Capping proteins regulate fungal development, DON-toxisome formation and virulence in Fusarium graminearum

Abstract

Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.

Keywords: Fusarium graminearum; actin cytoskeleton; capping protein; deoxynivalenol (DON); toxisome; virulence.

Figure 1

FgCapA interacts with FgMyoI and Tri1. (A) The interaction of FgCapA‐GFP and FgMyo1‐Flag was verified by the co‐immunoprecipitation (Co‐IP) assay. Total protein (Input) extracted from the strain bearing FgCapA‐GFP and FgMyo1‐Flag constructs or a single construct (FgCapA‐GFP or FgMyo1‐Flag) were subjected to SDS‐PAGE, and immunoblots were incubated with anti‐FLAG and anti‐GFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti‐Flag agarose and further detected with anti‐GFP antibody (Flag pull‐down panel). Protein samples were also detected with anti‐GAPDH antibody as a reference. (B) The interaction of FgCapA‐mCherry and Tri1‐GFP was verified by the Co‐IP assay. Protein samples were pulled down using anti‐GFP agarose and further detected with an anti‐mCherry antibody. Protein samples were also detected with anti‐GAPDH antibody as a reference. (C) FgCapA‐mCherry was partially colocalized with Tri1‐GFP on DON‐toxisomes at 48 h of incubation in trichothecene biosynthesis induction (TBI) medium. Localization is indicated with yellow arrows. Bar = 10 µm. (D) The interaction of FgCapA with Tri1 was confirmed by bimolecular fluorescence complementation (BiFC) assay. The constructs of YFP^N‐Tri1 and pFgCapA‐YFP^C were co‐transformed into Fusarium graminearum PH‐1 to generate the strain YFPN ‐Tri1 + FgCapA‐YFP^C. The strains bearing a single construct (YFP^N‐Tri1 or FgCapA‐YFP^C) were used as negative controls. The yellow fluorescent protein (YFP) signals in hyphae of each strain grown in the TBI medium were examined under a confocal microscope. DIC, differential interference contrast. Bar = 10 μm.

Figure 2

FgCapA and FgCapB form a heterodimer. (A) The CAPs interacted with each other in the yeast‐two‐hybrid assay. Serial concentrations of yeast cells transferred with the bait and prey constructs indicated in the figure were assayed for growth on SD−Leu−Trp−His−Ade plates. pGBKT7‐53 and pGADT7 were used as positive controls. Another pair of plasmids, pGBKT7‐Lam and pGADT7, were used as negative controls. Images were taken after 3 days of incubation at 30 °C. pGADT7 and pGBKT7 are abbreviated to AD and BD, respectively. (B) FgCapA‐GFP colocalized with FgCapB‐mCherry. Vegetative hyphae of dual‐labelled strains were observed under a confocal microscope after incubation in PDB medium for 24 h. DIC, differential interference contrast. Bar = 10 µm. (C) The interaction of FgCapA‐GFP and FgcapB‐mCherry was verified by the Co‐IP assay. Protein samples were pulled down using anti‐GFP agarose and further detected with an anti‐mCherry antibody. Protein samples were also detected with anti‐GAPDH antibody as a reference. (D) A three‐dimensional homology model of the Fusarium graminearum CAP heterodimer based on the structure of chicken CAPs (Protein Data Bank, accession code 1IZN).

Figure 3

Capping proteins interact with actin for actin organization in Fusarium graminearum. (A) and (B) FgCapA‐GFP and FgCapB‐GFP colocalized with the F‐actin reporter Lifeact‐RFP in the mature hyphae and hyphal tips. Vegetative hyphae of dual‐labelled strains were observed under a confocal microscope after incubation in potato dextrose broth (PDB) medium for 24 h. DIC, differential interference contrast. Bar = 10 µm. (C) and (D) The interaction of FgCapA‐GFP or FgCapB‐GFP and actin‐RFP was verified by the Co‐IP assay. Protein samples were pulled down using anti‐GFP agarose and further detected with an anti‐RFP antibody. Protein samples were also detected with anti‐GAPDH antibody as a reference. (E) Deletion of FgCapA or FgCapB affected the actin morphology. Actin cable and patch was observed by expressing Lifeact‐RFP in the wild‐type and mutants. Hyphae of labelled strains were grown in PDB for 24 h. Representative micrographs of actin patterns in each strain are shown. Bar = 10 µm.

Figure 4

Capping proteins are required for vegetative growth, asexual and sexual reproduction in Fusarium graminearum. (A) Transcriptional levels of the FgCAPA and FgCAPB genes in conidiation (carboxymethyl cellulose, CMC), sexual reproduction (carrot agar, CA), mycelium (potato dextrose agar, PDA), and trichothecene biosynthesis induction (TBI) media and during the plant infection process by RNA‐Seq. (B) Colony morphology of the wild‐type F. graminearum PH‐1, ΔFgcapA, ΔFgcapB and ΔΔFgcapA/B grown on PDA, CMC and minimal medium (MM) agar plates for 3 days at 25 °C. (C) Hyphal branching patterns and tip growth of the wild‐type and mutant strains grown on PDA for 1 day. Bar = 100 µm. (D) Conidial production of the wild‐type, mutant and complementation strains harvested from 4‐day‐old CMC cultures. (E) Average length of conidia of the wild‐type, mutant and complementation strains. Conidia produced by each strain were harvested after incubation in CMC medium for 4 days. Length of conidia was measured using the Nikon NIS‐Element D 4. 20 imaging software (n = 50 for each strain). (F) Conidial germination of the wild‐type, mutant and complementation strains in 2% sucrose solution. Bars denote standard deviations from three experiments. Columns labelled with the same letter are not significantly different according to the least significant difference (LSD) test at P = 0.05. (G) CAP mutants reduced the fungal sexual development. Strains grown on CA were self‐fertilized. Photographs of perithecia were taken after 3 weeks of incubation. Bar = 200 µm.

Figure 5

FgCAP gene deletion mutants have reduced endocytosis. Time‐course of FM4‐64 internalization via the endocytic pathway in mycelium and conidia of Fusarium graminearum wild‐type PH‐1 and mutant strains. Bar = 10 µm.

Figure 6

FgCAP gene deletion mutants have reduced sensitivities to abiotic stresses. (A) The sensitivity of Fusarium graminearum wild‐type PH‐1, mutant and complementation strains to 0.02% SDS (cell membrane‐damaging agent) and Congo red (cell wall‐damaging agent) at the final concentration of 0.2 g/L and 0.02% H₂O₂. (B) The inhibition of the mycelial growth rate was examined after each strain was incubated for 3 days on potato dextrose agar (PDA) supplemented with each stress compound. Bars denote standard deviations from three experiments. Columns labelled with the same letter are not significantly different according to the LSD test at P = 0.05.

Figure 7

F‐actin‐capping motifs are essential for the function of FgCapA. (A) Schematic architecture of FgCapA and FgCapB. The F‐actin‐capping motifs and their corresponding amino acid sequences are indicated. (B) F‐actin‐capping motifs were indispensable for FgCapA function on vegetative growth, but not for FgCapB. Fusarium graminearum wild‐type PH‐1, ΔFgcapA and the complementation strains ΔFgcapA‐C, ΔFgcapA‐C‐ΔA1 (lacking the F‐actin‐capping motif A1) and ΔFgcapA‐C‐ΔA2 (lacking the F‐actin‐capping motif A2); ΔFgcapB, ΔFgcapB‐C and ΔFgcapB‐C‐ΔB (lacking the F‐actin‐capping motif B) were grown on potato dextrose agar (PDA) for 3 days before imaging. (C) The subcellular localization of F‐actin‐capping motif truncated mutants, ΔFgcapA‐C‐ΔA1, ΔFgcapA‐C‐ΔA2 and ΔFgcapB‐C‐ΔB in the mycelia grown in potato dextrose broth (PDB). Bar = 10 µm. (D) Conidial production of the wild‐type, F‐actin‐capping motif truncated mutants, ΔFgcapA‐C‐ΔA1, ΔFgcapA‐C‐ΔA2 and ΔFgcapB‐C‐ΔB harvested from 4‐day‐old carboxymethyl cellulose (CMC) cultures. (E) Average length of conidia of the wild‐type and F‐actin‐capping motif truncated mutants. Conidia produced by each strain were harvested after incubation in CMC medium for 4 days. Length of conidia was measured using the Nikon NIS‐Element D 4. 20 imaging software (n = 50 for each strain). (F) Perithecium formation of F‐actin‐capping motif truncated mutants on carrot agar medium. Photographs of perithecia were taken after 3 weeks of incubation. Bar = 200 µm. (G) Endocytosis phenotype of the wild‐type PH‐1 and various complementation strains. Mycelia were stained with FM4‐64 for 10 min before imaging. Bar = 10 µm.

Figure 8

F‐actin‐capping motifs are indispensable for the interaction between FgCapA and actin. The interaction between ΔFgcapA‐C‐ΔA1 (A), ΔFgcapA‐C‐ΔA2 (B) or ΔFgcapA‐C‐ΔB (C) and actin‐RFP was examined by the co‐immunoprecipitation (Co‐IP) assay. Total protein (Input) extracted from the strain bearing FgCap‐GFP lacking individual F‐actin‐capping motif and actin‐RFP constructs or a single construct (FgCap‐GFP lacking individual F‐actin‐capping motif or Actin‐RFP) were subjected to SDS‐PAGE, and immunoblots were incubated with anti‐GFP and anti‐RFP antibodies, as indicated (Input panel). Each protein sample was pulled down using anti‐GFP agarose and further detected with anti‐RFP antibody (GFP pull‐down panel). Protein samples were also detected with anti‐GAPDH antibody as a reference.

Figure 9

Capping gene deletion mutants attenuated toxisome formation, DON production and virulence in planta. (A) Toxisome formation in Fusarium graminearum wild‐type PH‐1, ΔFgcapA, ΔFgcapB and F‐actin‐capping motif truncated mutants. All strains were labelled with Tri1‐GFP as a toxisome indicator and incubated in trichothecene biosynthesis induction (TBI) medium for 48 h before imaging. Bar = 10 µm. (B) The accumulation of the Tri1‐GFP protein in each strain was further determined by western blot assay using the anti‐GFP antibody. The protein samples were also detected with anti‐GAPDH antibody as a reference. (C) Relative mRNA expression level of TRI1, TRI5 and TRI6 in the strains tested. After culturing in TBI for 2 days, mycelia of each strain were harvested for mRNA extraction. The Actin was used as a reference gene. (D) DON production in the wild‐type, mutant and various complementation strains after 7 days of incubation in TBI. Bars denote standard deviations from three experiments. Columns labelled with the same letter are not significantly different according to the LSD test at P = 0.05. (E) Virulence of the wild‐type, mutant and various complementation strains on wheat heads. Infected wheat heads were examined at 15 days after inoculation with a conidial suspension of each strain. The inoculation sites are indicated as black dots. (F) Disease symptoms on young wheat leaves infected by the wild‐type, mutant and various complementation strains. The images were taken at 6 days post‐inoculation.