The AreA transcription factor mediates the regulation of deoxynivalenol (DON) synthesis by ammonium and cyclic adenosine monophosphate (cAMP) signalling in Fusarium graminearum

Abstract

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium graminearum, is harmful to humans and animals. Because different nitrogen sources are known to have opposite effects on DON production, in this study, we characterized the regulatory mechanisms of the AREA transcription factor in trichothecene biosynthesis. The ΔareA mutant showed significantly reduced vegetative growth and DON production in cultures inoculated with hyphae. Suppression of TRI gene expression and DON production by ammonium were diminished in the ΔareA mutant. The deletion of AREA also affected the stimulatory effects of arginine on DON biosynthesis. The AreA-green fluorescent protein (GFP) fusion complemented the ΔareA mutant, and its localization to the nucleus was enhanced under nitrogen starvation conditions. Site-directed mutagenesis showed that the conserved predicted protein kinase A (PKA) phosphorylation site S874 was important for AreA function, indicating that AreA may be a downstream target of the cyclic adenosine monophosphate (cAMP)-PKA pathway, which is known to regulate DON production. We also showed that AreA interacted with Tri10 in co-immunoprecipitation assays. The interaction of AreA with Tri10 is probably related to its role in the regulation of TRI gene expression. Interestingly, the ΔareA mutant showed significantly reduced PKA activity and expression of all three predicted ammonium permease (MEP) genes, in particular MEP1, under low ammonium conditions. Taken together, our results show that AREA is involved in the regulation of DON production by ammonium suppression and the cAMP-PKA pathway. The AreA transcription factor may interact with Tri10 and control the expression and up-regulation of MEP genes.

High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production

“…The F. graminearum global nitrogen regulator AreA carries a conserved putative PKA phosphorylation site and could thus be activated by CPK1 and CPK2 (Hou et al, 2015). The activated AreA could then regulate DON production through TRI10, one of the key transcription factors of the TRI cluster; with which AreA has been shown to interact (Hou et al, 2015;Peplow et al, 2003;Seong et al, 2009). …”

“…AreA in turn might be regulated by cAMP-PKA signalling (Hou et al, 2015). Generally, protein kinase A (PKA) is activated by cyclic 3',5'-adenosine monophosphate (cAMP) produced by adenylyl cyclases, which are regulated by G-proteins (Li et al, 2007).…”

The tri‐snRNP specific protein FgSnu66 is functionally related to FgPrp4 kinase in Fusarium graminearum

“…The FgSnu66 CT50 ‐3xFLAG and FgSnu66 N596 ‐GFP fusion constructs were generated by the yeast gap repair approach (Zhou et al, ) and co‐transformed into the wild‐type strain PH‐1. Total proteins were isolated from transformants expressing both FgSnu66 CT50 ‐3xFLAG and FgSnu66 N596 ‐GFP constructs and incubated with anti‐GFP beads (Sigma‐Aldrich, St. Louis, MO) as described (Hou et al, , Zhang et al, ). Western blots of total proteins and proteins eluted from anti‐GFP beads were detected with the anti‐GFP (Roche, USA) and anti‐FLAG (Sigma‐Aldrich, USA) antibodies as described (Liu et al, ).…”

Nuclear localization and relative stability of the zinc finger domain of TRI6 trichothecene regulator

“…1A; lower panel; see Supplementary Fig 3 for experimental detail). A previous immunoprecipitation study failed to detect the full-length TRI6 C-terminally fused to GFP in the total protein extract of the fusion gene overexpressor 5) , also suggesting instability of TRI6 within F. graminearum cells. The use of a different protein extraction reagent [TRIzol ® reagent (Fischer Thermo Scientific) in this study versus non-denaturing buffer in the previous study 5) ] may account for the successful detection of TRI6::EGFP in this study.…”