A mutant cotton fatty acid desaturase 2-1d allele causes protein mistargeting and altered seed oil composition.

BACKGROUND: Cotton (Gossypium sp.) has been cultivated for centuries for its spinnable fibers, but its seed oil also possesses untapped economic potential if, improvements could be made to its oleic acid content. RESULTS: Previous studies, including those from our laboratory, identified pima accessions containing approximately doubled levels of seed oil oleic acid, compared to standard upland cottonseed oil. Here, the molecular properties of a fatty acid desaturase encoded by a mutant allele identified by genome sequencing in an earlier analysis were analyzed. The mutant sequence is predicted to encode a C-terminally truncated protein lacking nine residues, including a predicted endoplasmic reticulum membrane retrieval motif. We determined that the mutation was caused by a relatively recent movement of a Ty1/copia type retrotransposon that is not found associated with this desaturase gene in other sequenced cotton genomes. The mutant desaturase, along with its repaired isozyme and the wild-type A-subgenome homoeologous protein were expressed in transgenic yeast and stably transformed Arabidopsis plants. All full-length enzymes efficiently converted oleic acid to linoleic acid. The mutant desaturase protein produced only trace amounts of linoleic acid, and only when strongly overexpressed in yeast cells, indicating that the missing C-terminal amino acid residues are not strictly required for enzyme activity, yet are necessary for proper subcellular targeting to the endoplasmic reticulum membrane. CONCLUSION: These results provide the biochemical underpinning that links a genetic lesion present in a limited group of South American pima cotton accessions and their rare seed oil oleic acid traits. Markers developed to the mutant desaturase allele are currently being used in breeding programs designed to introduce this trait into agronomic upland cotton varieties.

RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

MAIN CONCLUSION: Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Divergent Aspergillus flavus corn population is composed of prolific conidium producers: Implications for saprophytic disease cycle.

The ascomycete fungus Aspergillus flavus infects and contaminates corn, peanuts, cottonseed, and tree nuts with toxic and carcinogenic aflatoxins. Subdivision between soil and host plant populations suggests that certain A. flavus strains are specialized to infect peanut, cotton, and corn despite having a broad host range. In this study, the ability of strains isolated from corn and/or soil in 11 Louisiana fields to produce conidia (field inoculum and male gamete) and sclerotia (resting bodies and female gamete) was assessed and compared with genotypic single-nucleotide polymorphism (SNP) differences between whole genomes. Corn strains produced upward of 47× more conidia than strains restricted to soil. Conversely, corn strains produced as much as 3000× fewer sclerotia than soil strains. Aspergillus flavus strains, typified by sclerotium diameter (small S-strains, <400 µm; large L-strains, >400 µm), belonged to separate clades. Several strains produced a mixture (M) of S and L sclerotia, and an intermediate number of conidia and sclerotia, compared with typical S-strains (minimal conidia, copious sclerotia) and L-strains (copious conidia, minimal sclerotia). They also belonged to a unique phylogenetic mixed (M) clade. Migration from soil to corn positively correlated with conidium production and negatively correlated with sclerotium production. Genetic differences correlated with differences in conidium and sclerotium production. Opposite skews in female (sclerotia) or male (conidia) gametic production by soil or corn strains, respectively, resulted in reduced effective breeding population sizes when comparing male:female gamete ratio with mating type distribution. Combining both soil and corn populations increased the effective breeding population, presumably due to contribution of male gametes from corn, which fertilize sclerotia on the soil surface. Incongruencies between aflatoxin clusters, strain morphotype designation, and whole genome phylogenies suggest a history of sexual reproduction within this Louisiana population, demonstrating the importance of conidium production, as infectious propagules and as fertilizers of the A. flavus soil population.

Transcript profiling by microarray and marker analysis of the short cotton (Gossypium hirsutum L.) fiber mutant Ligon lintless-1 (Li1).

BACKGROUND: Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points. RESULTS: Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene. CONCLUSIONS: The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Microbiota of maize kernels as influenced by Aspergillus flavus infection in susceptible and resistant inbreds.

Background: Nearly everything on Earth harbors a microbiome. A microbiome is a community of microbes (bacteria, fungi, and viruses) with potential to form complex networks that involve mutualistic and antagonistic interactions. Resident microbiota on/in an organism are determined by the external environment, both biotic and abiotic, and the intrinsic adaptability of each organism. Although the maize microbiome has been characterized, community changes that result from the application of fungal biocontrol strains, such as non-aflatoxigenic Aspergillus flavus, have not. Methods: We silk channel inoculated field-grown maize separately with a non-aflatoxigenic biocontrol strain (K49), a highly toxigenic strain (Tox4), and a combination of both A. flavus strains. Two maize inbreds were treated, A. flavus-susceptible B73 and A. flavus-resistant CML322. We then assessed the impacts of A. flavus introduction on the epibiota and endobiota of their maize kernels. Results: We found that the native microbial communities were significantly affected, irrespective of genotype or sampled tissue. Overall, bacteriomes exhibited greater diversity of genera than mycobiomes. The abundance of certain genera was unchanged by treatment, including genera of bacteria (e.g., Enterobacter, Pantoea) and fungi (e.g., Sarocladium, Meyerozyma) that are known to be beneficial, antagonistic, or both on plant growth and health. Conclusion: Beneficial microbes like Sarocladium that responded well to A. flavus biocontrol strains are expected to enhance biocontrol efficacy, while also displacing/antagonizing harmful microbes.

Putative Core Transcription Factors Affecting Virulence in Aspergillus flavus during Infection of Maize.

Aspergillus flavus is an opportunistic pathogen responsible for millions of dollars in crop losses annually and negative health impacts on crop consumers globally. A. flavus strains have the potential to produce aflatoxin and other toxic secondary metabolites, which often increase during plant colonization. To mitigate the impacts of this international issue, we employ a range of strategies to directly impact fungal physiology, growth and development, thus requiring knowledge on the underlying molecular mechanisms driving these processes. Here we utilize RNA-sequencing data that are obtained from in situ assays, whereby Zea mays kernels are inoculated with A. flavus strains, to select transcription factors putatively driving virulence-related gene networks. We demonstrate, through growth, sporulation, oxidative stress-response and aflatoxin/CPA analysis, that three A. flavus strains with knockout mutations for the putative transcription factors AFLA_089270, AFLA_112760, and AFLA_031450 demonstrate characteristics such as reduced growth capacity and decreased aflatoxin/CPA accumulation in kernels consistent with decreased fungal pathogenicity. Furthermore, AFLA_089270, also known as HacA, eliminates CPA production and impacts the fungus's capacity to respond to highly oxidative conditions, indicating an impact on plant colonization. Taken together, these data provide a sound foundation for elucidating the downstream molecular pathways potentially contributing to fungal virulence.

Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus.

Sclerotium (female) fertility, the ability of a strain to produce ascocarps, influences internal morphological changes during sexual reproduction in Aspergillus flavus. Although sclerotial morphogenesis has been linked to secondary metabolite (SM) biosynthesis, metabolic and transcriptomic changes within A. flavus sclerotia during sexual development are not known. Successful mating between compatible strains may result in relatively high or low numbers of ascocarps being produced. Sclerotia from a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) were harvested immediately after crosses were made and every two weeks until 8 weeks of incubation, then subjected to targeted metabolomics (n = 106) and transcriptomics analyses (n = 80). Aflatoxin B1 production varied between Hi-Fert-Mated and Hi-Fert-Unmated sclerotia, while it remained low or was undetected in Lo-Fert-Mated and Lo-Fert-Unmated sclerotia. Profiling of 14 SMs showed elevated production of an aflavazole analog, an aflavinine isomer, and hydroxyaflavinine in Hi-Fert-Mated sclerotia at 4 to 8 weeks. Similarly, genes ayg1, hxtA, MAT1, asd-3, preA and preB, and genes in uncharacterized SM gene clusters 30 and 44 showed increased expression in Hi-Fert-Mated sclerotia at these time points. These results broaden our knowledge of the biochemical and transcriptional processes during sexual development in A. flavus.

Dataset for transcriptomic profiles associated with development of sexual structures in Aspergillus flavus.

Information on the transcriptomic changes that occur within sclerotia of Aspergillus flavus during its sexual cycle is very limited and warrants further research. The findings will broaden our knowledge of the biology of A. flavus and can provide valuable insights in the development or deployment of non-toxigenic strains as biocontrol agents against aflatoxigenic strains. This article presents transcriptomic datasets included in our research article entitled, "Development of sexual structures influences metabolomic and transcriptomic profiles in Aspergillus flavus" [1], which utilized transcriptomics to identify possible genes and gene clusters associated with sexual reproduction and fertilization in A. flavus. RNA was extracted from sclerotia of a high fertility cross (Hi-Fert-Mated), a low fertility cross (Lo-Fert-Mated), and unmated strains (Hi-Fert-Unmated and Lo-Fert-Unmated) of A. flavus collected immediately after crossing and at every two weeks until eight weeks of incubation on mixed cereal agar at 30 °C in continuous darkness (n = 4 replicates from each treatment for each time point; 80 total). Raw sequencing reads obtained on an Illumina NovaSeq 6000 were deposited in NCBI's Sequence Read Archive (SRA) repository under BioProject accession number PRJNA789260. Reads were mapped to the A. flavus NRRL 3357 genome (assembly JCVI-afl1-v2.0; GCA_000006275.2) using STAR software. Differential gene expression analyses, functional analyses, and weighted gene co-expression network analysis were performed using DESeq2 R packages. The raw and analyzed data presented in this article could be reused for comparisons with other datasets to obtain transcriptional differences among strains of A. flavus or closely related species. The data can also be used for further investigation of the molecular basis of different processes involved in sexual reproduction and sclerotia fertility in A. flavus.

Chromosome-Level Aspergillus flavus Strain CA14 Genome Assembly.

We report here a chromosome-level genome assembly of the aflatoxigenic fungus Aspergillus flavus strain CA14. This strain is the basis for numerous studies in fungal physiology and secondary metabolism. This full-length assembly will aid in subsequent genomics research.

Flavonoids Modulate the Accumulation of Toxins From Aspergillus flavus in Maize Kernels.

Aspergillus flavus is an opportunistic fungal pathogen capable of producing aflatoxins, potent carcinogenic toxins that accumulate in maize kernels after infection. To better understand the molecular mechanisms of maize resistance to A. flavus growth and aflatoxin accumulation, we performed a high-throughput transcriptomic study in situ using maize kernels infected with A. flavus strain 3357. Three maize lines were evaluated: aflatoxin-contamination resistant line TZAR102, semi-resistant MI82, and susceptible line Va35. A modified genotype-environment association method (GEA) used to detect loci under selection via redundancy analysis (RDA) was used with the transcriptomic data to detect genes significantly influenced by maize line, fungal treatment, and duration of infection. Gene ontology enrichment analysis of genes highly expressed in infected kernels identified molecular pathways associated with defense responses to fungi and other microbes such as production of pathogenesis-related (PR) proteins and lipid bilayer formation. To further identify novel genes of interest, we incorporated genomic and phenotypic field data from a genome wide association analysis with gene expression data, allowing us to detect significantly expressed quantitative trait loci (eQTL). These results identified significant association between flavonoid biosynthetic pathway genes and infection by A. flavus. In planta fungal infections showed that the resistant line, TZAR102, has a higher fold increase of the metabolites naringenin and luteolin than the susceptible line, Va35, when comparing untreated and fungal infected plants. These results suggest flavonoids contribute to plant resistance mechanisms against aflatoxin contamination through modulation of toxin accumulation in maize kernels.

Genetic Responses and Aflatoxin Inhibition during Co-Culture of Aflatoxigenic and Non-Aflatoxigenic Aspergillus flavus.

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus. Non-aflatoxigenic (Non-tox) A. flavus isolates are deployed in corn fields as biocontrol because they substantially reduce aflatoxin contamination via direct replacement and additionally via direct contact or touch with toxigenic (Tox) isolates and secretion of inhibitory/degradative chemicals. To understand touch inhibition, HPLC analysis and RNA sequencing examined aflatoxin production and gene expression of Non-tox isolate 17 and Tox isolate 53 mono-cultures and during their interaction in co-culture. Aflatoxin production was reduced by 99.7% in 72 h co-cultures. Fewer than expected unique reads were assigned to Tox 53 during co-culture, indicating its growth and/or gene expression was inhibited in response to Non-tox 17. Predicted secreted proteins and genes involved in oxidation/reduction were enriched in Non-tox 17 and co-cultures compared to Tox 53. Five secondary metabolite (SM) gene clusters and kojic acid synthesis genes were upregulated in Non-tox 17 compared to Tox 53 and a few were further upregulated in co-cultures in response to touch. These results suggest Non-tox strains can inhibit growth and aflatoxin gene cluster expression in Tox strains through touch. Additionally, upregulation of other SM genes and redox genes during the biocontrol interaction demonstrates a potential role of inhibitory SMs and antioxidants as additional biocontrol mechanisms and deserves further exploration to improve biocontrol formulations.

Genome Sequences of 20 Georeferenced Aspergillus flavus Isolates.

Several agricultural commodities can be infected by Aspergillus flavus, a fungus that can produce the carcinogen aflatoxin. Here, we report the whole-genome sequences for 20 georeferenced isolates collected from soil and corn under field conditions. This information contributes to an understanding of A. flavus population structure and dynamics in a field environment.

The Transcriptional Regulator Hbx1 Affects the Expression of Thousands of Genes in the Aflatoxin-Producing Fungus Aspergillus flavus.

In filamentous fungi, homeobox proteins are conserved transcriptional regulators described to control conidiogenesis and fruiting body formation. Eight homeobox (hbx) genes are found in the genome of the aflatoxin-producing ascomycete, Aspergillus flavus While loss-of-function of seven of the eight genes had little to no effect on fungal growth and development, disruption of hbx1, resulted in aconidial colonies and lack of sclerotial production. Furthermore, the hbx1 mutant was unable to produce aflatoxins B1 and B2, cyclopiazonic acid and aflatrem. In the present study, hbx1 transcriptome analysis revealed that hbx1 has a broad effect on A. flavus gene expression, and the effect of hbx1 increases overtime, impacting more than five thousand protein-coding genes. Among the affected genes, those in the category of secondary metabolism (SM), followed by that of cellular transport, were the most affected. Specifically, regarding the effect of hbx1 on SM, we found that genes in 44 SM gene clusters where upregulated while 49 were downregulated in the absence of hbx1, including genes in the SM clusters responsible for the synthesis of asparasone, piperazine and aflavarin, all known to be associated with sclerotia. In addition, our study revealed that hbx1 affects the expression of other transcription factor genes involved in development, including the conidiation central regulatory pathway and flb genes.

Characterization of BEAF mutations isolated by homologous recombination in Drosophila.

The Drosophila BEAF-32A and BEAF-32B proteins bind to the scs' insulator and to hundreds of other sites on Drosophila chromosomes. These two proteins are encoded by the same gene. We used ends-in homologous recombination to generate the null BEAF(AB-KO) allele and also isolated the BEAF(A-KO) allele that eliminates production of only the BEAF-32A protein. We find that the BEAF proteins together are essential, but BEAF-32B alone is sufficient to obtain viable flies. Our results show that BEAF is important for both oogenesis and development. Maternal or zygotic BEAF is sufficient to obtain adults, although having only maternal BEAF impairs female fertility. In the absence of all BEAF, a few fertile but sickly males are obtained. Using both a chromosomal position-effect assay and an enhancer-blocking assay, we find that BEAF is necessary for scs' insulator function. Lack of BEAF causes a disruption of male X polytene chromosome morphology. However, we did not find evidence that dosage compensation was affected. Position-effect variegation of the w(m4h) allele and different variegating y transgenes was enhanced by the knockout mutation. Combined with the effects on male X polytene chromosomes, we conclude that BEAF function affects chromatin structure or dynamics.

Aspergillus flavus Secondary Metabolites: More than Just Aflatoxins.

Aspergillus flavus is best known for producing the family of potent carcinogenic secondary metabolites known as aflatoxins. However, this opportunistic plant and animal pathogen also produces numerous other secondary metabolites, many of which have also been shown to be toxic. While about forty of these secondary metabolites have been identified from A. flavus cultures, analysis of the genome has predicted the existence of at least 56 secondary metabolite gene clusters. Many of these gene clusters are not expressed during growth of the fungus on standard laboratory media. This presents researchers with a major challenge of devising novel strategies to manipulate the fungus and its genome so as to activate secondary metabolite gene expression and allow identification of associated cluster metabolites. In this review, we discuss the genetic, biochemical and bioinformatic methods that are being used to identify previously uncharacterized secondary metabolite gene clusters and their associated metabolites. It is important to identify as many of these compounds as possible to determine their bioactivity with respect to fungal development, survival, virulence and especially with respect to any potential synergistic toxic effects with aflatoxin.

Whole genome comparison of Aspergillus flavus L-morphotype strain NRRL 3357 (type) and S-morphotype strain AF70.

Aspergillus flavus is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within A. flavus, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 µm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 µm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an A. flavus S morphotype, strain AF70. We provide a comprehensive comparison of the A. flavus S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.

The Drosophila boundary element-associated factors BEAF-32A and BEAF-32B affect chromatin structure.

Binding sites for the Drosophila boundary element-associated factors BEAF-32A and -32B are required for the insulator activity of the scs' insulator. BEAF binds to hundreds of sites on polytene chromosomes, indicating that BEAF-utilizing insulators are an important class in Drosophila. To gain insight into the role of BEAF in flies, we designed a transgene encoding a dominant-negative form of BEAF under GAL4 UAS control. This BID protein encompasses the BEAF self-interaction domain. Evidence is provided that BID interacts with BEAF and interferes with scs' insulator activity and that BEAF is the major target of BID in vivo. BID expression during embryogenesis is lethal, implying that BEAF is required during early development. Expression of BID in eye imaginal discs leads to a rough-eye phenotype, and this phenotype is rescued by a third copy of the BEAF gene. Expression of BID in salivary glands leads to a global disruption of polytene chromatin structure, and this disruption is largely rescued by an extra copy of BEAF. BID expression also enhances position-effect variegation (PEV) of the w(m4h) allele and a yellow transgene inserted into the pericentric heterochromatin of chromosome 2R, while a third copy of the BEAF gene suppresses PEV of both genes. These results support the hypothesis that BEAF-dependent insulators function by affecting chromatin structure or dynamics.

Carbon Dioxide Mediates the Response to Temperature and Water Activity Levels in Aspergillus flavus during Infection of Maize Kernels.

Aspergillus flavus is a saprophytic fungus that may colonize several important crops, including cotton, maize, peanuts and tree nuts. Concomitant with A. flavus colonization is its potential to secrete mycotoxins, of which the most prominent is aflatoxin. Temperature, water activity (aw) and carbon dioxide (CO2) are three environmental factors shown to influence the fungus-plant interaction, which are predicted to undergo significant changes in the next century. In this study, we used RNA sequencing to better understand the transcriptomic response of the fungus to aw, temperature, and elevated CO2 levels. We demonstrate that aflatoxin (AFB1) production on maize grain was altered by water availability, temperature and CO2. RNA-Sequencing data indicated that several genes, and in particular those involved in the biosynthesis of secondary metabolites, exhibit different responses to water availability or temperature stress depending on the atmospheric CO2 content. Other gene categories affected by CO2 levels alone (350 ppm vs. 1000 ppm at 30 °C/0.99 aw), included amino acid metabolism and folate biosynthesis. Finally, we identified two gene networks significantly influenced by changes in CO2 levels that contain several genes related to cellular replication and transcription. These results demonstrate that changes in atmospheric CO2 under climate change scenarios greatly influences the response of A. flavus to water and temperature when colonizing maize grain.

Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production.

Effects of Aspergillus flavus colonization of maize kernels under different water activities (aw; 0.99 and 0.91) and temperatures (30, 37°C) on (a) aflatoxin B1 (AFB1) production and (b) the transcriptome using RNAseq were examined. There was no significant difference (p=0.05) in AFB1 production at 30 and 37°C and 0.99 aw. However, there was a significant (p=0.05) increase in AFB1 at 0.91 aw at 37°C when compared with 30°C/0.99 aw. Environmental stress effects using gene ontology enrichment analysis of the RNA-seq results for increasing temperature at 0.99 and 0.91 aw showed differential expression of 2224 and 481 genes, respectively. With decreasing water availability, 4307 were affected at 30°C and 702 genes at 37°C. Increasing temperature from 30 to 37°C at both aw levels resulted in 12 biological processes being upregulated and 9 significantly downregulated. Decreasing aw at both temperatures resulted in 22 biological processes significantly upregulated and 25 downregulated. The interacting environmental factors influenced functioning of the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA). An elevated number of genes were co-regulated by both aw and temperature. An interaction effect for 4 of the 25 AFB1 genes, including regulatory and transcription activators occurred. For CPA, all 5 biosynthetic genes were affected by aw stress, regardless of temperature. The molecular regulation of A. flavus in maize is discussed.

Draft Genome Sequence of an Aflatoxigenic Aspergillus Species, A. bombycis.

Aspergillus bombycis was first isolated from silkworm frass in Japan. It has been reportedly misidentified as A. nomius due to their macro-morphological and chemotype similarities. We sequenced the genome of the A. bombycis Type strain and found it to be comparable in size (37 Mb), as well as in numbers of predicted genes (12,266), to other sequenced Aspergilli. The aflatoxin gene cluster in this strain is similar in size and the genes are oriented the same as other B- + G-aflatoxin producing species, and this strain contains a complete but nonfunctional gene cluster for the production of cyclopiazonic acid. Our findings also showed that the A. bombycis Type strain contains a single MAT1-2 gene indicating that this species is likely heterothallic (self-infertile). This draft genome will contribute to our understanding of the genes and pathways necessary for aflatoxin synthesis as well as the evolutionary relationships of aflatoxigenic fungi.