Histone H3 N-Terminal Lysine Acetylation Governs Fungal Growth, Conidiation, and Pathogenicity through Regulating Gene Expression in Fusarium pseudograminearum

Abstract

The acetylation of histone lysine residues regulates multiple life processes, including growth, conidiation, and pathogenicity in filamentous pathogenic fungi. However, the specific function of each lysine residue at the N-terminus of histone H3 in phytopathogenic fungi remains unclear. In this study, we mutated the N-terminal lysine residues of histone H3 in Fusarium pseudograminearum, the main causal agent of Fusarium crown rot of wheat in China, which also produces deoxynivalenol (DON) toxins harmful to humans and animals. Our findings reveal that all the FpH3^K9R, FpH3^K14R, FpH3^K18R, and FpH3^K23R mutants are vital for vegetative growth and conidiation. Additionally, FpH3K14 regulates the pathogen's sensitivity to various stresses and fungicides. Despite the slowed growth of the FpH3^K9R and FpH3^K23R mutants, their pathogenicity towards wheat stems and heads remains unchanged. However, the FpH3^K9R mutant produces more DON. Furthermore, the FpH3^K14R and FpH3^K18R mutants exhibit significantly reduced virulence, with the FpH3^K18R mutant producing minimal DON. In the FpH3^K9R, FpH3^K14R, FpH3^K18R, and FpH3^K23R mutants, there are 1863, 1400, 1688, and 1806 downregulated genes, respectively, compared to the wild type. These downregulated genes include many that are crucial for growth, conidiation, pathogenicity, and DON production, as well as some essential genes. Gene ontology (GO) enrichment analysis indicates that genes downregulated in the FpH3^K14R and FpH3^K18R mutants are enriched for ribosome biogenesis, rRNA processing, and rRNA metabolic process. This suggests that the translation machinery is abnormal in the FpH3^K14R and FpH3^K18R mutants. Overall, our findings suggest that H3 N-terminal lysine residues are involved in regulating the expression of genes with important functions and are critical for fungal development and pathogenicity.

Keywords: Fusarium crown rot; H3 N-terminal lysine residues; gene expression; histone acetylation; phytopathogen.

Figure 1

N-terminal sequence alignment of FpH3 with orthologs from representative species across fungi, plants, metazoans, and mammals. The alignment of N-terminal amino acid sequences of FpH3 and its orthologs from Fusarium graminearum, Magnaporthe oryzae, Verticillium dahlia, Sclerotinia sclerotiorum, Botrytis cinerea, Valsa mali, Colletotrichum orbiculare, Blumeria graminis, Ustilago maydis, Puccinia graminis, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and Human was performed using ClustalX 2.1 software. Conserved N-terminal lysines, mainly utilized for acetylation, are indicated by red arrows. Both the asterisk (*) and colon (:) represent conserved amino acids.

Figure 2

Defects in growth and conidiation in FpH3 N-terminal K to R mutants. (A) Three-day-old PDA cultures of the wild-type (CN23), FpH3^K9R (K9R), FpH3^K14R (K14R), FpH3^K18R (K18R), and FpH3^K23R (K23R) mutant strains. (B) Growth rate of CN23, K9R, K14R, K18R, and K23R strains. (C) Conidia numbers harvested from 3-day-old CMC cultures of CN23, K9R, K14R, K18R, and K23R strains. (D) Conidia length harvested from 3-day-old CMC cultures of CN23, K9R, K14R, K18R, and K23R strains. Different letters indicate significant differences based on ANOVA analysis with Duncan’s pair-wise comparison (p = 0.05).

Figure 3

Morphology characteristics and mycelial inhibition of the FpH3 K to R mutants under various cellular stresses. (A) Three-day-old cultures of the wild-type (CN23), FpH3^K9R (K9R), FpH3^K14R (K14R), FpH3^K18R (K18R), and FpH3^K23R (K23R) mutant strains grown on regular PDA and PDA supplemented with 0.02% SDS, 0.05% H₂O₂ (HO), 1.2 M NaCl (NC), or 300 μg mv⁻¹ Congo red (CR). (B) The percentage of mycelial growth inhibition of CN23, K9R, K14R, K18R, and K23R in PDA cultures with SDS, HO, NC, and CR compared to that in PDA cultures without stresses. Different letters denote significant differences based on ANOVA analysis with Duncan’s pair-wise comparison (p = 0.05).

Figure 4

Sensitivity of FpH3 K to R mutants to different fungicides. (A) Three-day-old cultures of the wild-type (CN23), FpH3^K9R (K9R), FpH3^K14R (K14R), FpH3^K18R (K18R), and FpH3^K23R (K23R) mutant strains grown on regular PDA and PDA supplemented with 0.25 μg mL⁻¹ tebuconazole (Teb), 0.08 μg mL⁻¹ fludioxonil (Flu), 0.25 μg mL⁻¹ phenamacril (Phe), or 0.25 μg mL⁻¹ carbendazim (Car). (B) The percentage of mycelial growth inhibition of CN23, K9R, K14R, K18R, and K23R in PDA cultures with Teb, Flu, Phe, and Car compared to that in PDA cultures without fungicides. Different letters denote significant differences based on ANOVA analysis with Duncan’s pair-wise comparison (p = 0.05).

Figure 5

Defects of FpH3 K to R mutants in plant infection. (A) Wheat seedlings inoculated with the wild-type (CN23), FpH3^K9R (K9R), FpH3^K14R (K14R), FpH3^K18R (K18R), and FpH3^K23R (K23R) mutant strains were photographed at 7 dpi. CK were inoculated with blank PDA cultures. (B) The lesion size on wheat seedling stems caused by CN23, K9R, K14R, K18R, and K23R strains. (C) Flowering wheat heads inoculated with CN23, K9R, K14R, K18R, and K23R strains were photographed at 14 dpi. CK were inoculated with sterile water. (D) The number of diseased spikelets caused by CN23, K9R, K14R, K18R, and K23R strains. (E) DON levels in diseased wheat spikelets inoculated with CN23, K9R, K14R, K18R, and K23R strains. Different letters denote significant differences based on ANOVA analysis with Duncan’s pair-wise comparison (p = 0.05).

Figure 6

RNA-seq analysis of K9R, K14R, K18R, and K23R mutants. (A) Venn diagram illustrating the number of genes upregulated in the FpH3^K9R (K9R), FpH3^K14R (K14R), FpH3^K18R (K18R), and FpH3^K23R (K23R) mutant strains. (B) Venn diagram illustrating the number of genes downregulated in the K9R, K14R, K18R, and K23R strains.

Figure 7

GO enrichment analysis of the upregulated genes in FpH3^K9R mutant (A), FpH3^K14R mutant (B), FpH3^K18R mutant (C), and FpH3^K23R mutant (D) strains.

Figure 8

GO enrichment analysis of the downregulated genes in the FpH3^K9R mutant (A), FpH3^K14R mutant (B), FpH3^K18R mutant (C), and FpH3^K23R mutant (D) strains.