RESUMO
The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.
Assuntos
Aflatoxinas/genética , Aspergillus flavus/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Reguladores/genética , Família Multigênica/genética , Metabolismo Secundário/genética , Fatores de Transcrição/genética , Aflatoxinas/biossíntese , Aspergillus flavus/patogenicidade , Perfilação da Expressão Gênica , Transcriptoma/genéticaRESUMO
Production of carcinogenic aflatoxins has been reported from members of Aspergillus section Flavi, Aspergillus section Nidulantes and a newly proposed Aspergillus section Ochraceorosei that consists of Aspergillus ochraceoroseus and A. rambellii. Unlike members of section Flavi, A. ochraceoroseus and A. rambellii have been shown to accumulate both aflatoxin (AF) and the aflatoxin precursor sterigmatocystin (ST). Alhough morphologically distinct from A. nidulans, molecular characterization of A. ochraceoroseus AF/ST genes and physiological characteristics of AF/ST production indicated that A. ochraceoroseus is more closely related to A. nidulans than to A. flavus. Knowing that the A. nidulans ST gene cluster is organized differently from the A. flavus AF gene cluster, we determined the genetic organization of the AF/ST biosynthetic cluster in A. ochraceoroseus. Sequencing of overlapping lambda clones and genomic PCR fragments obtained by gene-walking techniques demonstrated that the A. ochraceoroseus AF/ST gene cluster is organized much like the A. nidulans ST gene cluster except that the region from aflN to aflW is located directly upstream of aflC and in reverse orientation such that aflW represents the distal end and aflY the proximal end of the cluster. The A. ochraceoroseus cluster genes demonstrated 62-76% nucleotide identity to their A. nidulans ST cluster gene homologs. Transformation of an A. nidulans aflR mutant with the A. ochraceoroseus aflR restored ST production in A. nidulans transformants. PCR amplification of A. rambellii genomic DNA demonstrated that the AF/ST gene cluster is organized in the same manner as that of A. ochraceoroseus.
Assuntos
Aflatoxinas/genética , Aspergillus ochraceus/genética , Família Multigênica , Esterigmatocistina/biossíntese , Aflatoxinas/biossíntese , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Aspergillus nidulans/efeitos dos fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Aspergillus ochraceus/metabolismo , Northern Blotting , Ciclopentanos/farmacologia , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Variação Genética , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Ativação Transcricional/efeitos dos fármacosRESUMO
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.
