GTPase Rac Regulates Conidiation, AFB1 Production and Stress Response in Pathogenic Fungus Aspergillus flavus.

As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis, and immunosuppression. In this study, by constructing Rac-deleted mutants in Aspergillus flavus, it was found that the deletion of Rac gene led to the decline of growth and development, conidia production, AFB1 toxin synthesis, and seed infection ability of A. flavus. The deletion of Rac gene also caused the disappearance of A. flavus sclerotium, indicating that Rac is required for sclerotium formation in A. flavus. The sensitivity of Rac-deficient strains responding to cell wall stress and osmotic pressure stress increased when compared to A.flavus WT. The Western blot result showed that mitogen-activated serine/threonine-protein kinase Slt2 and mitogen-activated protein kinase Hog1 proteins were no longer phosphorylated in Rac-deficient strains of A. flavus, showing that Rac may be used as a molecular switch to control the Slt2-MAPK cascade pathway and regulate the osmotic Hog-MAPK cascade pathway in A. flavus in response to external stress. Altogether, these results indicated that Rac was involved in regulating the growth and development, conidia formation and AFB1 synthesis, and response to cell wall stress and osmotic pressure stress in A. flavus.

Cellular Apoptosis Induced by Deoxynivalenol.

Deoxynivalenol (DON) is synthesized by Fusarium species that frequently infect crops during storage, and it's harm risk to human is reflected in the consumption of infected food crops or indirectly through foods of animal origin. In this study, Hela and Chang liver cells were used to research the cellular apoptosis induced by deoxynivalenol. Cells were treated by DON toxin with a series of concentration and incubated for different time. MTT, fluorescence microscope, flow cytometer and Western blot methods were used to analyze the effect of DON on the cell apoptosis in vitro and in vivo systematically. The results showed that DON was toxic to the cells tested. After being treated by DON, the morphology of Chang livers and Hela cells changed significantly. The DON promoted apoptosis in a dose- and time-dependent manner. The activity of Caspase 3 was significantly increased in DON-induced apoptosis. Moreover, endogenous Glutathione (GSH) level in these cell lines was gradually decreased. In the early apoptosis progress, oxidative stress was induced by DON. When DON reached 10 µg/mL, a markedly increased content of Malondialdehyde (MDA) was detected in both Hela and Chang liver cells. Furthermore, an in vivo test indicated that DON had toxicity to mice by causing weight loss and swollen spleen, and significantly increased expression of AST and ALT. In conclusion, the DON was toxic to mice and could induce the apoptosis of tested cells undergoing a Caspase-3 related pathway.

The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus.

As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen-activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Δmsb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Δmsb2C ) in A. flavus. Using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down-regulated aflatoxin B1 (AFB1 ) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Δmsb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Δmsb2C . Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.