Asymmetrical lineage introgression and recombination in populations of Aspergillus flavus: Implications for biological control.

Aspergillus flavus is an agriculturally important fungus that causes ear rot of maize and produces aflatoxins, of which B1 is the most carcinogenic naturally-produced compound. In the US, the management of aflatoxins includes the deployment of biological control agents that comprise two nonaflatoxigenic A. flavus strains, either Afla-Guard (member of lineage IB) or AF36 (lineage IC). We used genotyping-by-sequencing to examine the influence of both biocontrol agents on native populations of A. flavus in cornfields in Texas, North Carolina, Arkansas, and Indiana. This study examined up to 27,529 single-nucleotide polymorphisms (SNPs) in a total of 815 A. flavus isolates, and 353 genome-wide haplotypes sampled before biocontrol application, three months after biocontrol application, and up to three years after initial application. Here, we report that the two distinct A. flavus evolutionary lineages IB and IC differ significantly in their frequency distributions across states. We provide evidence of increased unidirectional gene flow from lineage IB into IC, inferred to be due to the applied Afla-Guard biocontrol strain. Genetic exchange and recombination of biocontrol strains with native strains was detected in as little as three months after biocontrol application and up to one and three years later. There was limited inter-lineage migration in the untreated fields. These findings suggest that biocontrol products that include strains from lineage IB offer the greatest potential for sustained reductions in aflatoxin levels over several years. This knowledge has important implications for developing new biocontrol strategies.

Changes in the Fungal Microbiome of Maize During Hermetic Storage in the United States and Kenya.

Prior to harvest, maize kernels are invaded by a diverse population of fungal organisms that comprise the microbiome of the grain mass. Poor post-harvest practices and improper drying can lead to the growth of mycotoxigenic storage fungi and deterioration of grain quality. Hermetic storage bags are a low-cost technology for the preservation of grain during storage, which has seen significant adoption in many regions of Sub-Saharan Africa. This study explored the use of high-throughput DNA sequencing of the fungal Internal Transcribed Spacer 2 (ITS2) region for characterization of the fungal microbiome before and after 3 months of storage in hermetic and non-hermetic (woven) bags in the United States and Kenya. Analysis of 1,377,221 and 3,633,944 ITS2 sequences from the United States and Kenya, respectively, resulted in 251 and 164 operational taxonomic units (OTUs). Taxonomic assignment of these OTUs revealed 63 and 34 fungal genera in the US and Kenya samples, respectively, many of which were not detected by traditional plating methods. The most abundant genus was Fusarium, which was identified in all samples. Storage fungi were detected in the grain mass prior to the storage experiments and increased in relative abundance within the woven bags. The results also indicate that storage location had no effect on the fungal microbiome of grain stored in the United States, while storage bag type led to significant changes in fungal composition. The fungal microbiome of the Kenya grain also underwent significant changes in composition during storage and fungal diversity increased during storage regardless of bag type. Our results indicated that extraction of DNA from ground kernels is sufficient for identifying the fungi associated with the maize. The results also indicated that bag type was the most important factor influencing changes in fungal microbiome during storage. The results also support the recommended use of hermetic storage for reducing food safety risks, especially from mycotoxigenic fungi.

Comparative Histological and Transcriptional Analysis of Maize Kernels Infected with Aspergillus flavus and Fusarium verticillioides.

Aspergillus flavus and Fusarium verticillioides infect maize kernels and contaminate them with the mycotoxins aflatoxin, and fumonisin, respectively. Genetic resistance in maize to these fungi and to mycotoxin contamination has been difficult to achieve due to lack of identified resistance genes. The objective of this study was to identify new candidate resistance genes by characterizing their temporal expression in response to infection and comparing expression of these genes with genes known to be associated with plant defense. Fungal colonization and transcriptional changes in kernels inoculated with each fungus were monitored at 4, 12, 24, 48, and 72 h post inoculation (hpi). Maize kernels responded by differential gene expression to each fungus within 4 hpi, before the fungi could be observed visually, but more genes were differentially expressed between 48 and 72 hpi, when fungal colonization was more extensive. Two-way hierarchal clustering analysis grouped the temporal expression profiles of the 5,863 differentially expressed maize genes over all time points into 12 clusters. Many clusters were enriched for genes previously associated with defense responses to either A. flavus or F. verticillioides. Also within these expression clusters were genes that lacked either annotation or assignment to functional categories. This study provided a comprehensive analysis of gene expression of each A. flavus and F. verticillioides during infection of maize kernels, it identified genes expressed early and late in the infection process, and it provided a grouping of genes of unknown function with similarly expressed defense related genes that could inform selection of new genes as targets in breeding strategies.

Comparative genomics of maize ear rot pathogens reveals expansion of carbohydrate-active enzymes and secondary metabolism backbone genes in Stenocarpella maydis.

Stenocarpella maydis is a plant pathogenic fungus that causes Diplodia ear rot, one of the most destructive diseases of maize. To date, little information is available regarding the molecular basis of pathogenesis in this organism, in part due to limited genomic resources. In this study, a 54.8 Mb draft genome assembly of S. maydis was obtained with Illumina and PacBio sequencing technologies, and analyzed. Comparative genomic analyses with the predominant maize ear rot pathogens Aspergillus flavus, Fusarium verticillioides, and Fusarium graminearum revealed an expanded set of carbohydrate-active enzymes for cellulose and hemicellulose degradation in S. maydis. Analyses of predicted genes involved in starch degradation revealed six putative α-amylases, four extracellular and two intracellular, and two putative γ-amylases, one of which appears to have been acquired from bacteria via horizontal transfer. Additionally, 87 backbone genes involved in secondary metabolism were identified, which represents one of the largest known assemblages among Pezizomycotina species. Numerous secondary metabolite gene clusters were identified, including two clusters likely involved in the biosynthesis of diplodiatoxin and chaetoglobosins. The draft genome of S. maydis presented here will serve as a useful resource for molecular genetics, functional genomics, and analyses of population diversity in this organism.

Involvement of FST1 from Fusarium verticillioides in virulence and transport of inositol.

Fumonisin B1 (FB1), a polyketide mycotoxin produced by Fusarium verticillioides during the colonization of maize kernels, is detrimental to human and animal health. FST1 encodes a putative protein with 12 transmembrane domains; however, its function remains unknown. The FST1 gene is highly expressed by the fungus in the endosperm of maize kernels compared with the levels of expression in germ tissues. Previous research has shown that FST1 affects FB1 production, virulence, hydrogen peroxide resistance, hydrophobicity and macroconidia production. Here, we examine the phylogeny of FST1, its expression in a Saccharomyces cerevisiae strain lacking a functional myo-inositol transporter (ITR1) and the effect of amino acid changes in the central loop and C-terminus regions of FST1 on functionality. The results indicate that expression of FST1 in an ITR1 mutant strain restores growth on myo-inositol medium to wild-type levels and restores the inhibitory effects of FB1, suggesting that FST1 can transport both myo-inositol and FB1 into yeast cells. Our results with engineered FST1 also indicate that amino acids in the central loop and C-terminus regions are important for FST1 functionality in both S. cerevisiae and F. verticillioides. Overall, this research has established the first characterized inositol transporter in filamentous fungi and has advanced our knowledge about the global regulatory functions of FST1.

Transcriptome changes in Fusarium verticillioides caused by mutation in the transporter-like gene FST1.

BACKGROUND: Fusarium verticillioides causes an important seed disease on maize and produces the fumonisin group of mycotoxins, which are toxic to humans and livestock. A previous study discovered that a gene (FST1) in the pathogen affects fumonisin production and virulence. Although the predicted amino acid sequence of FST1 is similar to hexose transporters, previous experimental evidence failed to prove function. RESULTS: Three new phenotypes were identified that are associated with the FST1 mutant of F. verticillioides (Δfst1), namely reduction in macroconidia production, increased sensitivity to hydrogen peroxide, and reduced mycelial hydrophobicity. A transcriptome comparison of the wild type and strain Δfst1 grown on autoclaved maize kernels for six days identified 2677 genes that were differentially expressed. Through gene ontology analysis, 961 genes were assigned to one of 12 molecular function categories. Sets of down-regulated genes in strain Δfst1 were identified that could account for each of the mutant phenotypes. CONCLUSION: The study provides evidence that disruption of FST1 causes several metabolic and developmental defects in F. verticillioides. FST1 appears to connect the expression of several gene networks, including those involved in secondary metabolism, cell wall structure, conidiogenesis, virulence, and resistance to reactive oxygen species. The results support our hypothesis that FST1 functions within the framework of environmental sensing.

Tissue-specific gene expression in maize seeds during colonization by Aspergillus flavus and Fusarium verticillioides.

Aspergillus flavus and Fusarium verticillioides are fungal pathogens that colonize maize kernels and produce the harmful mycotoxins aflatoxin and fumonisin, respectively. Management practice based on potential host resistance to reduce contamination by these mycotoxins has proven difficult, resulting in the need for a better understanding of the infection process by these fungi and the response of maize seeds to infection. In this study, we followed the colonization of seeds by histological methods and the transcriptional changes of two maize defence-related genes in specific seed tissues by RNA in situ hybridization. Maize kernels were inoculated with either A. flavus or F. verticillioides 21-22 days after pollination, and harvested at 4, 12, 24, 48, 72, 96 and 120 h post-inoculation. The fungi colonized all tissues of maize seed, but differed in their interactions with aleurone and germ tissues. RNA in situ hybridization showed the induction of the maize pathogenesis-related protein, maize seed (PRms) gene in the aleurone and scutellum on infection by either fungus. Transcripts of the maize sucrose synthase-encoding gene, shrunken-1 (Sh1), were observed in the embryo of non-infected kernels, but were induced on infection by each fungus in the aleurone and scutellum. By comparing histological and RNA in situ hybridization results from adjacent serial sections, we found that the transcripts of these two genes accumulated in tissue prior to the arrival of the advancing pathogens in the seeds. A knowledge of the patterns of colonization and tissue-specific gene expression in response to these fungi will be helpful in the development of resistance.

Aspergillus flavus infection induces transcriptional and physical changes in developing maize kernels.

Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understanding host response to infection by the fungus, transcription of approximately 9000 maize genes were monitored during the host-pathogen interaction with a custom designed Affymetrix GeneChip® DNA array. More than 4000 maize genes were found differentially expressed at a FDR of 0.05. This included the up regulation of defense related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel by A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.

Localization, morphology and transcriptional profile of Aspergillus flavus during seed colonization.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize kernels pre-harvest, creating major human health concerns and causing substantial agricultural losses. Improved control strategies are needed, yet progress is hampered by the limited understanding of the mechanisms of infection. A series of studies were designed to investigate the localization, morphology and transcriptional profile of A. flavus during internal seed colonization. Results from these studies indicate that A. flavus is capable of infecting all tissues of the immature kernel by 96 h after infection. Mycelia were observed in and around the point of inoculation in the endosperm and were found growing down to the germ. At the endosperm-germ interface, hyphae appeared to differentiate and form a biofilm-like structure that surrounded the germ. The exact nature of this structure remains unclear, but is discussed. A custom-designed A. flavus Affymetrix GeneChip® microarray was used to monitor genome-wide transcription during pathogenicity. A total of 5061 genes were designated as being differentially expressed. Genes encoding secreted enzymes, transcription factors and secondary metabolite gene clusters were up-regulated and considered to be potential effector molecules responsible for disease in the kernel. Information gained from this study will aid in the development of strategies aimed at preventing or slowing down A. flavus colonization of the maize kernel.

Aflatoxins, fumonisins, and trichothecenes: a convergence of knowledge.

Plant pathogenic fungi Aspergillus flavus, Fusarium verticillioides, and Fusarium graminearum infect seeds of the most important food and feed crops, including maize, wheat, and barley. More importantly, these fungi produce aflatoxins, fumonisins, and trichothecenes, respectively, which threaten health and food security worldwide. In this review, we examine the molecular mechanisms and environmental factors that regulate mycotoxin biosynthesis in each fungus, and discuss the similarities and differences in the collective body of knowledge. Whole-genome sequences are available for these fungi, providing reference databases for genomic, transcriptomic, and proteomic analyses. It is well recognized that genes responsible for mycotoxin biosynthesis are organized in clusters. However, recent research has documented the intricate transcriptional and epigenetic regulation that affects these gene clusters. Significantly, molecular networks that respond to environmental factors, namely nitrogen, carbon, and pH, are connected to components regulating mycotoxin production. Furthermore, the developmental status of seeds and specific tissue types exert conditional influences during fungal colonization. A comparison of the three distinct mycotoxin groups provides insight into new areas for research collaborations that will lead to innovative strategies to control mycotoxin contamination of grain.

HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production and pathogenesis in Fusarium verticillioides.

In Fusarium verticillioides, a ubiquitous pathogen of maize, virulence and mycotoxigenesis are regulated in response to the types and amounts of carbohydrates present in maize kernels. In this study, we investigated the role of a putative hexokinase-encoding gene (HXK1) in growth, development and pathogenesis. A deletion mutant (Δhxk1) of HXK1 was not able to grow when supplied with fructose as the sole carbon source, and growth was impaired when glucose, sucrose or maltotriose was provided. Additionally, the Δhxk1 mutant produced unusual swollen hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the Δhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On maize kernels, the Δhxk1 mutant was substantially less virulent than the wild-type, but virulence on maize stalks was not impaired, possibly indicating a metabolic response to tissue-specific differences in plant carbohydrate content. Finally, disruption of HXK1 had a pronounced effect on fungal metabolites produced during colonization of maize kernels; the Δhxk1 mutant produced approximately 50 % less trehalose and 80 % less fumonisin B1 (FB1) than the wild-type. The reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic stress in the Δhxk1 mutant. In summary, this study links early events in carbohydrate sensing and glycolysis to virulence and secondary metabolism in F. verticillioides, and thus provides a new foothold from which the genetic regulatory networks that underlie pathogenesis and mycotoxigenesis can be unravelled and defined.

Gene expression profile and response to maize kernels by Aspergillus flavus.

Aspergillus flavus causes an ear rot of maize, often resulting in the production of aflatoxin, a potent liver toxin and carcinogen that impacts the health of humans and animals. Many aspects of kernel infection and aflatoxin biosynthesis have been studied but the precise effects of the kernel environment on A. flavus are poorly understood. The goal of this research was to study the fungal response to the kernel environment during colonization. Gene transcription in A. flavus was analyzed by microarrays after growth on kernels of the four developmental stages: blister (R2), milk (R3), dough (R4), and dent (R5). Five days after inoculation, total RNA was isolated from kernels and hybridized to Affymetrix Gene Chip arrays containing probes representing 12,834 A. flavus genes. Statistical comparisons of the expression profile data revealed significant differences that included unique sets of upregulated genes in each kernel stage and six patterns of expression over the four stages. Among the genes expressed in colonized dent kernels were a phytase gene and six putative genes involved in zinc acquisition. Disruption of the phytase gene phy1 resulted in reduced growth on medium containing phytate as the sole source of phosphate. Furthermore, growth of the mutant (Δphy1) was 20% of the wild-type strain when wound inoculated into maize ears. In contrast, no difference was detected in the amount of aflatoxin produced relative to fungal growth, indicating that phy1 does not affect aflatoxin production. The study revealed the genome-wide effects of immature maize kernels on A. flavus and suggest that phytase has a role in pathogenesis.

Functional characterization of fst1 in Fusarium verticillioides during colonization of maize kernels.

The putative hexose transporter gene fst1 in Fusarium verticillioides was identified previously by microarray analysis as a gene that was more highly expressed during colonization of autoclaved maize endosperm than germ. In contrast to a previous study, in which disruption of fst1 did not affect growth of the pathogen on autoclaved maize kernels, in the current study, we demonstrated that disruption of fst1 delayed growth and symptom development on wounded maize ears. Characterization of the fst1 promoter revealed that regulation of fst1 expression was similar to that of fumonisin biosynthetic (fum) genes; expression was highest during growth on endosperm tissue and repressed by elevated concentrations of ammonium in the growth medium. With a fluorescent tag attached to FST1, the protein localized transiently to the periphery of the cells near the plasma membrane and in vacuole-like structures, suggesting that membrane-localized FST1 was internalized and degraded in vacuoles. Expression of fst1 in a yeast strain lacking hexose transporter genes did not complement the yeast mutation, suggesting that FST1 does not transport glucose, fructose, or mannose. The results indicate a functional role for FST1 in pathogenesis during the colonization of living kernels.

Beyond aflatoxin: four distinct expression patterns and functional roles associated with Aspergillus flavus secondary metabolism gene clusters.

Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis has predicted that these species have the capacity to synthesize many more compounds. It has been possible to infer the presence of 55 gene clusters associated with secondary metabolism in Aspergillus flavus; however, only three metabolic pathways-aflatoxin, cyclopiazonic acid (CPA) and aflatrem-have been assigned to these clusters. To gain an insight into the regulation of and to infer the ecological significance of the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture medium and temperature, fungal development, colonization of developing maize seeds and misexpression of laeA, a global regulator of secondary metabolism. Hierarchical clustering analysis of expression profiles allowed us to categorize the gene clusters into four distinct clades. Gene clusters for the production of aflatoxins, CPA and seven other unknown compound(s) were identified as belonging to one clade. To further explore the relationships found by gene expression analysis, aflatoxin and CPA production were quantified under five different cell culture environments known to be conducive or nonconducive for aflatoxin biosynthesis and during the colonization of developing maize seeds. Results from these studies showed that secondary metabolism gene clusters have distinctive gene expression profiles. Aflatoxin and CPA were found to have unique regulation, but are sufficiently similar that they would be expected to co-occur in substrates colonized with A. flavus.

Use of the plant defense protein osmotin to identify Fusarium oxysporum genes that control cell wall properties.

Fusarium oxysporum is the causative agent of fungal wilt disease in a variety of crops. The capacity of a fungal pathogen such as F. oxysporum f. sp. nicotianae to establish infection on its tobacco (Nicotiana tabacum) host depends in part on its capacity to evade the toxicity of tobacco defense proteins, such as osmotin. Fusarium genes that control resistance to osmotin would therefore reflect coevolutionary pressures and include genes that control mutual recognition, avoidance, and detoxification. We identified FOR (Fusarium Osmotin Resistance) genes on the basis of their ability to confer osmotin resistance to an osmotin-sensitive strain of Saccharomyces cerevisiae. FOR1 encodes a putative cell wall glycoprotein. FOR2 encodes the structural gene for glutamine:fructose-6-phosphate amidotransferase, the first and rate-limiting step in the biosynthesis of hexosamine and cell wall chitin. FOR3 encodes a homolog of SSD1, which controls cell wall composition, longevity, and virulence in S. cerevisiae. A for3 null mutation increased osmotin sensitivity of conidia and hyphae of F. oxysporum f. sp. nicotianae and also reduced cell wall beta-1,3-glucan content. Together our findings show that conserved fungal genes that determine cell wall properties play a crucial role in regulating fungal susceptibility to the plant defense protein osmotin.

Silencing of the aflatoxin gene cluster in a diploid strain of Aspergillus flavus is suppressed by ectopic aflR expression.

Aflatoxins are toxic secondary metabolites produced by a 70-kb cluster of genes in Aspergillus flavus. The cluster genes are coordinately regulated and reside as a single copy within the genome. Diploids between a wild-type strain and a mutant (649) lacking the aflatoxin gene cluster fail to produce aflatoxin or transcripts of the aflatoxin pathway genes. This dominant phenotype is rescued in diploids between a wild-type strain and a transformant of the mutant containing an ectopic copy of aflR, the transcriptional regulator of the aflatoxin biosynthetic gene cluster. Further characterization of the mutant showed that it is missing 317 kb of chromosome III, including the known genes for aflatoxin biosynthesis. In addition, 939 kb of chromosome II is present as a duplication on chromosome III in the region previously containing the aflatoxin gene cluster. The lack of aflatoxin production in the diploid was not due to a unique or a mis-expressed repressor of aflR. Instead a form of reversible silencing based on the position of aflR is likely preventing the aflatoxin genes from being expressed in 649 x wild-type diploids. Gene expression analysis revealed the silencing effect is specific to the aflatoxin gene cluster.

Cloning and functional expression of the gene encoding an inhibitor against Aspergillus flavus alpha-amylase, a novel seed lectin from Lablab purpureus (Dolichos lablab).

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.

Glass-fiber disks provide suitable medium to study polyol production and gene expression in Eurotium rubrum.

Eurotium species often dominate the fungal population in stored grain and are responsible for spoilage. In this study we tested the usefulness of glass fiber disks to aid the analysis of growth, polyol content and gene expression in E. rubrum in response to various water activities. Growth measurements based on ergosterol content and conidial production indicated that E. rubrum grew as well at 0.86 aw as 0.98 aw. The rate of growth was considerably reduced at 0.83 aw and 0.78 aw. In contrast, under our conditions, Aspergillus flavus and A. nidulans were able to grow only in the highest water activity (0.98 aw). Mannitol was the predominant polyol in all three fungal species grown at 0.98 aw. When E. rubrum was grown at 0.86 aw or lower, glycerol comprised greater than 90% of the total polyols. After a shift from 0.86 aw to 0.98 aw, mannitol levels in E. rubrum increased to 89% of the total polyols within 24 h. Of six genes whose expression was measured by quantitative real-time PCR, three were affected by water activity. Expression of putative hydrophobin and mannitol dehydrogenase genes was higher at 0.98 aw than at 0.86 aw. A putative triacylglycerol lipase gene was expressed at higher levels in 0.86 aw.. The results of this study indicate that the disk method is suitable to study the effects of water activity on growth, polyol biosynthesis and gene expression in E. rubrum. The results also indicate the potential competitiveness of E. rubrum over A. flavus and A. nidulans in low water environments associated with stored grain.

Regulation of fumonisin biosynthesis in Fusarium verticillioides by a zinc binuclear cluster-type gene, ZFR1.

Fusarium verticillioides, a pathogen of maize, produces a class of mycotoxins called fumonisins in infected kernels. In this study, a candidate regulatory gene, ZFR1, was identified in an expressed sequence tag library enriched for transcripts expressed by F. verticillioides during fumonisin B(1) (FB(1)) biosynthesis. ZFR1 deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain. ZFR1 encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression of ZFR1 in colonized germ and degermed kernel tissues correlated with FB(1) levels. Overexpression of ZFR1 in zfr1 mutants restored FB(1) production to wild-type levels; however, FB(1) was not restored in an fcc1 (Fusarium C-type cyclin) mutant by overexpression of ZFR1. The results of this study indicate that ZFR1 is a positive regulator of FB(1) biosynthesis in F. verticillioides and suggest that FCC1 is required for ZFR1 function.