The high-affinity phosphodiesterase PdeH regulates development and aflatoxin biosynthesis in Aspergillus flavus

Abstract

Cyclic AMP signaling controls a range of physiological processes in response to extracellular stimuli in organisms. Among the signaling cascades, cAMP, as a second messenger, is orchestrated by adenylate cyclase (biosynthesis) and cAMP phosphodiesterases (PDEs) (hydrolysis). In this study, we investigated the function of the high-affinity (PdeH) and low-affinity (PdeL) cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus Aspergillus flavus, and found that instead of PdeL, inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation. However, the ΔpdeL/ΔpdeH mutant exhibited an enhanced phenotype defects, a similar phenotype defects to wild-type strain treated with exogenous cAMP. The activation of PKA activity was inhibited in the ΔpdeH or ΔpdeL/ΔpdeH mutant, both of whom exhibited increasing AF production. Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain, and affected pdeL transcription. Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol, while PdeL-GFP localized mainly to the nucleus. Overall, our results indicated that PdeH plays a major role, but has overlapping function with PdeL, in vegetative growth, development and AF biosynthesis in A. flavus.

Enhancement of Monascus Azaphilone Pigments Production by Activating the cAMP Signalling Pathway in Monascus Purpureus HJ11

“…Similar study has been reported in F. graminearum, the knockout of PDE gene pde1 resulted in increased production of secondary metabolite deoxynivalenol [23]. The deletion of PDE gene pdeH from A. avus led to an increased production of a atoxin to 110 mg/mL from 48 mg/mL [38].…”

Antioxidant‐related catalase CTA1 regulates development, aflatoxin biosynthesis, and virulence in pathogenic fungus Aspergillus flavus

“…The chloroform in sub-layer was dried in the fume hood overnight, and the precipitates were resuspended in 100 μl chloroform. TLC was used to analyse AF production, as described previously (Yang et al, 2017a). Briefly, 5 μl AF suspension was injected into a silica gel plate and separated by chromatographic solution including acetone: chloroform (1: 9, v/v).…”

“…About 100 μg of protein extracts derived from WT and mutants were then in-solution digested by trypsin (Promega) as previously described (Yang et al, 2017a). After trypsin digestion, an equal amount of the trypsindigested samples were diluted with 100 mM triethylammonium bicarbonate buffer and further processed according to the manufacturer's protocol for TMT 6plex isobaric label kit (Thermo Fisher Scientific).…”

“…The ChemiScopeseries 3300 Mini system (CLiNX, China) was used for signal detection. For ingel digestion, the gel was washed with deionized water, then cut into slices and digested with in-gel digestion methods as previously described (Yang et al, 2017a). Samples were completely dried with a vacuum centrifuge and stored at −80 C for further use.…”

AflSte20 Regulates Morphogenesis, Stress Response, and Aflatoxin Biosynthesis of Aspergillus flavus

“…For the thin-layer chromatography (TLC) analysis and high-performance liquid chromatography (HPLC) analysis of aflatoxins (AF) production, WT, ∆Aflste20, and ∆Aflste20 C strains (10 7 spores/mL) were inoculated into 10 mL YES liquid media at 29 • C in dark for seven days, (30µL spore suspension per plate). After that, aflatoxin was extracted using a method previously described by Yang et al [44]. The supernatant samples were analyzed by TLC with a solvent system (chloroform: acetone = 9:1) and detected by ultraviolet light.…”