The MAP kinase AflSlt2 modulates aflatoxin biosynthesis and peanut infection in the fungus Aspergillus flavus.

Aflatoxin contamination in food and feed products has been brought into sharp focus over the last few decades in the world. However, there is no effective strategy for solving the problem thus far. Therefore, basic research on the aflatoxin-producer Aspergillus flavus is an urgent need. The vital role of mitogen-activated protein kinases (MAPKs) in signal transduction has been documented in various pathogenic fungi, but their functions in A. flavus have rarely been investigated. Herein, we characterized the detailed function of one of these MAPKs, AflSlt2. Targeted deletion of AflSlt2 gene indicates that this kinase is required for vegetative growth, conidia generation, and sclerotium formation. The analysis of AflSlt2 deletion mutant revealed hypersensitivity to cell wall-damaging chemicals and resistance against hydrogen peroxide. Interestingly, the ability of the ΔAflSlt2 mutant to generate aflatoxins in medium was significantly increased compared to wild type. However, a pathogenicity assay indicated that the ΔAflSlt2 mutant was deficient in peanut infection. Site-directed mutation study uncovered that the function of AflSlt2 was dependent on the phosphorylated residues (Thr-186 and Tyr-188) within the activation loop and the phosphotransfer residue (Lys-52) within the subdomain II. Interestingly, an autophosphorylation mutant of AflSlt2 (AflSlt2R66S) displayed wild type-like phenotypes. Bringing these observations together, we propose that Slt2-MAPK pathway is involved in development, stress response, aflatoxin biosynthesis, and pathogenicity in A. flavus. This study may be useful to unveil the regulation mechanism of aflatoxin biosynthesis and provide strategy to control A. flavus contamination.

Detection of okadaic acid (OA) and tetrodotoxin (TTX) simultaneously in seafood samples using colloidal gold immunoassay.

Tetrodotoxin (TTX) is a neurotoxin mainly responsible for severe neurological illness, and okadaic acid (OA) is another important lipophilic toxin to humans. In this study, we developed a gold strip for simultaneous detection of OA and TTX in real seafood samples. In the assay, the prepared nanoparticles (about 40 nm) was applied to conjugate with specific monoclonal antibodies against OA and TTX, and the resulted mixtures were used to capture its corresponding toxin in test strip. OA and TTX conjugates were coated as two test lines on the nitrocellulose membrane, and goat anti-mouse IgG was used to form the control line, forming three lines on the test strip. The visual detection limits (vLOD) of this immunoassay for OA and TTX were 0.75 and 15 ng/mL, respectively, and no cross reactions were observed in the process of detection. The visual assay for OA and TTX detection could be finished within 10 min. This study might provide a feasible method and good understanding for rapidly simultaneous detection for toxins based on immunoassay.