VeA is associated with the response to oxidative stress in the aflatoxin producer Aspergillus flavus

Abstract

Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms to counteract and survive the stress in the presence of ROS. In many fungi, the HOG signaling pathway is crucial for the oxidative stress response as well as for osmotic stress response. This study revealed that while the osmotic stress response is only slightly affected by the master regulator veA, this gene, also known to control morphological development and secondary metabolism in numerous fungal species, has a profound effect on the oxidative stress response in the aflatoxin-producing fungus Aspergillus flavus. We found that the expression of A. flavus homolog genes involved in the HOG signaling pathway is regulated by veA. Deletion of veA resulted in a reduction in transcription levels of oxidative stress response genes after exposure to hydrogen peroxide. Furthermore, analyses of the effect of VeA on the promoters of cat1 and trxB indicate that the presence of VeA alters DNA-protein complex formation. This is particularly notable in the cat1 promoter, where the absence of VeA results in abnormally stronger complex formation with reduced cat1 expression and more sensitivity to ROS in a veA deletion mutant, suggesting that VeA might prevent binding of negative transcription regulators to the cat1 promoter. Our study also revealed that veA positively influences the expression of the transcription factor gene atfB and that normal formation of DNA-protein complexes in the cat1 promoter is dependent on AtfB.

FIG 1

Effect of osmotic stress on A. flavus colony growth and conidiation. (A) Photographs of A. flavus 70S, psl82, and ΔveA point-inoculated cultures incubated for 5 days. The medium was supplemented with 0.6 M NaCl, 0.7 M KCl, or 1.0 M sorbitol to generate osmotic stress. (B) Colony growth measured as colony diameter. (C) Quantification of conidial production. Cores (16 mm in diameter) were taken 1 cm from the center of the plates and homogenized in water. Spores were counted with a hemocytometer. Error bars represent standard errors. Experiments were carried out with three replicates. Different letters indicate samples that are significantly different (P ≤ 0.05).

FIG 2

Effect of osmotic stress on sclerotial production. Microscopic examination of sclerotia. Sclerotial production on top agar-inoculated A. flavus 70S, psl82, andΔveA cultures was observed after 5 days of incubation at 30°C. The medium was supplemented with 0.6 M NaCl, 0.7 M KCl, or 1.0 M sorbitol to generate osmotic stress. Images (×32) were captured using an upright Leica MZ75 stereomicroscope.

FIG 3

veA is necessary for a normal response to oxidative stress in A. flavus. Photographs of point-inoculated A. flavus 70S, psl82, and ΔveA strains growing on YGT supplemented with increasing amounts of hydrogen peroxide. Culture numbers: 1, 70S; 2, psl82; and 3, ΔveA mutant. Cultures were incubated at 30°C for 3 days.

FIG 4

veA is required for wild-type expression levels of A. flavus oxidative stress response genes. Relative expression levels of A. flavus genes involved in the oxidative stress response 6 and 24 h after addition of 15 mM hydrogen peroxide. The relative expression levels were calculated by the method described by Kenneth and Schmittgen (26), and all values were normalized to the expression of the A. flavus 18S rRNA gene and to the greatest expression, considered 100.

FIG 5

The formation of protein-DNA complexes at the cat1 and trxB promoters is influenced by VeA. (A) Schematic representation of promoter regions of the cat1 and trxB genes used in EMSA. The cat1 promoter region was divided into two fragments designated cat1-1 and cat1-2. The trxB promoter region was also divided into two fragments, designated trxB-1 and trxB-2. (B and C) DNA-protein complex formation on cat1-2 and trxB-2 promoters by EMSA using 70S, psl82, or ΔveA strain protein extracts with or without H₂O₂ treatment. Cell extracts enriched in nuclear proteins were prepared as described in Materials and Methods. Five micrograms of enriched nuclear protein extracts was added to a ³²P-labeled promoter probe for each gene.

FIG 6

Analysis of AtfB binding to the cat1 and trxB promoters and its relationship with VeA. Shown are results from EMSA of AtfB binding in the cat1-2 (A to C) and trxB-2 (D to E) promoter fragments using 70S or ΔveA mutant protein extracts with or without H₂O₂ treatment. Enriched nuclear protein extracts were prepared as described in Materials and Methods. Five micrograms of enriched nuclear protein extracts was added to a ³²P-labeled promoter probe for each gene. Anti-AtfB antibodies (YSR) or preimmune serum was added to determine whether these could block protein-DNA interaction (shift inhibition). (A) cat1-2 probe and 70S protein extracts. (B) cat1-2 probe and ΔveA protein extracts. (C) cat1-2 probe and anti-AtfB antibodies alone (without protein extracts). (D) trxB-2 probe and 70S protein extracts. (E) trxB-2 probe and ΔveA protein extracts.

FIG 7

The presence of a VeA antibody did not prevent protein-DNA complex formation at the cat1 and trxB promoters in shift inhibition EMSA. Shown are results from EMSA of VeA binding in the cat1-2 and trxB-2 promoter fragments using 70S or ΔveA mutant protein extracts with or without H₂O₂ treatment. Enriched nuclear protein extracts were prepared as described in Materials and Methods. Five micrograms of enriched nuclear protein extracts was added to a ³²P-labeled promoter probe for each gene. Anti-VeA antibodies or preimmune serum was added to determine whether these could block protein-DNA interaction (shift inhibition). (A) cat1-2 probe and 70S protein extracts. (B) cat1-2 probe and ΔveA protein extracts. (C) trxB-2 probe and 70S protein extracts. (D) trxB-2 probe and ΔveA protein extracts.