Abstract
The interaction between plants and pathogenic microorganisms is a multifaceted process mediated by both plant- and pathogen-derived molecules, including proteins, metabolites, and lipids. Large-scale proteome analysis can quantify the dynamics of proteins, biological pathways, and posttranslational modifications (PTMs) involved in the plant–pathogen interaction. Mass spectrometry (MS)-based proteomics has become the preferred method for characterizing proteins at the proteome and sub-proteome (e.g., the phosphoproteome) levels. MS-based proteomics can reveal changes in the quantitative state of a proteome and provide a foundation for understanding the mechanisms involved in plant–pathogen interactions. This review is intended as a primer for biologists that may be unfamiliar with the diverse range of methodology for MS-based shotgun proteomics, with a focus on techniques that have been used to investigate plant–pathogen interactions. We provide a summary of the essential steps required for shotgun proteomic studies of plants, pathogens and plant–pathogen interactions, including methods for protein digestion, identification, separation, and quantification. Finally, we discuss how protein PTMs may directly participate in the interaction between a pathogen and its host plant.
Highlights
As a multifaceted process, plant–pathogen interactions have been extensively researched from both the pathogen and plant viewpoints
Technological advances in Mass spectrometry (MS)-based proteomics have significantly accelerated the characterization of pathogen–host protein interactions [11] and while gel-based methods for intact protein separation and identification remain in use for specialized applications [12], shotgun analysis is well established as the dominant proteomics platform [13]
Recent advances in MS-based proteomics and bioinformatics tools enable the robust profiling of plant and pathogenic microbe samples to an unprecedented depth
Summary
Plant–pathogen interactions have been extensively researched from both the pathogen and plant viewpoints. The increase in genomic and transcriptome studies has advanced the understanding of the pathogenicity strategies employed by pathogens and the immune responses in plants [2–5]. These approaches have limitations and cannot monitor post-transcriptional processes [6]. Technological advances in MS-based proteomics have significantly accelerated the characterization of pathogen–host protein interactions [11] and while gel-based methods for intact protein separation and identification remain in use for specialized applications [12], shotgun analysis is well established as the dominant proteomics platform [13] The success of these approaches depends on various factors, including the methods used for isolation, digestion, separation, identification, and quantification of proteins [14]. Harsh conditions, including mechanical force, must be applied for the disruption of the thickened cell walls in these structures [25]
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