Desiccation-tolerant plants are able to survive for extended periods of time in the absence of water. The molecular understanding of the mechanisms used by these plants to resist droughts can be of great value for improving drought tolerance in crops. This understanding is especially relevant in an environment that tends to increase the number and intensity of droughts. The combination of certain microorganisms with drought-sensitive plants can improve their tolerance to water scarcity. One of these bacteria is Microbacterium sp. 3J1, an actinobacteria able to protect pepper plants from drought. In this study, we supplemented drought-tolerant and drought-sensitive plant rhizospheres with Microbacterium sp. 3J1 and analyzed their proteomes under drought to investigate the plant-microbe interaction. We also compare this root proteome with the proteome found in desiccation-tolerant plants. In addition, we studied the proteome of Microbacterium sp. 3J1 subjected to drought to analyze its contribution to the plant-microbe interaction. We describe those mechanisms shared by desiccation-tolerant plants and sensitive plants protected by microorganisms focusing on protection against oxidative stress, and production of compatible solutes, plant hormones, and other more specific proteins.

Green Pepper Capsicum annuum L. cv. Maor plants inoculated and non-inoculated with the soil bacteria Microbacterium sp. 3J1 and then, they were subjected to a dehydration treatment to simulate drought conditions.

In addition, Microbacterium sp. 3J1 cultures were grown in tryptic soy broth (TSB) supplemented with 5% or 50% (w/v) PEG 8000 to simulate different drought conditions.

Soluble proteins of both, plants (roots) and bacterial cultures were isolated and precipitated by adding 50% (v/v) ice cold trichloroacetic acid (TCA). Total soluble protein were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PGAGE) and geles images were analyzed by PDQuest Basic Software (Bio-Rad, Hercules, CA, USA). Spots differentially expressed were selected by two different criteria: Qualitative spots (presence/absence), and quantitative spots (p = ≤ 0.05) with a fold difference of 2 compared to the control.

The spots containing the proteins of interest were cut using the EXQuest Spot Cutter (Bio-Rad). Gel pieces containing the proteins of interest were digested with Trypsin Gold Mass Spectrometry Grade (Promega) and tryptic digests of each spot were subjected to Mass Spectometry for protein identification. The MS and tandem MS (MS/MS) spectra were obtained using an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics) and processed using ProteinScape v3.1.3 (Bruker-Daltonics).

The peptide mass fingerprint and MS/MS search were performed on NCBI database for Viridiplantae and Capsicum annuum searching for plant proteins. Bacterial proteins of Microbacterium sp. 3J1 were searched in databases for Microbacteriae, Micrococci and Actinobacteria. SwissProt database was used for all organisms, using MASCOT 2.4.0 software integrated together with ProteinScape software (Bruker-Daltonics).