Gregory R. OBrian

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.

Identification of genes differentially expressed during aflatoxin biosynthesis in Aspergillus flavus and Aspergillus parasiticus.

A complex regulatory network governs the biosynthesis of aflatoxin. While several genes involved in aflatoxin production are known, their action alone cannot account for its regulation. Arrays of clones from an Aspergillus flavus cDNA library and glass slide microarrays of ESTs were screened to identify additional genes. An initial screen of the cDNA clone arrays lead to the identification of 753 unique ESTs. Many showed sequence similarity to known metabolic and regulatory genes; however, no function could be ascribed to over 50% of the ESTs. Gene expression analysis of Aspergillus parasiticus grown under conditions conducive and non-conductive for aflatoxin production was evaluated using glass slide microarrays containing the 753 ESTs. Twenty-four genes were more highly expressed during aflatoxin biosynthesis and 18 genes were more highly expressed prior to aflatoxin biosynthesis. No predicted function could be ascribed to 18 of the 24 genes whose elevated expression was associated with aflatoxin biosynthesis.

Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus

BackgroundAn available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent.ResultsThe efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study.ConclusionWe employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations.To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.

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.

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.

Comparative analysis of BiP gene expression in maize endosperm

Binding protein (BiP) is the endoplasmic reticulum member of the highly conserved HSP70 (heat shock protein 70) family of molecular chaperones. We have isolated and characterized two different BiP cDNA clones corresponding to genes expressed in immature kernels. These two cDNAs share extensive sequence similarity but map to unlinked loci in the maize genome. A comparison of the aa sequences predicted from the cDNA clones revealed only six aa differences between them. Investigation of gene-specific expression was carried out by RNA gel blot analysis. RNAs corresponding to both cDNA clones were present in increased amounts in the endosperm of floury-2 (fl2), Mucronate (Mc) and Defective endosperm-B30 (De*-B30) maize mutants, which produce abnormal storage proteins. Similar increases in RNAs corresponding to both probes were detected in cells treated with either of two agents that interfere with protein folding, azetidine-2-carboxylic acid (AZC) and tunicamycin. Investigation of the genomic complexity of the BiP genes by Southern blot analysis revealed several cross-hybridizing bands. These results are suggestive that the BiP genes expressed in endosperm are coordinately regulated members of a more complex maize BiP multigene family.

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.

Cloning and Sequencing of a Second Ribosome-Inactivating Protein Gene from Maize (Zea mays L.)

Toxic proteins are among the wide array of antibiotic compounds produced by plants. Some of the most potent plant toxins belong to a class of translational inhibitors called RIPs (Stirpe et al., 1992). RIPs act as N-glycosidases to remove a specific adenine from the large rRNA, thereby irreversibly inactivating the ribosome. A maize Rip gene encodes a highly expressed seed-specific RIP (also called b-32 or proRIP) that accumulates under the control of the transcriptional activator, Opaque-2, as a nontoxic, singlechain zymogen (Soave et al., 1981; Walsh et al., 1991; Bass et al., 1992). During germination the RIP is proteolytically cleaved to an active form with two noncovalently linked chains of 8 and 16 kD (Walsh et al., 1991). A genomic library was screened with a cDNA clone for the seed RIP. This led to the isolation of a genomic clone for a second, nonallelic Rip gene from maize. The sequence of a 963-bp segment within this clone is described in Table I. This region includes an ORF that predicts a 31-kD polypeptide with 73% amino acid identity with maize seed proRIP. Among the residues shared are the five active site cleft residues found in a11 plant RIPs for which data are available (Bass et al., 1992). Genomic Southern analysis and recombinant inbred restriction fragment length polymorphism mapping results are consistent with a two-member Rip gene family. One member corresponds to the seed-specific Rip gene, which has been previously mapped by segregation analysis of allelic protein isoforms to chromosome 8L (Soave et al., 1981). The Rip gene described here corresponds to a second member that can be placed on chromosome 7L (Bass et al., 1990). The existence of multiple RIPs, and in some cases organ-specific isoforms, has also been observed in severa1 other plant species (Stirpe et al., 1992). We propose that the seed-specific Rip gene be designated Rip3:l for the first member of the maize Rip multigene family and that the gene described here be designated Rip3:2 (Mundy et al., 1994). Alignment of the deduced amino acid sequences of the two maize RIPs reveals little similarity in the regions cor-

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.

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.