From Affinity to Proximity Techniques to Investigate Protein Complexes in Plants.

The study of protein-protein interactions (PPIs) is fundamental in understanding the unique role of proteins within cells and their contribution to complex biological systems. While the toolkit to study PPIs has grown immensely in mammalian and unicellular eukaryote systems over recent years, application of these techniques in plants remains under-utilized. Affinity purification coupled to mass spectrometry (AP-MS) and proximity labeling coupled to mass spectrometry (PL-MS) are two powerful techniques that have significantly enhanced our understanding of PPIs. Relying on the specific binding properties of a protein to an immobilized ligand, AP is a fast, sensitive and targeted approach used to detect interactions between bait (protein of interest) and prey (interacting partners) under near-physiological conditions. Similarly, PL, which utilizes the close proximity of proteins to identify potential interacting partners, has the ability to detect transient or hydrophobic interactions under native conditions. Combined, these techniques have the potential to reveal an unprecedented spatial and temporal protein interaction network that better understands biological processes relevant to many fields of interest. In this review, we summarize the advantages and disadvantages of two increasingly common PPI determination techniques: AP-MS and PL-MS and discuss their important application to plant systems.

In silico and in vitro approaches allow the identification of the Prosystemin molecular network.

Tomato Prosystemin (ProSys), the precursor of Systemin, a small peptidic hormone, is produced at very low concentration in unchallenged plants, while its expression greatly increases in response to several different stressors triggering an array of defence responses. The molecular mechanisms that underpin such a wide array of defence barriers are not fully understood and are likely correlated with the intrinsically disordered (ID) structure of the protein. ID proteins interact with different protein partners forming complexes involved in the modulation of different biological mechanisms. Here we describe the ProSys-protein network that shed light on the molecular mechanisms underpinning ProSys associated defence responses. Three different approaches were used. In silico prediction resulted in 98 direct interactors, most clustering in phytohormone biosynthesis, transcription factors and signal transduction gene classes. The network shows the central role of ProSys during defence responses, that reflects its role as central hub. In vitro ProSys interactors, identified by Affinity Purification-Mass Spectrometry (AP-MS), revealed over three hundred protein partners, while Bimolecular Fluorescent Complementation (BiFC) experiments validated in vivo some interactors predicted in silico and in vitro. Our results demonstrate that ProSys interacts with several proteins and reveal new key molecular events in the ProSys-dependent defence response of tomato plant.

Corrigendum to "In silico and in vitro approaches allow the identification of the Prosystemin molecular network" [Comput Struct Biotechnol J 21 (2023) 212-223].

[This corrects the article DOI: 10.1016/j.csbj.2022.12.006.].

Colonization of Solanum melongena and Vitis vinifera Plants by Botrytis cinerea Is Strongly Reduced by the Exogenous Application of Tomato Systemin.

Plant defense peptides are able to control immune barriers and represent a potential novel resource for crop protection. One of the best-characterized plant peptides is tomato Systemin (Sys) an octadecapeptide synthesized as part of a larger precursor protein. Upon pest attack, Sys interacts with a leucine-rich repeat receptor kinase, systemin receptor SYR, activating a complex intracellular signaling pathway that leads to the wound response. Here, we demonstrated, for the first time, that the direct delivery of the peptide to Solanum melongena and Vitis vinifera plants protects from the agent of Grey mould (Botrytis cinerea). The observed disease tolerance is associated with the increase of total soluble phenolic content, the activation of antioxidant enzymes, and the up-regulation of defense-related genes in plants treated with the peptide. Our results suggest that in treated plants, the biotic defense system is triggered by the Sys signaling pathway as a consequence of Sys interaction with a SYR-like receptor recently found in several plant species, including those under investigation. We propose that this biotechnological use of Sys, promoting defense responses against invaders, represents a useful tool to integrate into pest management programs for the development of novel strategies of crop protection.

Rapid Identification of Protein-Protein Interactions in Plants.

Enzyme-enzyme interactions can be discovered by affinity purification mass spectrometry (AP-MS) under in vivo conditions. Tagged enzymes can either be transiently transformed into plant leaves or stably transformed into plant cells prior to AP-MS. The success of AP-MS depends on the levels and stability of the bait protein, the stability of the protein-protein interactions, and the efficiency of trypsin digestion and recovery of tryptic peptides for MS analysis. Unlike in-gel-digestion AP-MS, in which the gel is cut into pieces for several independent trypsin digestions, we uses a proteomics-based in-solution digestion method to directly digest the proteins on the beads following affinity purification. Thus, a single replicate within an AP-MS experiment constitutes a single sample for LC-MS measurement. In subsequent data analysis, normalized signal intensities can be processed to determine fold-change abundance (FC-A) scores by use of the SAINT algorithm embedded within the CRAPome software. Following analysis of co-sublocalization of "bait" and "prey," we suggest considering only the protein pairs for which the intensities were more than 2% compared with the bait, corresponding to FC-A values of at least four within-biological replicates, which we recommend as minimum. If the procedure is faithfully followed, experimental assessment of enzyme-enzyme interactions can be carried out in Arabidopsis within 3 weeks (transient expression) or 5 weeks (stable expression). © 2019 The Authors. Basic Protocol 1: Gene cloning to the destination vectors Alternate Protocol: In-Fusion or Gibson gene cloning protocol Basic Protocol 2: Transformation of baits into the plant cell culture or plant leaf Basic Protocol 3: Affinity purification of protein complexes Basic Protocol 4: On-bead trypsin/LysC digestion and C18 column peptide desalting and concentration Basic Protocol 5: Data analysis and quality control.

Tomato Plants Treated with Systemin Peptide Show Enhanced Levels of Direct and Indirect Defense Associated with Increased Expression of Defense-Related Genes.

Plant defense peptides represent an important class of compounds active against pathogens and insects. These molecules controlling immune barriers can potentially be used as novel tools for plant protection, which mimic natural defense mechanisms against invaders. The constitutive expression in tomato plants of the precursor of the defense peptide systemin was previously demonstrated to increase tolerance against moth larvae and aphids and to hamper the colonization by phytopathogenic fungi, through the expression of a wealth of defense-related genes. In this work we studied the impact of the exogenous supply of systemin to tomato plants on pests to evaluate the use of the peptide as a tool for crop protection in non-transgenic approaches. By combining gene expression studies and bioassays with different pests we demonstrate that the exogenous supply of systemin to tomato plants enhances both direct and indirect defense barriers. Experimental plants, exposed to this peptide by foliar spotting or root uptake through hydroponic culture, impaired larval growth and development of the noctuid moth Spodoptera littoralis, even across generations, reduced the leaf colonization by the fungal pathogen Botrytis cinerea and were more attractive towards natural herbivore antagonists. The induction of these defense responses was found to be associated with molecular and biochemical changes under control of the systemin signalling cascade. Our results indicate that the direct delivery of systemin, likely characterized by a null effect on non-target organisms, represents an interesting tool for the sustainable protection of tomato plants.