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.

Keywords: DON production; Gibberella zeae; TRI6 expression; ammonium suppression; nitrogen metabolism.

Figure 1

AREA expression and its role in the regulation of the TRI gene. (A) Relative expression level of AREA in complete medium (CM) cultures with 50 mm ammonium (NH₄⁺; arbitrarily set to unity) or nitrate (NO₃ ⁻) as the sole nitrogen source. (B) The expression level of TRI5 and TRI6 in cultures of the wild‐type strain PH‐1 and ΔareA mutant grown under nitrogen starvation (NS) or ammonium suppression (AS; arbitrarily set to unity) conditions.

Figure 2

Defects of the ΔareA mutant in response to different nitrogen sources. (A) Cultures of the wild‐type strain PH‐1 and ΔareA mutant grown on complete medium (CM) with 50 mm sodium nitrate (NO₃ ^–), glutamate (GLU) or glutamine (GLN) and 5 or 50 mm ammonium phosphate (NH₄ ⁺) as the nitrogen source. (B) Quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) assays of the nitrate (FGSG_01947) and nitrite (FGSG_08402) reductase genes in PH‐1 and ΔareA mutant grown under nitrogen starvation (NS) and ammonium suppression (AS; arbitrarily set to 1) conditions.

Figure 3

Stimulation of deoxynivalenol (DON) production by arginine is affected by AREA. (A) The expression levels of TRI5, TRI6 and TRI10 in the wild‐type strain PH‐1 and ΔareA mutant in liquid DON‐inducing medium with 5 mm nitrate or arginine. For each gene, the expression level in nitrate cultures was arbitrarily set to unity. (B) The expression levels of TRI5 and TRI6 in DON‐inducing liquid cultures (with arginine) of PH‐1 and ΔareA mutant with 0 or 50 mm ammonium added at the fourth day. Mean and standard deviations were calculated with results from three independent biological replicates.

Figure 4

Expression and subcellular localization of AreA‐GFP fusion proteins. (A) Conidia of the ΔareA/ AREA‐GFP transformant AC6 were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and examined by differential interference contrast (DIC) or epifluorescence microscopy with or without incubation for 1 h under nitrogen starvation (NS) conditions. (B) Germlings of transformant AC6 were stained with DAPI and examined by DIC or epifluorescence microscopy with or without incubation for 1 h under NS conditions. CM, complete medium; GFP, green fluorescent protein. Bar, 10 μm.

Figure 5

Site‐directed mutagenesis analysis with the AREA gene. (A) The AreA protein is predicted to have three conserved nuclear localization signal (NLS) sequences (NLS1, 238–244; NLS2, 287–290; NLS3, 716–725), one consensus protein kinase A (PKA) (S874) and one mitogen‐activated protein kinase (MAPK) (S658) phosphorylation site, and one zinc figure domain (ZF, 686–710). (B) Four‐day‐old potato dextrose agar (PDA) cultures of the wild‐type strain PH‐1, ΔareA mutant, ΔareA/ AREA^ΔNLS1‐GFP (RL10 and RL11), ΔareA/ AREA^ΔNLS2‐GFP (RL12 and RL13), ΔareA/ AREA^ΔNLS3‐GFP (RE8 and RE15), ΔareA/ AREA^ΔS874‐GFP (RP2 and RP4), ΔareA/ AREA^{ΔS657 ‐658}‐GFP (RM3 and RM6) and ΔareA/ AREA^S874A‐GFP (RPA6 and RPA8) transformants, and complementation strain (AC6). (C) Wheat heads inoculated with the same set of strains were examined for scab symptoms at 14 days post‐inoculation (dpi). (D) Germlings of transformants RE8 and RE15 were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) and examined by differential interference contrast (DIC) or epifluorescence microscopy. GFP, green fluorescent protein. Bar, 10 μm. (E) Western blot analysis with total proteins isolated from vegetative hyphae of PH‐1 and ΔareA/ AREA^ΔNLS3‐GFP transformants RE8 and RE15. The AreA‐GFP band was detected with the anti‐GFP antibody. Detection with an anti‐H3 antibody was used as the control to show that similar amount of proteins were loaded in each lane.

Figure 6

Assays for the expression levels of three ammonium permease genes by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). (A) The expression levels of MEP1, MEP2 and MEP3 were compared between the wild‐type strain PH‐1 and ΔareA mutant, which were cultured in medium with 0.5 mm (low) or 50 mm (high) ammonium. For each gene, the relative expression level in the ΔareA mutant was arbitrarily set to unity. (B) The expression level of each MEP gene in cultures of PH‐1 or ΔareA mutant with 0.5 mm ammonium was compared with that of cultures with 50 mm ammonium (arbitrarily set to unity). Mean and standard deviation were calculated with data from three independent biological replicates.

Figure 7

Mitogen‐activated protein kinase (MAPK) phosphorylation and protein kinase A (PKA) activity assays. (A) Assays for the activation of Mgv1 and Gpmk1 MAPKs. Total proteins were isolated from vegetative hyphae of the wild‐type PH‐1, ΔareA mutant and complementation strain (AC6). The phosphorylation of Mgv1 (46 kDa) and Gpmk1 (42 kDa) was detected with the anti‐TpEY antibody. The expression level of Gpmk1 was detected with the anti‐Pmk1 antibody. (B) PKA activities were assayed with proteins isolated from hyphae of PH‐1 and ΔareA mutant using the PepTag A1 PKA substrate peptide. Whereas phosphorylated peptides migrated towards the anode (+), unphosphorylated peptides migrated towards the cathode (–) on a 0.8% agarose gel. N, non‐phosphorylated sample control; P, phosphorylated sample control.

Figure 8

Co‐immunoprecipitation assays for the interactions between Tri10 and AreA. Total proteins (Total) isolated from vegetative hyphae of PH‐1, transformant AT61 (expressing the AREA‐3 × FLAG and TRI6‐GFP constructs) and transformant AT02 (expressing the AREA‐3 × FLAG and TRI10‐GFP constructs) and proteins eluted (Elution) from anti‐FLAG M2 beads were separated on 10% sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel. After transfer to nitrocellulose membranes, the presence of AreA‐3 × FLAG, Tri10‐GFP or Tri6‐GFP fusion proteins was detected with the anti‐FLAG and anti‐GFP antibodies. Whereas the Tri6‐GFP band was not detectable in transformant AT61, the 74‐kDa Tri10‐GFP band was detected in total proteins and proteins eluted from anti‐FLAG beads in transformant AT02. GFP, green fluorescent protein.