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Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus

ABSTRACT: Aspergillus flavus and A. parasiticus are two of the most important aflatoxin-producing species that contaminate agricultural commodities worldwide. Both species are heterothallic and undergo sexual reproduction in laboratory crosses. Here, we examine the possibility of interspecific matings between A. flavus and A. parasiticus. These species can be distinguished morphologically and genetically, as well as by their mycotoxin profiles. Aspergillus flavus produces both B aflatoxins and cyclopiazonic acid (CPA), B aflatoxins or CPA alone, or neither mycotoxin; Aspergillus parasiticus produces B and G aflatoxins or the aflatoxin precursor O-methylsterigmatocystin, but not CPA. Only four out of forty-five attempted interspecific crosses between compatible mating types of A. flavus and A. parasiticus were fertile and produced viable ascospores. Single ascospore strains from each cross were isolated and were shown to be recombinant hybrids using multilocus genotyping and array comparative genome hybridization. Conidia of parents and their hybrid progeny were haploid and predominantly monokaryons and dikaryons based on flow cytometry. Multilocus phylogenetic inference showed that experimental hybrid progeny were grouped with naturally occurring A. flavus L strain and A. parasiticus. Higher total aflatoxin concentrations in some F1 progeny strains compared to midpoint parent aflatoxin levels indicate synergism in aflatoxin production; moreover, three progeny strains synthesized G aflatoxins that were not produced by the parents, and there was evidence of putative allopolyploidization in one strain. These results suggest that hybridization is an important diversifying force resulting in the genesis of novel toxin profiles in these agriculturally important species.

ORGANISM(S): Zea mays Aspergillus parasiticus Aspergillus oryzae Aspergillus caelatus Aspergillus flavus Aspergillus nomiae Aspergillus tamarii Aspergillus parasiticus x Aspergillus flavus

PROVIDER: GSE61981 | GEO | 2015/10/01

SECONDARY ACCESSION(S): PRJNA262861

REPOSITORIES: GEO

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Project description:Aspergillus flavus is the major producer of carcinogenic aflatoxins in crops worldwide. Natural populations of A. flavus show tremendous variation in aflatoxin production some of which can be attributed to extreme environmental conditions (e.g., drought), differential regulation of the aflatoxin biosynthetic pathway, missing cluster genes or loss-of-function mutations. Understanding the evolutionary processes that generate genetic diversity in A. flavus may also explain quantitative and qualitative differences in aflatoxigenicity. Several population studies provide indirect evidence of recombination in the aflatoxin gene cluster and genome-wide, using multilocus genealogical approaches. More recently A. flavus has been shown to be functionally heterothallic and capable of sexual reproduction in laboratory crosses. In the present study, we characterize the progeny from nine A. flavus crosses and show that crossovers in the aflatoxin cluster coincide with inferred recombination blocks and hotspots in natural populations, which suggests that recombination in the cluster is primarily driven by sex. Moreover, we show that a single crossover event in the cluster can restore aflatoxigenicity, which is significant as mycotoxin production in A. flavus is highly heritable. aCGH was used to corroborate inferences from cluster-based MLSTs and to possibly identify additional crosovers within the cluster. aCGH comparison between 3 strains of A. flavus: 2 parental (P) and 1 progeny (F1) analyzed at the probe level. A total of 9 trio comparisons were made from a total of 18 isolates analyzed by aCGH. Trio comparisons are as follows: IC278 (P), IC1179 (P) and IC1650 (F1); IC201 (P), IC310 (P) and IC1719 (F1); IC307 (P), IC308 (P) and IC1751 (F1); IC277 (P), IC311 (P) and IC1766 (F1); IC277 (P), IC311 (P) and IC1775 (F1); IC244 (P), IC277 (P) and IC2205 (F1); IC244 (P), IC277 (P) and IC2207 (F1); IC301 (P), IC1179 (P) and IC2171 (F1); and finally IC244 (P), IC277 (P) and IC2209 (F1).

Project description:Aflatoxins are carcinogenic fungal secondary metabolites. Levels of aflatoxins in agricultural commodities are stringently regulated by many countries. A cluster of genes is responsible for aflatoxin biosynthesis by Aspergillus flavus and other closely related species. Expression of the clustered aflatoxin genes is governed by a complex network of regulatory mechanisms. To better understand the molecular events that are associated with aflatoxin production, transcription profiling by microarray analyses which compared three independent aflatoxigenic A. flavus strains to individual isogenic progenies that no longer produced aflatoxins after serial transfers was carried out. Twenty-two significantly differentially expressed features were identified. After physical mapping using the A. oryzae genome sequence as the reference, the number of unique genes was reduced to 16. Compared to the parental strains, changes in the aflatoxin gene expression levels in the progenies were not significant, which suggests that the inability to produce aflatoxins is not caused by decreased expression. The only gene showing higher expression levels in the progenies is homologous to glutathione S-transferease genes. Overexpression of this gene, named hcc, at six- to nine-fold in an aflatoxigenic A. flavus did not cause discernible changes in colony morphology or aflatoxin production. Loss of aflatoxin production after serial transfers may not result from a single event but caused by multiple factors. Keywords: Compartiave hybridization toxigenic and atoxigenic lines of Aspergillus Aspergillus flavus NRRL 29459, NRRL 29474, and NRRL 29490 are aflatoxigenic strains originated from soil collection in a peanut field (Terrell Co., Georgia, USA). Strains 459B-20-2, 474A-20, and 499A-20 were nonaflatoxigenic isolates obtained after 20 serial transfers of the parental strains on potato dextrose agar slants (Horn and Dorner 2002). Comparsions in each experiment consisted of one aflatoxigenic parental strain and one nonaflatoxigenic progeny, compared after 48- or 72-hr growth. Each comparison was repeated with duplicate dye-flip.

Project description:Diverse expression patterns for secondary metabolism gene clusters from Aspergillus flavus under different environmental conditions and in genetic mutants: Insights into regulation of cyclopiazonic acid along with aflatoxin Species of Aspergillus produce a diverse array of secondary metabolites, and recent genomic analysis predicts 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 A. flavus. Presumably, secondary metabolites play important roles in the ecology of the producing species, but functions for most secondary metabolites remain unknown. Only three metabolic pathways have been associated with the predicted clusters in A. flavus. These include aflatoxin, cyclopiazonic acid (CPA), and aflatrem. To gain insight into the regulation of, and infer ecological significance for the 55 secondary metabolite gene clusters predicted in A. flavus, we examined their expression over 28 diverse conditions. Variables included culture media and temperature, fungal development, colonization of developing maize seeds, and misexpression of laeA, the 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 non-conducive for aflatoxin biosynthesis and during 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 similar enough that they would be expected to co-occur in commodities colonized with A. flavus.

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Enhanced diversity and aflatoxigenicity in interspecific hybrids of Aspergillus flavus and Aspergillus parasiticus

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