aflJ. Northern blot analysis showed that 5E6 transformants containing both aflR and aflJ had five times more pksA transcripts and four times more nor-1 transcripts than 5E6 transformants containing gpdA::aflR alone. Further, aflJ transcription was regulated by aflR. Over-expression of aflR resulted in elevated aflJ transcription. aflJ appears to modulate the regulation of early genes in aflatoxin biosynthesis.

Elucidation of veA-dependent genes associated with aflatoxin and sclerotial production in Aspergillus flavus by functional genomics.

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.

Aspergillus flavus expressed sequence tags and microarray as tools in understanding aflatoxin biosynthesis.

Aflatoxins are the most toxic and carcinogenic naturally occurring mycotoxins. They are produced primarily byAspergillus flavus andA. parasiticus. In order to better understand the molecular mechanisms that control aflatoxin production, identification of genes usingA. flavus expressed sequence tags (ESTs) and microarrays is currently being performed. Sequencing and annotation ofA. flavus ESTs from a normalizedA. flavus cDNA library identified 7,218 unique EST sequences. Genes that are putatively involved in aflatoxin biosynthesis, regulation and signal transduction, fungal virulence or pathogenicity, stress response or antioxidation, and fungal development were identified from these ESTs. Microarrays containing over 5,000 uniqueA. flavus gene amplicons were constructed at The Institute for Genomic Research. Gene expression profiling under aflatoxin-producing and non-producing conditions using this microarray has identified hundreds of genes that are potentially involved in aflatoxin production. Further investigations on the functions of these genes by gene knockout experiments are underway. This research is expected to provide information for developing new strategies for controlling aflatoxin contamination of agricultural commodities.

Transformation of Aspergillus flavus to study aflatoxin biosynthesis.

Aflatoxin contamination of agricultural commodities continues to be a serious problem in the United States. Breeding for resistant genotypes has been unsuccessful and detoxification of food sources is not economically feasible. New strategies for control may become apparent once more is known about the biosynthesis and regulation of aflatoxin. Although the biosynthetic pathway of aflatoxin has been extensively studied, little is known about the regulation of the individual steps in the pathway. We have developed a genetic transformation system for Aspergillus flavus that provides a new and expedient approach to studying the biosynthesis of aflatoxin and its regulation. Through the use of this genetic transformation system, genes for aflatoxin biosynthesis can be identified and isolated by the complementation of aflatoxin negative mutants. In this paper we discuss molecular strategies for studying the regulation and biosynthesis of aflatoxin.

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media.

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.

Overexpression of aflR Leads to Upregulation of Pathway Gene Transcription and Increased Aflatoxin Production in Aspergillus flavus.

The aflatoxin biosynthetic pathway regulatory gene, aflR, encodes a putative 47-kDa protein containing a zinc cluster DNA binding motif. It is required for the transcription of all of the characterized aflatoxin pathway genes in both Aspergillus flavus and Aspergillus parasiticus. The objective of this study was to examine the effects of aflR overexpression on temporal gene expression, aflatoxin production, and nitrate inhibition of aflatoxin biosynthesis in A. flavus. An inducible expression construct was made by fusing the coding region of aflR to the promoter region of the A. flavus adh1 gene. This construct was transformed into A. flavus 656-2 (FGSC A1010), a strain mutated at the aflR locus. Strain 656-2 containing the adh1(p)::aflR construct had induced transcription of two early aflatoxin pathway genes, nor-1 and pksA, and produced wild-type concentrations of aflatoxin in a temporal pattern similar to that of wild-type strains of A. flavus. Strains 656-2 and 86-10 (FGSC A1009) an aflatoxigenic strain, were transformed with a construct containing the constitutive promoter gpdA driving aflR. Transformants of these strains constitutively expressed aflR, fas-1A, pksA, nor-1, and omtA but did not constitutively produce aflatoxin. Strain 86-10 containing the gpdA(p)::aflR construct produced 50 times more aflatoxin than 86-10, but the temporal pattern of aflatoxin production was the same as for 86-10, and aflatoxin production was also induced by sucrose. The addition of 10 g of nitrate per liter to sucrose low salts medium inhibited aflatoxin production by both strain 86-10 and a transformant of 86-10 containing the gpdA(p)::aflR construct, indicating that nitrate inhibition of aflatoxin biosynthesis does not occur solely at the level of aflR transcription. These studies show that constitutive overexpression of the pathway transcriptional regulatory gene aflR leads to higher transcript accumulation of pathway genes and increased aflatoxin production but that the initiation of aflatoxin biosynthesis is not solely regulated by the transcriptional activities of the biosynthetic pathway.

The alcohol dehydrogenase gene adh1 is induced in Aspergillus flavus grown on medium conducive to aflatoxin biosynthesis.

An Aspergillus flavus cDNA library was screened by differential hybridization to isolate clones corresponding to genes that are actively transcribed under culture conditions conducive to aflatoxin biosynthesis. One clone with a 1.28-kb insert was isolated, and its nucleotide sequence was determined. The nucleotide sequence of this clone had 75% DNA identity to those of the alcohol dehydrogenase genes from Aspergillus nidulans, and the putative polypeptide translated from the cDNA sequence had 82% similarity with the amino acid sequences of the A. nidulans proteins. Thus, this gene has been designated adh1. Southern hybridization analysis of genomic DNA from A. flavus indicated that there was one copy of the adh1 gene. Northern (RNA) hybridization analysis indicated that the adh1 transcript accumulated in culture medium conducive to aflatoxin production and the timing of accumulation of adh1 transcripts was similar to that for aflatoxin. Fusion of the promoter region of adh1 to a beta-glucuronidase reporter gene indicated that accumulation of the adh1 transcript was the result of transcriptional activation. These molecular data support previous physiological evidence that showed the importance of carbohydrate metabolism during aflatoxin biosynthesis.

Drainage effects on stream nitrate-N and hydrology in south-central Minnesota (USA).

Excessive nitrate-N in south-central Minnesota ditches and streams is related to land-use change, and may be contributing to the development of the zone of hypoxia in the Gulf of Mexico. Intensive land-use (agricultural management) has progressively increased as subsurface drainage has improved crop productivity over the past 25 years. We have examined water at varying scales for delta18O and, nitrate-N concentrations. Additionally, analysis of annual peak flows, and channel geomorphic features provided a measure of hydrologic change. Laboratory and field results indicate that agricultural drainage has influenced riverine source waters, concentrations of nitrate-N, channel dimensions and hydrology in the Blue Earth River (BER) Basin. At the mouth of the BER shallow ground water comprises the largest source water component. The highest nitrate-N concentrations in the BER and tributaries typically occurred in May and June and ranged from 7-34 mg L(-1). Peak flows for the 1.01-2-yr recurrence intervals increased by 20-to-206% over the past 25 years. Geomorphic data suggest that small channels (ditches) were entrenched by design, whereas, natural that are disconnected from an accessible riparian corridor. Frequent access to a functioning riparian zone is important for denitrification.

Expression profile analysis of wild-type and fcc1 mutant strains of Fusarium verticillioides during fumonisin biosynthesis.

Fusarium verticillioides produces a group of mycotoxins known as fumonisins that are associated with a variety of mycotoxicoses in humans and animals. In this study, DNA microarrays were constructed with expressed sequence tags (ESTs) from F. verticillioides. To identify genes with patterns of expression similar to the fumonisin biosynthetic (FUM) genes, the microarray was probed with labeled cDNAs originating from a wild-type strain and a fcc1 mutant grown on maize and in a defined medium adjusted to either pH 3 or pH 8. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to the FUM ESTs. These results provide candidate genes with potential roles in fumonisin biosynthesis.