Effect of H2A.Z deletion is rescued by compensatory mutations in Fusarium graminearum

Abstract

Fusarium head blight is a destructive disease of grains resulting in reduced yields and contamination of grains with mycotoxins worldwide; Fusarium graminearum is its major causal agent. Chromatin structure changes play key roles in regulating mycotoxin biosynthesis in filamentous fungi. Using a split-marker approach in three F. graminearum strains INRA156, INRA349 and INRA812 (PH-1), we knocked out the gene encoding H2A.Z, a ubiquitous histone variant reported to be involved in a diverse range of biological processes in yeast, plants and animals, but rarely studied in filamentous fungi. All ΔH2A.Z mutants exhibit defects in development including radial growth, sporulation, germination and sexual reproduction, but with varying degrees of severity between them. Heterogeneity of osmotic and oxidative stress response as well as mycotoxin production was observed in ΔH2A.Z strains. Adding-back wild-type H2A.Z in INRA349ΔH2A.Z could not rescue the phenotypes. Whole genome sequencing revealed that, although H2A.Z has been removed from the genome and the deletion cassette is inserted at H2A.Z locus only, mutations occur at other loci in each mutant regardless of the genetic background. Genes affected by these mutations encode proteins involved in chromatin remodeling, such as the helicase Swr1p or an essential subunit of the histone deacetylase Rpd3S, and one protein of unknown function. These observations suggest that H2A.Z and the genes affected by such mutations are part or the same genetic interaction network. Our results underline the genetic plasticity of F. graminearum facing detrimental gene perturbation. These findings suggest that intergenic suppressions rescue deleterious phenotypes in ΔH2A.Z strains, and that H2A.Z may be essential in F. graminearum. This assumption is further supported by the fact that H2A.Z deletion failed in another Fusarium spp., i.e., the rice pathogen Fusarium fujikuroi.

Fig 1. Developmental defects in INRA349 mutants.

(A) and (B). Radial growth in FgINRA349 wild-type, ΔH2A.Z, ΔH2A.Z::H2A.Z, and OE:H2A.Z grown from a central 3 mm-diameter plug (A) or 100 spores (B) on CM agar for three and four days at 25°C in the dark. WT = wild-type I349; Δ = deletion mutant I349ΔH2A.Z; +H2A.Z = I349ΔH2A.Z::H2A.Z mutant; OE = I349OE:H2A.Z; dpi = days post-inoculation. (C). Germination rates of FgINRA349 wild-type, ΔH2A.Z, ΔH2A.Z::H2A.Z, and OE:H2A.Z after eight hours of incubation. (D). Fitted kinetics of sporulation followed for eight days. Black = wild-type I349; dashed red = I349ΔH2A.Z; dashed blue = I349ΔH2A.Z::H2A.Z mutant; dashed purple = I349OE:H2A.Z. For (C) and (D), letters indicate statistically significant curve groups after Kruskal-Wallis testing and Tukey-Kramer correction for multiple testing (p < 0.05).

Fig 2. Abiotic stress resistance of INRA349 and its mutants.

(A) and (B) Radial growth in FgINRA349 wild-type, ΔH2A.Z, ΔH2A.Z::H2A.Z, and OE:H2A.Z grown from a central 3 mm-diameter plug on CM agar supplemented with NaCl 1M, KCl 1M, H₂O₂ 5 mM, H₂O₂ 15 mM, or not supplemented for three (A) and six (B) days at 25°C in the dark. WT = wild-type I349; Δ = I349ΔH2A.Z; +H2A.Z = I349ΔH2A.Z::H2A.Z; OE = I349OE:H2A.Z. (C) and (D). Fitted radial growth kinetics followed for six days for wild-type I349 (C) and I349ΔH2A.Z (D). Black = CM; red = CM + NaCl 1M; blue = CM + KCl 1M; purple = CM + H₂O₂ 5 mM; green = CM + H₂O₂ 15 mM. Letters indicate statistically significant curve groups after Kruskal-Wallis testing and Tukey-Kramer correction for multiple testing (p < 0.05).

Fig 3. Fitted kinetics of production of DON and 15-ADON by INRA349, INRA156, PH-1, and their respective mutants grown in liquid MS cultures.

(A) FgINRA349 wild-type (black), ΔH2A.Z (red), ΔH2A.Z::H2A.Z (blue), and OE:H2A.Z (green). (B) FgINRA156 wild-type (black) and its four ΔH2A.Z, numbered #1 to #4 (red, blue, purple, and green, respectively). (C) PH-1 wild-type (black) and its ΔH2A.Z mutant (red). Toxin yields are expressed in micrograms of toxins per milligram of dry biomass. Letters indicate statistically significant curve groups after Kruskal-Wallis testing and Tukey-Kramer correction for multiple testing (p < 0.05).

Fig 4. Domain architecture of proteins affected by compensatory mutations in I349ΔH2A.Z and I349ΔH2A.Z:H2A.Z.

(A) Essential subunit of Rpd3S complex (FGRAMPH1_01G23597; 1,225 aa). The annotation “Ile673fs” indicates the position of a frameshift starting at position 673 of the protein in I349ΔH2A.Z (an isoleucine in wild-type). (B) Swr1 (FGRAMPH1_01G18675; 1,691 aa). The annotation “His852Pro” indicates the replacement of a histidine at position 852 by a proline in I349ΔH2A.Z:H2A.Z. Domain accession numbers in NCBI CDD/SPARCKLE database: TNG2 superfamily = CL34876, PHD = CL22851, PHD2_PHF12_Rco1 = CD15534, HAS = PFAM07529, DEXQc_SRCAP = CD18003, SF2_C_SNF = CD18793. Architectures are displayed with SnapGene Viewer 5.0.6.

Fig 5. Growth defects in INRA156 and PH-1 mutants.

WT = wild-type; Δ = deletion mutants ΔH2A.Z (numbered 1 to 4 for those obtained in INRA156 background); dpi = days post-inoculation. (A). Radial growth in FgINRA156 and FgPH-1 wild-type, and their ΔH2A.Z mutants, grown from a central 3 mm-diameter plug on CM agar for three and four days at 25°C in the dark. (B). Effect of abiotic stresses on radial growth in FgINRA156 and FgPH-1 wild-type and their ΔH2A.Z mutants, grown from a central 3 mm-diameter plug on CM agar supplemented with NaCl 1M, KCl 1M, H₂O₂ 5 mM, H₂O₂ 15 mM, or not supplemented for six days at 25°C in the dark.

Fig 6. Asexual sporulation and formation of perithecia in INRA156 and PH-1 ΔH2A.Z mutants.

(A). Fitted kinetics of sporulation for INRA156 wild-type and four ΔH2A.Z mutants. Black = wild-type; red = I156ΔH2A.Z#1; blue = I156ΔH2A.Z#2; purple = I156ΔH2A.Z#3; green = I156ΔH2A.Z#4. (B). Fitted kinetics of sporulation for PH-1 wild-type and PH-1ΔH2A.Z mutants. Black = wild-type; red = PH-1ΔH2A.Z. Letters indicate statistically significant curve groups after Kruskal-Wallis testing and Tukey-Kramer correction for multiple testing (p < 0.05). (C). Formation of perithecia on carrot agar by INRA156 and PH-1 wild-type and corresponding ΔH2A.Z mutants. Top lane: macroscopic view; bottom lane: microscopic view (x40). Pictures were taken four days after induction of sexual differentiation.