Flavohemoglobins are widely distributed in both prokaryotes and eukaryotes. These proteins are involved in reducing nitric oxide levels. Deletion of the Aspergillus nidulans flavohemoglobin gene fhbA induced sexual development and decreased sterigmatocystin production. Supplementation with a nitric oxide-releasing compound promoted cleistothecial formation and increased nsdD and steA expression, indicating that nitric oxide induces sexual development. This is the first study on the effect of nitric oxide on morphogenesis and secondary metabolism in fungi.Nitric oxide (NO) is a signaling compound of great importance in biological systems (7,10,21). This molecule can also cause nitrosative stress which is potentially remediated by the widely distributed flavohemoglobins (FHbs) found in both eukaryotes and prokaryotes (4, 7). Extensive research on FHb proteins from bacteria and yeast revealed their structure, function, and mechanism of action (3,7,17). Earlier studies have shown that FHbs in organisms such as Saccharomyces cerevisiae, Alcaligenes eutrophus, and Escherichia coli share similar steady-state NO dioxygenation kinetics (7). Through a dioxygenase-mediated reaction, FHbs, in the presence of molecular O 2 , converts NO into nontoxic nitrate ions. FHb proteins contain a hemoglobin-like domain with a noncovalently bound heme B protein and a reductase domain with binding sites for FAD and NAD(P)H. It is known that fhb genes are activated by various agents, such as nitrate, nitrite, NO, and NO-releasing agents (5,7,8,11,20,24). In aspergilli, the conversion of NO to NO 3 Ϫ by FHbs, Fhb1 and Fhb2 in Aspergillus oryzae (30) and FhbA and FhbB in the filamentous fungus model Aspergillus nidulans, has been demonstrated (24). The present work involves the study of the role of FHbs and NO in fungal development and secondary metabolism.Fungal strains and growth conditions. Aspergillus nidulans Cib08 (biA1 yA2), CibA (biA1 yA2 ⌬fhbA::argB), CibB (biA1 yA2 ⌬fhbB::argB) (described in reference 24), and FGSCA4 were used in this study. The strains were cultured on glucose minimum medium (GMM) plus the appropriate supplements for the corresponding auxotrophic markers (13). Medium was supplemented with 1.5 mM diethylenetriamine-NoNoate (Sigma), a NO-releasing compound, after sterilization when indicated. Solid medium was prepared by adding 10 g/liter agar. Strains were stored as 30% glycerol stocks at Ϫ80°C. Morphological studies.Plates containing 25 ml of solid GMM plus the appropriate supplements were top agar inoculated with 5 ϫ 10 6 spores per plate of medium. The cultures were wrapped and incubated at 37°C in the dark. Cores (8-mm diameter) were collected from each spread plate and examined for cleistothecium production. Ethanol (70%) was sprayed on the core surface to facilitate the visualization of cleistothecia under the microscope. The experiments included five replicates and were repeated twice, with similar results. mRNA analysis. Total RNA was isolated from mycelia at 48 h and 72 h after inoculation as previously ...

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.