MAPK pathway‐related tyrosine phosphatases regulate development, secondary metabolism and pathogenicity in fungus Aspergillus flavus

Abstract

Mitogen-activated protein kinase (MAPK) cascades are highly conserved in eukaryotic cells and are known to play crucial roles in the regulation of various cellular processes. However, compared with kinase-mediated phosphorylation, dephosphorylation catalysed by phosphatases has not been well characterized in filamentous fungi. In this study, we identified five MAPK pathway-related phosphatases (Msg5, Yvh1, Ptp1, Ptp2 and Oca2) and characterized their functions in Aspergillus flavus, which produces aflatoxin B 1 (AFB 1), one of the most toxic and carcinogenic secondary metabolites. These five phosphatases were identified as negative regulators of MAPK (Slt2, Fus3 and Hog1) pathways. Deletion of Msg5 and Yvh1 resulted in significant defects in conidiation, sclerotia formation, aflatoxin production and crop infection. Additionally, double knockout mutants (ΔMsg5/ΔPtp1, ΔMsg5/ΔPtp2 and ΔMsg5/ΔOca2) displayed similar defects to those observed in the ΔMsg5 single mutant, indicating that Msg5 plays a major role in the regulation of development and pathogenicity in A. flavus. Importantly, we found that the active site at C439 is essential for the function of the Msg5 phosphatase. Furthermore, the MAP kinase Fus3 was found to be involved in the regulation of development, aflatoxin biosynthesis and pathogenicity, and its conserved phosphorylation residues (Thr and Tyr) were critical for the full range of its functions in A. flavus. Overall, our results reveal that MAPK related tyrosine phosphatases play important roles in the regulation of development, secondary metabolism and pathogenicity in A. flavus, and could be developed as potential targets for preventing damage caused by this fungal pathogen.

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

“…In A. flavus , phosphatases have been proven to be involved in multiple stresses response [ 38 , 39 ]. To explore the role of putative phosphatase Ssu72 in response to osmotic and oxidative stresses, the strains (WT, Δ ssu72 and Δ ssu72-Com ) were cultured on PDA medium supplemented with different stress reagents.…”

“…As one of the most toxic secondary metabolites in nature, aflatoxin contamination poses a serious threat to food safety, human and animal health [ 45 ]. Recent studies have revealed that kinases and phosphatases play vital roles in regulation of aflatoxin production in A. flavus [ 39 , 46 , 47 ]. Here, we observed that aflatoxin production was dramatically reduced in Δ ssu72 mutant, and this finding is in accordance with the down-regulation of aflatoxin biosynthesis regulatory and structural genes ( Figure 6 ).…”

“…Tyrosine phosphatases are well conserved and play critical roles in fungal pathogenicity in filamentous fungi [ 24 , 39 ]. In F. graminearum , homologues of Ssu72 have been demonstrated to be essential for plant infection [ 34 ].…”

“…Mitogen-activated protein kinase (MAPK) related phosphatases have been identified to be involved in multiple stresses response by regulating the phosphorylation level of MAP kinases (Hog1, Slt2 and Fus3) in A. flavus [ 39 ]. In this study, we found that the putative phosphatase Ssu72 is critical to respond to osmotic stress ( Figure 8 A).…”

“…RNA extraction and cDNA synthesis were conducted according to the published references in our lab [ 39 ]. Mycelia were collected from PDA and WKM media cultured for 48 h, then TRIzol reagent (Biomarker Technologies, Beijing, China) was used for total RNA isolation.…”