The project commenced with the cultivation of wild-type Arabidopsis thaliana plants in soil, from which the rhizosphere was sampled at the one-month growth mark. Protocols were established to purify exosomes, small vesicles measuring between 30 nm and 150 nm, though this process proved challenging due to the presence of humic acids, a significant contaminant in soil. The presence of exosomes in the rhizosphere samples of A. thaliana was confirmed through Transmission Electron Microscopy (TEM), which revealed a higher concentration of exosomes compared to a control bulk soil sample (unplanted soil). The exosomes found in bulk soil reflect both historical and current microbial activity.
To facilitate the purification of exosomes secreted by plant roots, A. thaliana was then grown in hydroponic conditions, allowing its roots to come into direct contact with a nutrient solution, thus releasing the secreted exosomes into this solution. The initial hydroponic system involved growing individual A. thaliana plants in 50 ml falcon tubes. Confirmation of exosome production and secretion by the roots in the hydroponic medium was again achieved using Transmission Electron Microscopy (TEM).
A small quantity of extracellular vesicles (EVs) was purified from both the rhizosphere and hydroponic conditions, but this yielded insufficient RNA for processing using standard Next Generation Sequencing (NGS) library protocols. Gel electrophoresis of the RNA showed a mixed-size RNA population ranging from 30 nt to 150 nt. This prompted the development of a protocol to specifically identify the RNA sequences of this mixed-size population obtained from a low quantity of exosomes. A homemade NGS library protocol was validated for RNA samples derived from exosomes purified from both soil and hydroponic conditions. This protocol was optimized to include a 12 nt Unique Molecular Identifier (UMI), allowing for the tagging of each original RNA molecule within the exosomes. Consequently, this innovation enabled the differentiation of duplicate artifacts and sequencing errors resulting from the polymerase reagents during NGS library prep and sequencing itself.
To enhance exosome production from plants grown in hydroponic settings, homemade hydroponic systems with capacities of 500 ml and 4 liters were constructed, allowing for the cultivation of batches of up to 24 plants (Fablab, University Rennes 1). The 4-liter hydroponic system was utilized to grow wheat, an agricultural plant of interest. Specific protocols were developed to purify exosomes from this 4-liter system. The integrity of the exosomes was confirmed using cryo-TEM. The exosomes derived from wheat underwent RNA and proteomic analysis and were also employed as a reagent to explore their effects on modulating the expression of a bacterial strain found in the rhizosphere.
Pilot analyses of the RNA content from exosomes extracted from both wheat and A. thaliana revealed the presence of microRNA, various species of non-coding RNA, and partial coding RNA. A bioinformatic search for the identified microRNAs from A. thaliana exosomes was conducted across 283 bacterial strains from its rhizosphere to identify potential bacterial gene candidates targeted by these microRNAs. A corresponding analysis for wheat is currently underway.
Recent studies have indicated that miPEPs (very short peptides) can influence the expression of specific microRNAs. An investigation was conducted using the pathosystem Brassica napus (rapeseed) and its pathogen Plasmodiophora brassicae to examine the impact of miPEP on microRNA expression and the subsequent effects on pathogen infection in plants.
Additionally, interkingdom communication via microRNA was explored in a plant-fungus system, suggesting that microRNA may traverse between plants using fungi as a transport network.
The findings from this project are being compiled in several manuscripts currently in preparation.
