Global Proteomic Analysis of Lysine Crotonylation in the Plant Pathogen Botrytis cinerea

Abstract

Lysine crotonylation (Kcr), a recently discovered post-translational modification, plays a key role in the regulation of diverse cellular processes. Botrytis cinerea is a destructive necrotrophic fungal pathogen distributed worldwide with broad ranging hosts. However, the functions of Kcr are unknown in B. cinerea or any other plant fungal pathogens. Here, we comprehensively evaluated the crotonylation proteome of B. cinerea and identified 3967 Kcr sites in 1041 proteins, which contained 9 types of modification motifs. Our results show that although the crotonylation was largely conserved, different organisms contained distinct crotonylated proteins with unique functions. Bioinformatics analysis demonstrated that the majority of crotonylated proteins were distributed in cytoplasm (35%), mitochondria (26%), and nucleus (22%). The identified proteins were found to be involved in various metabolic and cellular processes, such as cytoplasmic translation and structural constituent of ribosome. Particularly, 26 crotonylated proteins participated in the pathogenicity of B. cinerea, suggesting a significant role for Kcr in this process. Protein interaction network analysis demonstrated that many protein interactions are regulated by crotonylation. Furthermore, our results show that different nutritional conditions had a significant influence on the Kcr levels of B. cinerea. These data represent the first report of the crotonylome of B. cinerea and provide a good foundation for further explorations of the role of Kcr in plant fungal pathogens.

Keywords: Botrytis cinerea; LC-MS/MS; crotonylome; fungal pathogenicity; lysine crotonylation.

FIGURE 1

Properties of the Kcr peptides in B. cinerea. (A) Crotonylation sequence motifs for ±10 amino acids surrounding the Kcr sites. (B) Heatmap of the amino acid compositions of the Kcr sites demonstrating the frequency of certain amino acids around the modified lysine. Red indicates high frequency and green means low frequency. (C) Probabilities of Kcr in the structures of beta-strand, alpha-helix, and coil. (D) Predicted surface accessibility of Kcr sites.

FIGURE 2

Conservation analysis of lysine crotonylated proteins. (A) Number of orthologous crotonylproteins in eight organisms or cell lines with reported crotonylomes. (B) A pie chart of conservation of crotonylproteins in Oryza sativa, Nicotiana tabacum, Human peripheral blood, Human Hela cells, Human H1299 cells, Human A549 cells, Danio rerio, and Carica papaya. Classification was performed as follows: Completely conserved group, 8 orthologs; Well conserved group, 6 to 7 orthologs; Conserved group, 3 to 5 orthologs; Poorly conserved group, 1 to 2 orthologs; Novel group, 0 orthologs.

FIGURE 3

Functional classification of lysine crotonylated proteins based on GO and subcellular location in B. cinerea. GO classification of crotonylated proteins based on biological process (A), cellular component (B), and molecular function (C). (D) Subcellular localization classification of the crotonylated proteins.

FIGURE 4

GO-based enrichment analysis of crotonylated proteins according to biological process (A), molecular function (B), and cellular component (C).

FIGURE 5

Three-dimensional structure of Bccpr1 (A) and Bcglr1 (B) with identified crotonylation site. The structure was derived from PDB database. The crotonylated lysine residues and adjacent arginine residues were indicated by blue and red colored sticks, respectively.

FIGURE 6

Protein interaction networks of the crotonylation proteins in B. cinerea.

FIGURE 7

Kcr levels of B. cinerea under different physiological conditions. (A) Immunoblot analysis of crotonylated proteins with pan anti-Kcr antibody of B. cinerea cultivated in liquid YEPD or MM. The loading control by Coomassie blue staining was used to ensure that equal amounts of protein were loaded in each lane. (B) Protein extracts containing Bcpck1-GFP were immunoprecipitated (IP) using anti-GFP beads. The isolated proteins were resolved by SDS-PAGE. Anti-GFP and anti-Kcr antibodies were used to detect Bcpck1-GFP and its crotonylated isoform, respectively.