Quantitative Acetylome Analysis of Differentially Modified Proteins in Virulence-Differentiated Fusarium oxysporum f. sp. cucumerinum Isolates during Cucumber Colonization

Abstract

Fusarium oxysporum f. sp. cucumerinum (Foc) is a prominent pathogen that adversely affects cucumber (Cucumis sativus) production. In the pathogen's parasitic lifestyle, the pathogenesis and virulence evolution may be regulated by lysine acetylation, as demonstrated in many living organisms. However, its specific function in Foc remains poorly understood. In this study, the acetylome profiles of a mild virulence strain (foc-3b) and its derived virulence-enhanced strain (Ra-4) were analyzed before and post-inoculation on cucumber plants. In total, 10,664 acetylation sites were identified corresponding to 3874 proteins, and 45 conserved acetylation motifs were detected. Through comparison of the acetylomes, numerous differentially lysine-acetylated proteins were enriched in energy metabolism and protein processing processes, indicating the critical role of lysine acetylation during the transition from the saprotrophic lifestyle to the parasitic lifestyle. Comparative acetylome analyses on the two virulence-differentiated strains revealed that several differentially lysine-acetylated proteins were involved in pathways of defense response and energy metabolism. Ra-4 showed enhanced energy metabolism compared to foc-3b. This indicates that robust metabolic activity is required to achieve high virulence and facilitating adaptive evolution. Additionally, faster host responses are supported by an ample energy supply enhancing virulence. Thus, lysine acetylation plays a crucial role in the pathogenesis and virulence evolution of Foc.

Keywords: Cucumis sativus; lysine acetylation; pathogenesis; proteome; virulence evolution.

Figure 1

Repeatability tests of Fusarium oxysporum f. sp. cucumerinum samples: (A) Principal component analysis; (B) heatmap of Pearson correlation; (C) distribution of relative standard deviation. The samples were from F. oxysporum f. sp. cucumerinum strains foc-3b (W) and Ra-4 (I) during the saprophytic lifestyle in potato dextrose broth cultures (0 h) or parasitic lifestyle at 6 h post-inoculation (6 h) on Cucumis sativus roots.

Figure 2

Comprehensive analysis of all identified lysine-acetylated peptides in Fusarium oxysporum f. sp. cucumerinum strains foc-3b and Ra-4: (A) Mass error distribution; (B) proteins modifications distribution; (C) peptide length (amino acids) distribution.

Figure 3

Motif-X intensity map of all identified lysine-acetylated sites in Fusarium oxysporum f. sp. cucumerinum strains foc-3b and Ra-4. On the right side, the interpretation of reference colors for frequency (from red to green), distance, and position.

Figure 4

Volcano plot of differentially lysine-acetylated sites in Fusarium oxysporum f. sp. cucumerinum under different comparisons: (A) W_6h versus W_0h; (B) I_6h versus I_0h; (C) I_0h versus W_0h; (D) I_6h versus W_6h. Interpretation of the reference colors: orange = up-regulated, green = down-regulated, grey = unchanged. W_0h: wild-type strain foc-3b before inoculation. I_0h: Virulence-induced strain Ra-4 before inoculation. W_6h: wild-type strain foc-3b at 6 h post-inoculation. I_6h: Virulence-induced strain Ra-4 6 h post-inoculation.

Figure 5

Fusarium oxysporum f. sp. cucumerinum Gene Ontology (GO) functional description of differentially lysine-acetylated proteins across various comparisons: (A) W_6h versus W_0h; (B) I_6h versus I_0h; (C) I_0h versus W_0h; (D) I_6h versus W_6h. Numbers above the bar charts indicate the lysine-acetylated proteins. W_0h: wild-type strain foc-3b before inoculation. I_0h: Virulence-induced strain Ra-4 before inoculation. W_6h: wild-type strain foc-3b at 6 h post-inoculation. I_6h: Virulence-induced strain Ra-4 at 6 h post-inoculation.

Figure 6

Enrichment analysis of differentially lysine-acetylated proteins in Fusarium oxysporum f. sp. cucumerinum for the comparison between 6 h post-inoculation (W_6h) and pre-inoculation (W_0h) in the wild-type strain foc-3b: (A) Gene Ontology (GO); (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway; (C) protein domain.

Figure 7

Enrichment analysis of differentially lysine-acetylated proteins in Fusarium oxysporum f. sp. cucumerinum for the comparison between 6 h post-inoculation (I_6h) and pre-inoculation (I_0h) in the virulence-enhanced strain Ra-4: (A) Gene Ontology (GO); (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway; (C) protein domain.

Figure 8

Enrichment analysis of differentially lysine-acetylated proteins in Fusarium oxysporum f. sp. cucumerinum for the comparison of the virulence-enhanced strain Ra-4 (I_0h) versus the wild-type strain foc-3b (W_0h) before inoculation: (A) Gene Ontology (GO); (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway; (C) protein domain.

Figure 9

Enrichment analysis of differentially lysine-acetylated proteins in Fusarium oxysporum f. sp. cucumerinum for the comparison of the virulence-enhanced strain Ra-4 (I_6h) versus the wild-type strain foc-3b (W_6h) 6 h post-inoculation: (A) Gene Ontology (GO); (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway; (C) protein domain.

Figure 10

Protein–protein interaction network of differentially lysine-acetylated modification sites in Fusarium oxysporum f. sp. cucumerinum for the comparison: (A) W_6h versus W_0h; (B) I_6h versus I_0h; (C) I_0h versus W_0h; (D) I_6h versus W_6h. Interpretation of the reference colors: red = up-regulated, green = down-regulated. W_0h: wild-type strain foc-3b before inoculation. I_0h: Virulence-induced strain Ra-4 before inoculation. W_6h: wild-type strain foc-3b at 6 h post-inoculation. I_6h: Virulence-induced strain Ra-4 at 6 h post-inoculation.