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. 2018 Mar 10;10(3):117.
doi: 10.3390/toxins10030117.

G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus

Yinghang Liu  1 , Kunlong Yang  2 , Qiuping Qin  3   4 , Guinan Lin  5 , Tianran Hu  6 , Zhangling Xu  7 , Shihua Wang  8
Affiliations

G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus

Yinghang Liu et al. Toxins (Basel). .

Abstract

The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus Aspergillus flavus. GanA, GpaB and FadA have homologues in other fungal species, while GaoC is a novel one. Here, we showed that the loss function of gpaB displayed a defect in conidiophore formation and considerably reduced expression levels of conidia-specific genes brlA and abaA. A decreased viability of cell wall integrity stress and oxidative stress were also found in the ∆gpaB mutant. More importantly, aflatoxin (AF) biosynthesis and infection on crop seeds were severely impaired in the gpaB-deficient mutant. Further analyses demonstrated that the intracellular cAMP levels significantly reduced in the gpaB-deficient mutant compared to wildtype strains. Additionally, an alteration of PKA activities in the ∆gpaB mutant was also found. Overall, our results indicated that GpaB played diverse roles in asexual sporulation, AF biosynthesis and virulence by regulating cAMP signaling in Aspergillus flavus.

Keywords: G protein; cAMP; fungal virulence; sclerotia.

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Conflict of interest statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Figures

Figure 1
Figure 1
Characterization of G protein α subunits in A. flavus. (A) Phylogenetic analysis of the four A. flavus Gα proteins (FadA, GpaB, GanA and AFLA_124830) with characterized fungal Gα subunits. (B) Amino acid sequence alignments of the four Gα proteins (FadA, GpaB, GanA and AFLA_124830) of A. flavus. The conserved amino acid residues are shown in black, while the similar ones are shaded in gray.
Figure 2
Figure 2
Deletion strategy and confirmation of the mutants used in this study. (A) Deletion and complement strategy for ∆gpaB strains; (B) Diagnostic PCR was performed to confirm the gene deletion and complemented strains. The gpaB ORF was confirmed by primers gpaB/OF and gpaB/OR; fragment AP was confirmed by primers gpaB/p1 and P801; while fragment APC was confirmed by primers gpaB/p1 and ptrA/R; (C) RT-PCR verification of gpaB gene deletion. The β-tubulin gene was used as a reference.
Figure 3
Figure 3
The ΔgpaB mutant was altered in pigmentation and vegetative growth. (A) Colony morphology of the WT, ΔgpaB and gpaBC strains after being grown on PDA, YGT and YES agar plates for four days at 37 °C. (B) Statistical analysis of the colony diameter of the indicated strains on different media. ** indicates significantly different between the wildtype and mutant strains (p ≤ 0.01), as assessed by one-way ANOVA and adjusted with Turkey’s multiple comparison test. The experiments were conducted with four replicates for the indicated strain and were repeated three times.
Figure 4
Figure 4
gpaB is involved in conidiation and sclerotia formation in A. flavus. (A) Colony and conidiophore morphology among the WT, ΔgpaB and gpaBC were observed after being grown on PDA agar medium for five days in the dark; (B) The number of conidia of the indicated strains was measured after being grown on PDA agar for five days; (C) Sclerotia formation of the indicated strains grown on sclerotia-inducing Wickerham (WKM) medium was detected. To visualize the sclerotia, 75% ethanol was sprayed on the WKM plates to remove the conidia; (D) The number of sclerotial was counted as in (C). Error bars represent the standard deviation from four replicates, and asterisks, “***” or “*”, represent significant differences compared to the wildtype according to the t-test with p < 0.001 and p < 0.05, respectively. The experiments were conducted with four replicates for the indicated strain and were repeated three times.
Figure 5
Figure 5
ΔgpaB is altered in its sensitivities to cell wall integrity stress and oxidative stress. (A) Colony phenotype of the indicated strains after being grown under cell wall integrity stress triggered by adding 100 µg/mL Calcofluor White (CFW) or 200 µg/mL Congo-Red (CR), for four days; (B) Statistical analysis of mycelia of the growth inhibition rate of the indicated strains under cell wall stress; (C) Colony phenotype of the indicated strains after being grown under oxidative stress triggered by 3 mM hydrogen peroxide (H2O2); (D) Statistical analysis of mycelia of the growth inhibition rate of the indicated strains under oxidative stress. Error bars represent the standard deviation from four replicates, and asterisks “***” represent significant differences compared to the wildtype according to the t-test with p < 0.001. The experiments were conducted with four replicates for the indicated strain and were repeated three times.
Figure 6
Figure 6
gpaB is required for AF biosynthesis. (A) AF production was measured by thin-layer chromatography (TLC) after being grown in YES medium in the dark at 29 °C for five days; (B) The amount of AF production in YES medium was qualified by Gene Tools analysis system software. The double asterisks “***” represent significant differences at p < 0.001. Stnd represents the AFB1 standard. The experiments were conducted with three replicates for the indicated strain and were repeated three times.
Figure 7
Figure 7
Disruption of gpaB leads to a significant reduction in seed infection. (A) Pathogenicity test of gpaB mutant on maize kernels; (B) Conidia production of gpaB mutant on maize kernels; (C) Detection of AF production of gpaB mutant on maize kernels by TLC. Error bars represent the standard deviation from three replicates, and triple asterisks “***” represent significant differences compared to the wildtype according to the t-test with p < 0.001. Stnd represents the AFB1 standard. The experiments were conducted with three replicates for the indicated strain and were repeated three times.
Figure 8
Figure 8
gpaB is involved in the regulation of intracellular cAMP levels and PKA activities in A. flavus. (A) Deletion of gpaB leads to decreased accumulation of cAMP levels in A. flavus; (B) Disruption of gpaB leads to a change of the cAMP-dependent protein kinase activities in A. flavus. A 0.8% agarose gel was used to separate protein samples. Phosphorylated peptides migrated towards the cathode (+), while nonphosphorylated peptides migrated towards the anode (−). P, phosphorylated sample control; N, non-phosphorylated sample control. The triple asterisks “***” represent significant differences compared to the wildtype according to the t-test with p < 0.001. The experiments were conducted with three replicates for the indicated strain and were repeated three times.
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