Abstract
Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of canonical subunits and various regulators of G-protein signaling (RGS) proteins, plays an important role in transducing signals under environmental stress. Earlier studies demonstrated that Gα and Gβ subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in Gβ deletion mutant ∆Fvgbb1. Notably, FvFlbA2 has a negative role in FB1 regulation. While many fungi contain a single copy of FlbA, F. verticillioides harbors two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While ∆FvflbA1 deletion mutant exhibited no significant defects, ∆FvflbA2 and ∆FvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays determined that FvFlbA paralogs interact with key heterotrimeric G protein components, which in turn will lead altered G-protein-mediated signaling pathways that regulate FB1 production and asexual development in F. verticillioides.
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Acknowledgements
This research was supported in part by the Agriculture and Food Research Initiative Competitive Grants Program Grant (2013-68004-20359) from the USDA National Institute of Food and Agriculture. We also would like to acknowledge China Scholarship Council support (201906850058) for Z. Zhou. The authors declare no conflict of interest.
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Supplementary file 1: Fig. S1. Sequence alignment FlbA ortholog proteins in select fungal species. We aligned protein sequences of Fusarium verticillioides FvFlbA1, F. verticillioides FvFlbA2, Saccharomyces cerevisiae ScSst2, A. nidulans AnFlbA, Neurospora crassa NcFlbA, and Magnaporthe oryzae MoRgs1. Identical and similar sequences were displayed in the box. Fig. S2. Split marker approach employed in FLBA generating gene deletion mutants in F. verticillioides. (A) ΔFvflbA1 mutant was generated by replacing FLBA1 gene with a hygromycin B phosphotransferase gene (HPH). (B) FvFLBA2 gene was replaced by a hygromycin gene to generate a ΔFvflbA2 mutant. (C) ΔFvflbA2/A1FvFLBA1 was generated by replacing FLBA1 gene with a geneticin gene (GEN) in ΔFvflbA2 background. (D) Mutants ΔFvflbA1, ΔFvflbA1-Com, ΔFvflbA2/A1 were subjected to qPCR using FvFLBA1 gene primer. Relative expression was normalized to F. verticillioides β-tubulin gene (FVEG_04081). (E) FvFLBA2 gene transcription level was examined in WT, ΔFvflbA2, ΔFvflbA2-Com, ΔFvflbA2/A1 strains. Fig. S3. Relative FB1 expression in wild-type and FvflbA mutants. FB1 was extracted from supernatant (2ml) of 7-day incubation at 150 RPM in myro liquid culture. In details, YEPD mycelial samples (3 dpi) were harvested through Miracloth (EMD Millipore). Subsequently, mycelia (0.3g) were inoculated into 100 ml myro liquid medium at room temperature. (PDF 240 KB)
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Yan, H., Zhou, Z. & Shim, W.B. Two regulators of G-protein signaling (RGS) proteins FlbA1 and FlbA2 differentially regulate fumonisin B1 biosynthesis in Fusarium verticillioides. Curr Genet 67, 305–315 (2021). https://doi.org/10.1007/s00294-020-01140-5
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DOI: https://doi.org/10.1007/s00294-020-01140-5