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Integrating state-of-the-art tandem mass spectrometry with de novo-assisted database peptide sequencing as a viable strategy for the discovery of peptide proteolytic cleavage products (PCPs) in plant-microbe interactions.

Small peptides that are proteolytic cleavage products (PCPs) less than 100 amino acids are emerging as key signaling molecules that mediate cell-to-cell communication and biological processes that occur between and within plants, fungi, and bacteria. Yet, the discovery and characterization of these molecules is largely overlooked. Today, selective enrichment and subsequent characterization by mass spectrometry (MS)-based sequencing offers the greatest potential for their comprehensive characterization, however qualitative and quantitative performance metrics are rarely captured. Herein, we addressed this need by benchmarking the performance of an enrichment strategy optimized specifically for small PCPs using state-of-the-art de novo-assisted peptide sequencing. As a case study, we implemented this approach to identify PCPs from different root and foliar tissues of the hybrid poplar Populus X canescens 717-1B4 in interaction with the ectomycorrhizal basidiomycete Laccaria bicolor. In total, we identified 1660 and 2870 Populus and L. bicolor unique PCPs, respectively. Qualitative results supported the identification of well-known PCPs, like the mature form of the Photosystem II complex 5 kDa protein (~ 3 kDa). A total of 157 PCPs were determined to be significantly more abundant in root tips with established ectomycorrhiza when compared to root tips without established ectomycorrhiza and extramatrical mycelium of L. bicolor. These PCPs mapped to 64 Populus proteins and 69 L. bicolor proteins in our database, with several of them previously implicated in biologically relevant associations between plant and fungus.