Evaluation of Hypovirus Infection on the Vesicular Protein Expression Pattern of Cryphonectria parasitica by TMT-Based Proteomics Analysis

Abstract

Hypovirus infection is known to reduce the pathogenicity of Cryphonectria parasitica, the causative agent of chestnut blight. Isoforms derived from a viral protein p48 have been discovered in host mitochondria and vesicles, which may contribute to virulence attenuation, as reported in earlier work using two-dimensional electrophoresis (2-DE). In this study, a total of 1739 fungal proteins were identified in fungal vesicles through Tandem Mass Tag (TMT)-based quantitative proteomics. The infection of CHV1-EP713 was associated with 75 up-regulated and 201 down-regulated proteins, predominantly involved in vesicular transport process and related cellular functions, including protein folding, membrane fusion, retrograde transport, autophagy, and ER stress responses. The down-regulation of calnexin, COPI, ArfGAP, importin-β, and Atg8 is consistent with impairments in protein folding, retrograde transport, and autophagy. Meanwhile, the up-regulation of clathrin, dynamin, Vps10p, HSP70, and t-SNAREs indicated enhanced trafficking to vacuoles and increased stress response activity. Overall, our findings indicate that hypoviral infection is associated with extensive alterations in the vesicular transport system of C. parasitica, likely mediated through changes in the abundance of multiple key protein regulators. These alterations may underlie attenuation of virulence by impacting crucial cellular processes.

Keywords: Cryphonectria parasitica; differential proteomic analysis; hypovirus; vesicle-mediated intracellular transport.

Figure 1

An overview of the differential protein expression is provided, comparing the vesicular proteins of EP155 and EP713. (A) Electron microscope observation of C. parasitica vesicle sample; (B) Electrophoresis analysis of C. parasitica vesicle protein samples.

Figure 2

Volcano plot showing the distribution of differentially expressed proteins between EP713 and EP155.

Figure 3

The analysis focuses on protein distribution and enrichment based on GO annotation results. (A) The Level 2 GO annotation bar chart shows the functional distribution of vesicle proteins in C. parasitica; (B) The expression of EP713 changed significantly in key GO categories such as metabolic process, catalytic activity, and binding function; (C) Hierarchical GO enrichment analysis of strain EP713 showing enrichment in metabolic processes and catalytic activity, supporting the hypothesis of viral modulation of host metabolic functions and stress responses.

Figure 4

KEGG Annotation and Enrichment Analysis. (A) The histogram of KEGG annotations shows the distribution of proteins across different pathways, with a significant number of proteins engaged in amino acid, carbohydrate, energy, and nucleotide metabolism, as well as in processes that maintain protein homeostasis, signal transduction, and genetic information processing; (B) KEGG enrichment analysis reveals enrichment trends in metabolic pathways in the EP713 strain, such as peroxisome, galactose metabolism, and starch and sucrose metabolism; (C) The distribution of differentially expressed proteins shows significant down-regulation in key pathways, including metabolism, protein synthesis, cellular signaling, and genetic information processing, in EP713.

Figure 5

CHV1-induced alterations in key trafficking components, including calnexin, COPI, ArfGAP, importin-β, Atg8, HSP70, clathrin, dynamin, and SNAREs, reconfigure the vesicular network.