Mechanism of validamycin A inhibiting DON biosynthesis and synergizing with DMI fungicides against Fusarium graminearum

Abstract

Deoxynivalenol (DON) is a vital virulence factor of Fusarium graminearum, which causes Fusarium head blight (FHB). We recently found that validamycin A (VMA), an aminoglycoside antibiotic, can be used to control FHB and inhibit DON contamination, but its molecular mechanism is still unclear. In this study, we found that both neutral and acid trehalase (FgNTH and FgATH) are the targets of VMA in F. graminearum, and the deficiency of FgNTH and FgATH reduces the sensitivity to VMA by 2.12- and 1.79-fold, respectively, indicating that FgNTH is the main target of VMA. We found FgNTH is responsible for vegetative growth, FgATH is critical to sexual reproduction, and both of them play an important role in conidiation and virulence in F. graminearum. We found that FgNTH resided in the cytoplasm, affected the localization of FgATH, and positively regulated DON biosynthesis; however, FgATH resided in vacuole and negatively regulated DON biosynthesis. FgNTH interacted with FgPK (pyruvate kinase), a key enzyme in glycolysis, and the interaction was reduced by VMA; the deficiency of FgNTH affected the localization of FgPK under DON induction condition. Strains with a deficiency of FgNTH were more sensitive to demethylation inhibitor (DMI) fungicides. FgNTH regulated the expression level of FgCYP51A and FgCYP51B by interacting with FgCYP51B. Taken together, VMA inhibits DON biosynthesis by targeting FgNTH and reducing the interaction between FgNTH and FgPK, and synergizes with DMI fungicides against F. graminearum by decreasing FgCYP51A and FgCYP51B expression.

Keywords: Fusarium graminearum; DMI fungicides; acid trehalase; deoxynivalenol; neutral trehalase; validamycin A.

FIGURE 1

FgNTH and FgATH regulate the sensitivity to validamycin A (VMA) in Fusarium graminearum. (a) Phylogenetic analysis of NTH and ATH from Rhizoctonia solani, Saccharomyces cerevisiae, and F. graminearum. Abbreviations and accession numbers of the sequences are as follows: ScNTH1 (NP_010284.1), ScNTH2 (NP_009555.1), and ScATH (NP_015351.1) from S. cerevisiae; RsNTH (ELU40049.1), RsATH1 (ELU42236.1), and RsATH2 (ELU42026.1) from R. solani; FgNTH (FGSG_09895) and FgATH (FGSG_05622) from F. graminearum. The amino acid sequences of NTH and ATH were analysed using the neighbour‐joining method with the MEGA 8.0 program. (b, c, d) Colony morphology and mycelial growth of the wild‐type strain PH‐1, the single‐deletion mutants ΔFgNTH and ΔFgATH, the complemented strains of ΔFgNTHC and ΔFgATHC, the overexpression strains OEFgNTH and OEFgATH, and the double‐deletion mutant ΔFgNTHΔFgATH on potato dextrose agar (PDA) and complete medium (CM) at 25 °C for 3 days. (e, f) Sensitivity of all strains to VMA on Czapek medium without carbon source and on Czapek medium containing trehalose as sole carbon source, respectively. Each strain was cultured on potato dextrose agar for 2 days and then transferred agar plugs (5 mm in diameter) containing mycelia from the colony margin onto the plates with VMA. Colony diameters were determined and pictures were taken after incubation for 4 days. (g) and (h) Inhibition rate of 10 μg/ml VMA to all strains on Czapek medium without carbon source and on Czapek medium containing trehalose as sole carbon source. Each test was independently performed three times. The data were analysed using by one‐way analysis of variance (ANOVA) and means were compared by the least significant difference at p < .05. The statistics and bar graphs were produced using GraphPad Prism v. 8.2

FIGURE 2

Roles of FgNTH and FgATH in modulating sexual reproduction and their subcellular localization in Fusarium graminearum. (a) The colocalization of FgNTH and FgATH with cell nucleus. (b) The colocalization of FgNTH and FgATH with vacuoles. The fusion strains FgNTH‐GFP and FgATH‐GFP were cultured in YEPD liquid medium for 1 day at 25 °C to harvest mycelia and in CMC medium for 3 days at 25 °C to produce conidia. The mycelia and conidia were stained with 4,6‐diamidino‐2‐phenylindole (DAPI) or 7‐amino‐4‐chloro‐methylcoumarin (CMAC) to observe fluorescence using a Leica TCS SP5 confocal microscope. GFP fusion protein: green fluorescence; DAPI and CAMC: blue fluorescence. Bar = 10 μm. (c) The localization of FgNTH in the mutant ΔFgATH. (d) The localization of FgATH in the mutant ΔFgNTH. The mycelia and conidia of strain ΔFgATH FgNTH‐GFP were harvested and stained with CMAC and DAPI, respectively, as described above. Bar = 10 μm. (e) Conidial morphology. Conidia were produced in CMC medium and stained with calcofluor white (CFW) to observe the septa using an Olympus IX‐71 inverted fluorescence microscope. Left image, bright field; right image, ultravioletlight excitation. Bar = 10 μm. (f) Conidial germination. Conidia were cultured in YEPD liquid medium for 8 hr and stained with CFW to observe the germination using an Olympus IX‐71 inverted fluorescence microscope. Top image, bright field; bottom image, ultraviolet light excitation. Bar = 20 μm. (g) Sexual reproduction. All strains were incubated on carrot medium for 14 days to produce perithecia. The perithecia and ascospores were observed using a Nikon SMZ25 fluorescence stereomicroscope and an Olympus IX‐71 inverted fluorescence microscope, respectively. Top image, perithecia, bar = 1 mm; bottom image, asci and ascospores, bar = 50 μm

FIGURE 3

FgNTH and FgATH regulate virulence and deoxynivalenol (DON) biosynthesis in Fusarium graminearum. (a) Virulence on wheat heads 14 days after inoculation. The central spikelet of each wheat head was inoculated with 10 μl of conidial suspension. The controls were inoculated with sterile double‐distilled water. (b) Virulence on wheat coleoptiles 10 days after inoculation. The coleoptiles of 3‐day‐old wheat seedlings were cut and inoculated with 2 μl of conidial suspension. The controls were inoculated with sterile double‐distilled water. (c) Number of diseased spikelets. For the pathogenicity assay on wheat heads, each strain was inoculated to 15 wheat heads and the test was repeated twice. (d) Lesion length. For the pathogenicity assay on wheat coleoptiles, each strain was inoculated to 30 wheat coleoptiles and the test was repeated three times. (e) DON production. (f) The relative expression of FgTRI1, FgTRI5, and FgTRI6. (g) Pyruvate production. (h) The relative expression levels of FgNTH, FgATH, and FgPK. Fresh mycelia of each strain cultured in GYEP liquid medium for 3 days at 28 °C were harvested and used for determining the production of DON and pyruvate. These mycelia were also used for total RNA extraction and quantitative reverse transcription PCR assays. FgActin was used as the reference. (i) The fluorescence of fusion protein FgTRI1‐GFP. (j) The fluorescence of fusion protein FgTRI5‐GFP. The mycelia of fusion strains FgTRI1‐GFP, FgTRI5‐GFP, ΔFgNTH FgTRI1‐GFP, ΔFgNTH FgTRI5‐GFP, ΔFgATH FgTRI1‐GFP, and ΔFgATH‐FgTRI5‐GFP were cultured in GYEP liquid medium for 1 day at 28 °C and harvested for observing fluorescence using a Leica TCS SP5 confocal microscope. Bar = 10 μm. All assays were repeated three times independently. The data were analysed using one‐way analysis of variance (ANOVA) and means were compared by the least significant difference at p < .05. The statistics and bar graphs were produced using GraphPad Prism v. 8.2

FIGURE 4

Validamycin A (VMA) reduces the interaction of FgNTH with FgPK in Fusarium graminearum. (a) VMA decreases the expression of FgNTH and FgATH. The wild‐type strain PH‐1 was cultured in GYEP liquid medium with 1, 10, and 100 μg/ml VMA at 28 °C for 3 days, and then used for quantitative reverse transcription PCR assays. (b) Yeast two‐hybrid analysis of the interaction between FgNTH and FgGPI, FgNTH, and FgPK. Different concentrations of the labelled yeast transformants were assayed for growth on SD−Trp−Leu−His plates. (c) Verification of FgNTH and FgPK interaction by coimmunoprecipitation (Co‐IP) assay. Total proteins (input) extracted from the strain containing FgNTH‐3 × FLAG FgPK‐GFP constructs or a single construct (FgNTH‐3 × FLAG or FgPK‐GFP) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and immunoblots were incubated with monoclonal anti‐GFP and monoclonal anti‐FLAG antibodies as indicated (upper image). In addition, each protein sample was pulled down using anti‐GFP antibodies with magnetic beads and further detected with monoclonal anti‐GFP and monoclonal anti‐FLAG antibodies (lower image). The protein samples were also detected with monoclonal anti‐actin antibody as a reference. (d) Verification of FgNTH and FgGPI interaction by Co‐IP assay. (e) VMA decreases the expression of FgPK. The fusion strains were cultured in GYEP liquid medium with or without 10 μg/ml VMA for 3 days at 28 °C, and were harvested for extracting the total proteins. Each lane was loaded with10 ng of total proteins for SDS‐PAGE, and further detected with monoclonal anti‐GFP antibody and monoclonal anti‐GAPDH antibody as a loading control. (f) FgNTH affects the localization of FgPK in GYEP medium. The fusion strains were cultured in YEPD or GYEP liquid medium for 3 days at 28 °C and then observed for fluorescence. Bar = 10 μm. (g) The location of FgPK‐GFP. The fusion strains were cultured in GYEP liquid medium with or without 10 μg/ml VMA for 3 days at 28 °C and were harvested to observe fluorescence. Bar = 10 μm. (h) VMA reduces the interaction between FgNTH and FgPK in YEPD medium. (i) VMA reduces the interaction between FgNTH and FgPK in GYEP medium. The fusion strain FgNTH‐3 × FLAG FgPK‐GFP was cultured in YEPD liquid medium with or without 10 μg/ml VMA for 1 day at 25 °C or in GYEP liquid medium with or without 10 μg/ml VMA for 3 days at 28 °C, and fresh mycelia were harvested for Co‐IP assay. All assays were repeated three times independently. The data were analysed using one‐way analysis of variance (ANOVA), and means were compared by the least significant difference at p < .05. The statistics and bar graphs were produced using GraphPad Prism v.8.2

FIGURE 5

FgNTH regulates the sensitivity to tebuconazole in Fusarium graminearum. (a) Sensitivity of the mutant strains of FgNTH and FgATH to tebuconazole and carbendazim. The sensitivity of each strain to tebuconazole and carbendazim was determined on potato dextrose agar (PDA) based on mycelial growth inhibition and EC₅₀ values. The concentration of tebuconazole was set to 0, 0.01, 0.05, 0.25, 0.625, and 1.25 μg/ml. The concentration of carbendazim was set to 0, 0.2, 0.4, 0.6, 0.8, and 1.0 μg/ml. (b) The EC₅₀ values of each strain to tebuconazole. (c) The EC₅₀ values of each strain to carbendazim. (d) FgNTH and FgATH affect the expression of FgCYP51. (e) FgCYP51 expression in FgCYP51 deletion mutants. Each strain was cultured in YEPD liquid medium for 2 days at 25 °C and was used for quantitative reverse transcription PCR assays. (f) The location of FgCYP51 in deletion mutants of FgNTH and FgATH. The fusion strains were cultured in YEPD liquid medium for 1 day at 25 °C and then the fluorescence was observed using a Leica TCS SP5 confocal microscope. Bar = 10 μm. (g) The interaction of FgNTH and FgCYP51B. Total proteins were eluted from anti‐GFP beads and detected with monoclonal anti‐GFP and monoclonal anti‐FLAG antibodies, respectively. Protein samples were detected with anti‐GAPDH antibody as a reference. Each test was independently determined three times. The data were analysed using one‐way analysis of variance (ANOVA) and means were compared by the least significant difference at p < .05. The statistics and bar graphs were produced using GraphPad Prism v. 8.2

FIGURE 6

A schematic summary of validamycin A (VMA) inhibiting deoxynivalenol (DON) biosynthesis and synergizing with DMI fungicides against Fusarium graminearum. VMA inhibits FgNTH activity and reduces the expression level of FgNTH, which in turn reduces the interaction intensity between FgNTH and FgPK, interferingwith the glycolysis pathway to produce pyruvate and thus reducing DON biosynthesis. FgNTH can interact with FgCYP51B to reduce the expression level of FgCYP51A and FgCYP51B, resulting in the increasedsensitivity to DMIs in F. graminearum