Submitted to: Journal of Biomolecular Techniques
Publication Type: Abstract Only
Publication Acceptance Date: 5/30/2006
Publication Date: 7/14/2006
Citation: Thannhauser, T.W., Yang, Y., Zhang, S., Damasceno, C., Sherwood, R., Rose, J. 2006. Itraq based protein quantitative analysis of the cell wall proteome of pathogen-infected tomato leaves. Journal of Biomolecular Techniques. 17:40.
Technical Abstract: The goal of this work is to use an iTRAQ-based shotgun approach to identify qualitative and quantitative changes in the extracellular proteome, or secretome, of tomato leaves following infection with the oomycete pathogen Phytophthora infestans. Proteins that are secreted by plants and microbes into the plant cell wall, or apoplast, play a fundamental role in establishing and determining the outcome of plant-microbe interactions. However, substantial portions of these 'secretomes' remain unidentified and little is known about wall-localized proteins which are up/down-regulated during plant-pathogen interactions. Phytophthora infestans-infected tomato leaves and uninfected control leaves were vacuum infiltrated in with a salt buffer and surfactant, and apoplastic proteins were removed by centrifugation, avoiding contamination with intercellular proteins. Proteins (100 g) from each state were reduced, denatured, MMTS blocked and digested by trypsin prior to labeling with iTRAQ 114 and 117 reagents respectively. The labeled tryptic peptides were pooled and separated on a SCX column (PolySulfoethyl A). Ten SCX fractions were used for nanoLC/MS/MS analysis. A hybrid linear ion trap (4000 Q Trap) was operated in an information-dependent acquisition (IDA) mode. The resulting IDA files were used for the database searches using ProQuant and ProGroup view software. A total of 113 distinct tomato proteins were identified, 19 of which were up-regulated during infection and 6 of which showed a 4 to 8 fold change in abundance. The results demonstrate that the iTRAQ shotgun approach is a valuable technique for analysis of the plant cell wall proteome, a notoriously recalcitrant sub cellular compartment. The present study will provide the foundation for an extensive catalog of plant secreted proteins, many of which will have anti-microbial properties and will help elucidate the complex interactions between pathogens and their hosts proceduring pathogenesis.