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. 2020 Nov 13;12(11):717.
doi: 10.3390/toxins12110717.

Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus

Guang Yang  1 , Xiaohong Cao  1 , Ling Qin  1 , Lijuan Yan  1 , Rongsheng Hong  1 , Jun Yuan  1 , Shihua Wang  1
Affiliations

Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus

Guang Yang et al. Toxins (Basel). .

Abstract

The RNA polymerase II (Pol II) transcription process is coordinated by the reversible phosphorylation of its largest subunit-carboxy terminal domain (CTD). Ssu72 is identified as a CTD phosphatase with specificity for phosphorylation of Ser5 and Ser7 and plays critical roles in regulation of transcription cycle in eukaryotes. However, the biofunction of Ssu72 is still unknown in Aspergillus flavus, which is a plant pathogenic fungus and produces one of the most toxic mycotoxins-aflatoxin. Here, we identified a putative phosphatase Ssu72 and investigated the function of Ssu72 in A. flavus. Deletion of ssu72 resulted in severe defects in vegetative growth, conidiation and sclerotia formation. Additionally, we found that phosphatase Ssu72 positively regulates aflatoxin production through regulating expression of aflatoxin biosynthesis cluster genes. Notably, seeds infection assays indicated that phosphatase Ssu72 is crucial for pathogenicity of A. flavus. Furthermore, the Δssu72 mutant exhibited more sensitivity to osmotic and oxidative stresses. Taken together, our study suggests that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity in A. flavus, and may provide a novel strategy to prevent the contamination of this pathogenic fungus.

Keywords: A. flavus; aflatoxins; pathogenicity; phosphatase; ssu72.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Identification of putative phosphatase Ssu72 in A. flavus. (A) Phylogenetic tree analysis of putative Ssu72 phosphatases from different organisms. The tree was generated by MEGA 5.1 software with Neighbour-joining and bootstrap method. (B) Domain structure analysis of putative Ssu72 phosphatase from different fungi. Protein structure was characterized by SMART and drew using DOG 1.0 software. The Ssu72-like phosphatase domain is shown in blue.
Figure 2
Figure 2
Construction and verification of ssu72 mutants. (A) Strategy for deletion and complementation of phosphatase ssu72 gene by using homologous recombination. (B) PCR analysis of deletion and complementation strains. ORF: open reading frame; AP: 5′UTR+pyrG; BP: 5′UTR+pyrG. (C) RT-PCR analysis was used to detect the expression levels of ssu72 in different strains. Actin was used as reference gene. (D) qRT-PCR was performed to detect the transcript levels of ssu72 in different strains. ND means not detected.
Figure 3
Figure 3
The function of ssu72 in vegetative growth in A. flavus. (A) The colony phenotype of WT, Δssu72 and Δssu72-Com strains grown on YES, PDA and YGT media for 5 days. All strains were cultured at 37 °C. (B) Colony diameter of all strains was measured on different media. Significant difference was analyzed by t test. ** p < 0.01 stands for significant difference. (C) Microscopic examination of mycelial tips in WT, Δssu72 and Δssu72-Com strains, scale bars = 200 μm. Each experiment was repeated at least three times. Standard deviation is indicated by error bars.
Figure 4
Figure 4
Defects of conidiation in Δssu72 mutant. (A) Morphology analysis of WT, Δssu72 and Δssu72-Com strains grown on PDA medium at 37 °C for 5 days. (B) Statistical analysis of the amount of conidia produced on PDA medium. (C) Conidiophores of all strains were observed by light microscope, scale bars = 200 μm. (D) Transcript levels of conidia key genes (brlA and abaA) in different strains after cultured for 48 h. Actin was used as reference gene. (** p < 0.01 means significant difference by t test.) Each experiment was repeated at least three times. Standard deviation is indicated by error bars.
Figure 5
Figure 5
Phosphatase Ssu72 is essential for sclerotia formation. (A) Sclerotia formation of WT and ssu72 mutants on WKM medium after 7 days. (B) Amount of sclerotia produced in different strains on WKM medium. (C) Expression levels of sclerotia formation key genes (nsdC and nsdD) in WT, Δssu72 and Δssu72-Com strains cultured for 48 h. Actin was used as reference gene. (** p ≤ 0.01 means significant difference by t test.) ND means not detected. Each experiment was repeated at least three times. Standard deviation is indicated by error bars.
Figure 6
Figure 6
Analysis of aflatoxin production in the WT and Δssu72 mutants. (A) Aflatoxins in the YES liquid medium were extracted in YES liquid medium for 6 days at 29 °C, and TLC was used to detect the aflatoxin production in each strain. S means AFB1 standard. The concentration of the AFB1 standard is 0.1 mg/mL. (B) Gene Tools software was used for quantification analysis of AFB1 as in (A). (C) Relative expression levels of aflatoxin biosynthesis cluster genes cultured for 48 h. Actin was used as reference gene. (** p ≤ 0.01). Each experiment was repeated at least three times. Standard deviation is indicated by error bars.
Figure 7
Figure 7
Analysis of seeds infection of WT, Δssu72 and Δssu72-Com strains. (A) Sterile peanut and maize seeds were infected with WT, Δssu72 and Δssu72-Com strains and cultured at 29 °C for 5 days. Mock means a blank control, peanuts and maize seeds were treated with the same amount of water instead of conidia. (B) Quantification analysis of conidia collected from the infected seeds. (C) TLC was used to detect the AFB1 production from the infected seeds. (D) Quantification of AFB1 as in (C). (** p ≤ 0.01). Each experiment was repeated at least three times. Standard deviation is indicated by error bars.
Figure 8
Figure 8
The Ssu72 is involved in osmotic and oxidative stresses response. (A) Colony morphology of WT, Δssu72 and Δssu72-Com strains on PDA media added with 1 M NaCl or 5 mM H2O2 for 4 days. (B) Growth inhibition rate of all strains under osmotic and oxidative stresses. [inhibition of growth rate = (the diameter of untreated strain- the diameter of treated strain)/(the diameter of untreated strain) × 100%]. (** p ≤ 0.01 means significant difference by t test.) Each experiment was repeated at least three times. Standard deviation is indicated by error bars.
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References

    1. Zhang F., Huang L., Deng J., Tan C., Geng L., Liao Y., Yuan J., Wang S. A Cell Wall Integrity-Related MAP Kinase Kinase Kinase AflBck1 Is Required for Growth and Virulence in Fungus Aspergillus flavus. Mol. Plant Microbe Interact. 2020;33:680–692. doi: 10.1094/MPMI-11-19-0327-R. - DOI - PubMed
    1. Wang X., Zha W., Liang L., Fasoyin O.E., Wu L., Wang S. The bZIP Transcription Factor AflRsmA Regulates Aflatoxin B(1) Biosynthesis, Oxidative Stress Response and Sclerotium Formation in Aspergillus flavus. Toxins. 2020;12:271. doi: 10.3390/toxins12040271. - DOI - PMC - PubMed
    1. Fasoyin O.E., Yang K., Qiu M., Wang B., Wang S., Wang S. Regulation of Morphology, Aflatoxin Production, and Virulence of Aspergillus flavus by the Major Nitrogen Regulatory Gene areA. Toxins. 2019;11:718. doi: 10.3390/toxins11120718. - DOI - PMC - PubMed
    1. Yang K., Liu Y., Wang S., Wu L., Xie R., Lan H., Fasoyin O.E., Wang Y., Wang S. Cyclase-Associated Protein Cap with Multiple Domains Contributes to Mycotoxin Biosynthesis and Fungal Virulence in Aspergillus flavus. J. Agric. Food Chem. 2019;67:4200–4213. doi: 10.1021/acs.jafc.8b07115. - DOI - PubMed
    1. Chang P.K., Zhang Q., Scharfenstein L., Mack B., Yoshimi A., Miyazawa K., Abe K. Aspergillus flavus GPI-anchored protein-encoding ecm33 has a role in growth, development, aflatoxin biosynthesis, and maize infection. Appl. Microbiol. Biotechnol. 2018;102:5209–5220. doi: 10.1007/s00253-018-9012-7. - DOI - PubMed

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