Fig 1.
Identification of the FgPfn in Fusarium graminearum.
(A) Phylogenetic and domain analysis of the profilins. Maximum Likelihood (ML) method, J Le and Gascuel (LG) model, and 1000 replicates were used to construct the phylogenetic tree by MEGA7. The domains were drawn with the Batch SMART in TBtools. (B) Domain and interaction sites analysis. With the CDD database online service function, the domains and actin-binding sites of FgPfn were drawn by DOG 2.0. The actin-binding sites include 11 amino acid residues in F62/A76/R77/D79/R86/G91/I111/A112/G113/T117/S121.
Fig 2.
FgPfn is essential for vegetative growth.
(A) Growth phenotype of ΔFgPfn mutants. The colony morphology was photographed after the 3d inoculation of each strain on PDA, CM, MM, and V8 medium. (B) The colony morphology was photographed after the 20d inoculation of mutants on PDA, CM, MM, and V8 medium. (C) Mycelial growth rate of each strain was measured after the 3d growth on PDA, CM, MM, and V8 medium. Bars with the same letter indicate no significant difference according to the least significant difference (LSD) test at p < 0.05. (D) Mycelial growth rate of each mutant was measured after the 20d growth on PDA, CM, MM, and V8 medium. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05.
Fig 3.
Deletion of FgPfn causes defects in asexual and sexual development.
(A) Deletion of FgPfn results in conidial morphological defects. Conidia were examined by differential interference contrast (DIC) microscopy. The septum of conidia was stained with Calcofluor white (CFW) and photographed with an inverted fluorescent microscope at 20×. The nuclei of conidia were stained with 4’,6-DiAmidino-2-PhenylIndole (DAPI) for 30 min and photographed with an inverted fluorescent microscope at 20×. Bar = 10 μm. (B) Sexual development of ΔFgPfn. Fresh mycelia of each strain were inoculated on carrot agar medium (CA) plates and cultivated in the dark at 25°C, in which PH-1 and complementation strain were cultured for 6d, ΔFgPfn mutants were inoculated in advance and cultured for 15d. All strains were scraped aerial mycelium simultaneously and treated with 2.5% Tween-20. After being cultivated under fluorescent light for 7-14d, the perithecia were photographed using a stereo microscope. Bar = 1 000 μm. Then the perithecia were crushed on the slides to observe the asci. Bar = 10 μm. (C) The average conidia length of each strain was measured with 100 conidia, which was three repeated times. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05. (D) The number of conidial septa of each strain was counted after staining with CFW, and then the proportion of conidia with different septate numbers to the total number was calculated. Count 100 conidia for each strain and repeat three times. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05.
Table 1.
Conidiation and conidia germination of ΔFgPfn mutants.
Fig 4.
Sensitivity of ΔFgPfn mutants to different stress factors.
(A) The colony morphology of ΔFgPfn mutants on different mediums. PH-1, ΔFgPfn mutants, and complementation strain were inoculated on PDA medium containing 1.2 M NaCl, 1.2 M KCl, 0.05% SDS, 1.2 M Sorbitol, and 0.03% Congo red at 25°C. The colony morphology of PH-1 and complementation strain was photographed after the 3d of growth, which was 20d in ΔFgPfn. (B) The inhibition rates of PH-1, ΔFgPfn mutants, and complementation strain with different stress factors. The hyphal growth inhibition rates of different stress factors on PH-1, ΔFgPfn mutants, and complementation strain were calculated with the colony diameters grown on PDA as control, where PH-1 and complementation mutant grew for 3d, and ΔFgPfn grew for 20d. Inhibition rates = (Average diameters of control group—Average diameters of treatment group)/Average diameters of control group × 100%. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05.
Fig 5.
FgPfn deletion mutant attenuates pathogenicity and DON content.
(A) Pathogenicity assays of ΔFgPfn mutants on wheat heads. At the blooming stage of wheat, 10 μL 1×106 /mL conidia suspension was evaluated by point-inoculating in flowering wheat heads. After 14d, the incidence was investigated and the disease grade was counted. The wheat variety was Huaimai 20. (B) Proportion of disease grade, reflecting the severity of the disease: 0, disease free; 1, disease proportion is less than 25%; 3, disease proportion is between 25% and 50%; 5, disease proportion is between 50% and 75%; 7, disease proportion is more than75%. (C) Pathogenicity assays of ΔFgPfn mutants on wheat coleoptiles. Add 2 μL 1×106 /mL conidia suspension were inoculated on the injured wheat coleoptile for 7d to observe the incidence and take photos. The wheat variety was Huaimai 33. (D) The average length of lesion on wheat coleoptile infected by each strain was measured after 7d post-inoculation. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05. (E) DON content assay of ΔFgPfn mutants. After the 7d of TBI culture, the DON content in the wild-type PH-1, ΔFgPfn mutants, and complementation strain were determined. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05. (F) Relative gene expression level of TRI1, TRI4, TRI5, TRI6 and TRI12 in the strains tested. After the 36h culture in TBI, mycelia of each strain were harvested for RNA extraction. The GAPDH was used as a reference gene. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05.
Fig 6.
FgPfn deletion mutant disrupts toxisome formation and DON transfer.
(A) Toxisome formation in ΔFgPfn. All strains were labeled with Tri1-GFP as a toxisome indicator and photographed under a confocal microscope after 36h of TBI culture. Bar = 10 μm. (B) The localization of Tri12-GFP in the PH-1 and ΔFgPfn mutant. All strains were labeled with Tri12-GFP and photographed under a confocal microscope after the 36h of TBI culture. Bar = 10 μm. (C) The association between toxisome and F-actin. Strain PH-1 co-expressing TRI1-RFP and LifeAct-GFP fusion proteins was photographed under a confocal microscope after 36h of TBI culture. Bar = 10 μm. (D) Actin organization in YEPD and TBI cultures. After the 36h of YEPD or TBI culture, the organization of actin marked by LifeAct-GFP in wild-type strain PH-1 and ΔFgPfn were photographed under a confocal microscope. Bar = 10 μm.
Fig 7.
FgPfn regulates the sensitivity to carbendazim and contributes to the organization of microtubules.
(A) ΔFgPfn mutants significantly increased the sensitivity to carbendazim. PH-1 and ΔFgPfn-C strains were able to grow after the 3d of 0.8 μg/mL carbendazim treatment, while the ΔFgPfn mutants were still unable to grow at 20d. (B) Mycelial growth inhibition of each strain by carbendazim quantified. (C) FgPfn contributes to microtubule organization. 105 /mL conidia were inoculated in YEPD medium for 12h at 25°C, and then carbendazim with a final concentration of 0.4 μg/mL was added for another 12h. white lines indicate differences. Microtubule organization was examined under a confocal microscope. Bar = 10 μm. (D) Analysis of the interaction of FgPfn with Fgα1 by Co-IP assay. The Fgα1-GFP fusion plasmid was transformed into PH-1 and ΔFgPfn-C containing a 3×Flag tag, respectively. To explore the effect of carbendazim on the interaction, strains were cultured in YEPD 36h, and carbendazim with a final concentration of 2 μg/mL was added for another 12h. Total protein samples were first incubated with anti-GFP agarose beads (GFP-Trap Magnetic Agarose beads, ChromoTek, Redmond, WA, USA) following the manufacturer’s protocol. Then, 10 μL protein samples eluted from beads were analyzed by western blot. (E) Analysis of the interaction of FgPfn with Fgβ2 by Co-IP assay. The Fgβ2-GFP fusion plasmid was transformed into PH-1 and ΔFgPfn-C containing a 3×Flag tag, respectively. To explore the effect of carbendazim on the interaction, strains were cultured in YEPD 36h, and carbendazim with a final concentration of 2 μg/mL was added for another 12h. Total protein samples were first incubated with anti-GFP agarose beads following the manufacturer’s protocol. Then, 10 μL protein samples eluted from beads were analyzed by western blot. (F) The content of the Fgβ2-GFP protein in both PH-1 and ΔFgPfn mutant each strain was determined by western blot assay with the anti-GFP antibody. The protein samples were also detected with anti-GAPDH antibody as a reference. (G) Analysis of the interaction of FgPfn with Fgβ2 by BiFC assay. The strains bearing a single construct (FgPfn-GFPN with GFPC or GFPN with Fgβ2-GFPC) were used as the negative control. The GFP signals in the hyphae of each strain were examined under a confocal microscope. Bar = 10 μm.
Fig 8.
FgPfn is required for the FgMyo5-FgAct cytoskeleton organization.
(A) ΔFgPfn mutants significantly increased the sensitivity to phenamacril. PH-1 and ΔFgPfn-C grew for the 3d under 0.5 μg/mL phenamacril treatment and the ΔFgPfn mutants grew for the 20d. (B) Mycelial growth inhibition of each strain by phenamacril quantified. (C) Deletion of FgPfn affected the FgMyo5 localization. The localization of FgMyo5 was observed by expressing FgMyo5-GFP in the wild-type and mutant strains. All strains were grown in YEPD for 12h and photographed under a confocal microscope. White arrows indicate differences. Bar = 10 μm. (D) Deletion of FgPfn affected the actin organization. Actin cable and patch were observed by expressing LifeAct-GFP in the wild-type and mutant strains. All strains were grown in YEPD for 12h and photographed under a confocal microscope. White arrows indicate differences. Bar = 10 μm. (E) The content of the FgMyo5 and FgAct in both PH-1 and ΔFgPfn mutant was determined by western blot assay with the anti-GFP antibody and anti-Actin antibody. The protein samples were also detected with anti-GAPDH antibody as a reference. (F) Analysis of the interaction of FgMyo5 with FgAct in ΔFgPfn. The FgMyo5-GFP fusion plasmid was transformed into PH-1 and ΔFgPfn, respectively. Total protein samples were first incubated with anti-GFP agarose beads following the manufacturer’s protocol. Then 10 μL protein samples eluted from beads were analyzed by western blot.
Fig 9.
FgPfn interacts with both FgMyo5 and FgAct.
(A) Analysis of the interaction of FgPfn with FgMyo5 by Co-IP assay. The FgMyo5-GFP fusion plasmid was transformed into PH-1 and ΔFgPfn-C containing a 3×Flag tag, respectively. To explore the effect of phenamacril on the interaction, strains were cultured in YEPD 36h, and phenamacril with a final concentration of 2 μg/mL was added for another 12h. Total protein samples were first incubated with anti-GFP agarose beads following the manufacturer’s protocol. Then, 10 μL protein samples eluted from beads were analyzed by western blot. (B) Analysis of the interaction of FgPfn with FgMyo5 by BiFC assay. The strains bearing a single construct (FgPfnt-GFPN with GFPC or GFPN with FgMyo5-GFPC) were used as the negative control. The GFP signals in the hyphae of each strain were examined under a confocal microscope. Bar = 10 μm. (C) Analysis of the interaction of FgPfn with FgAct by Co-IP assay. The FgAct-GFP fusion plasmid was transformed into PH-1 and ΔFgPfn-C containing a 3×Flag tag, respectively. To explore the effect of phenamacril on the interaction, strains were cultured in YEPD 36h, and phenamacril with a final concentration of 2 μg/mL was added for another 12h. Total protein samples were first incubated with anti-GFP agarose beads following the manufacturer’s protocol. Then, 10 μL protein samples eluted from beads were analyzed by western blot. (D) Analysis of the interaction of FgPfn with FgAct by BiFC assay. The strains bearing a single construct (FgAct-GFPN with GFPC or GFPN with FgPfn-GFPC) were used as the negative control. The GFP signals in the hyphae of each strain were examined under a confocal microscope. Bar = 10 μm.
Fig 10.
The protein integrity is necessary for FgPfn interaction with FgAct.
(A) The subcloning motifs of FgPfn and FgAct were indicated. FgPfn and FgAct were divided into 7 and 9 motifs, respectively. Each cDNA of FgPfn, which lacked one motif, was cloned into pGADT7, and each cDNA of FgAct, which lacked one motif, was cloned into pGBKT7 plasmid, respectively. (B) Analysis of the interaction of FgPfn and FgAct subclone motifs. (C) The amino acid reversal mutations on FgPfn were indicated. FgPfn were divided into 9 motifs, reversed the order of the amino acid sequence of each segment respectively, and then cloned the 9 mutations of FgPfn into pGADT7 plasmid to confirm interaction with FgAct by Y2H. (D) Analysis of the interaction of FgPfn amino acid reversal mutations with FgAct by Y2H assay.
Fig 11.
Sequence alignment and identification of conserved residues of profilins.
(A) The sequence alignment of profilins among FgPfn, FfPfn, MoPfn, and ScPfn. The amino acids with the same polarity or acidity and basicity were annotated in red font. Identical amino acids were annotated with red shading. (B) Analysis of the interaction of FgPfn, FfPfn, MoPfn, and ScPfn with FgAct by Y2H assay. (C) The sequence alignment of profilins among ascomycetes and basidiomycetes. The amino acids with the same chemical properties were annotated in red font. Identical amino acids were annotated with red shading. (D) Analysis of the interaction of FgPfn amino acid mutations with FgAct by Y2H assay.
Fig 12.
Conserved amino acids were required for the function of FgPfn.
(A) Growth phenotypes of different mutants. Mycelial growth was photographed after the 3d culture of each mutant on PDA. (B) The colony diameter of each mutant strain was measured after the 3d growth on PDA. Bars with the same letter indicate no significant difference according to the LSD test at p < 0.05. (C) Mutations in conserved residues caused defects in actin organization. The images of actin organization were taken after the 24h culture of each mutant in YEPD. Bar = 10 μm.
Fig 13.
The schematic illustration of the interaction and function of FgPfn in FgPfn-dependent cytoskeleton.
The interaction between FgPfn with Fgα1 and Fgβ2 contributes to microtubule organization and the sensitivity to carbendazim. FgPfn regulated the FgMyo5-FgAct cytoskeleton and the sensitivity to phenamacril by participating in the organization and interaction of FgMyo5 and FgAct. FgPfn regulates the formation of toxisomes by participating in the reorganization of actin induced in TBI. In this way, FgPfn regulates the vegetative growth, sexual reproduction and pathogenicity of the filamentous fungus Fusarium graminearum.