Insights into Bacterial Extracellular Vesicle Biogenesis, Functions, and Implications in Plant-Microbe Interactions.

Plant-microbe interactions play a crucial role in shaping plant health and survival. In recent years, the role of extracellular vesicles (EVs) in mediating intercellular communication between plants and microbes has emerged as an intriguing area of research. EVs serve as important carriers of bioactive molecules and genetic information, facilitating communication between cells and even between different organisms. Pathogenic bacteria leverage extracellular vesicles (EVs) to amplify their virulence, exploiting their cargo rich in toxins and virulence factors. Conversely, beneficial microbes initiate EV secretion to stimulate plant immune responses and nurture symbiotic relationships. The transfer of EV-packed small RNAs (sRNAs) has been demonstrated to facilitate the modulation of immune responses. Furthermore, harnessing the potential of EVs holds promise for the development of innovative diagnostic tools and sustainable crop protection strategies. This review highlights the biogenesis and functions of EVs in bacteria and their importance in plant defense, and paves the way for future research in this exciting field.

Extraction and Purification of Outer Membrane Vesicles and Their Associated RNAs.

Outer membrane vesicles (OMVs), produced by Gram negative-bacteria and sRNAs, are key players in cell-to-cell communication and interactions of bacteria with the environment. OMVs act as information carriers and encapsulate various molecules such as proteins, lipids, metabolites, and RNAs. OMVs and sRNAs play a broad range of functions from pathogenesis to stress resistance, to biofilm formation and both mediate interkingdom signaling. Various studies indicate that there is a mechanism of intercellular communication mediated by OMV-derived bacterial RNAs that is conserved among certain bacterial species. Here we describe methods for the extraction and purification of vesicles produced by Gram-negative bacteria, such as Pseudomonas brassicacearum and Escherichia coli, and address methods for the extraction of OMVs-derived sRNA and techniques for the analysis of sRNAs.

Interactions and Insertion of Escherichia coli Hfq into Outer Membrane Vesicles as Revealed by Infrared and Orientated Circular Dichroism Spectroscopies.

The possible carrier role of Outer Membrane Vesicles (OMVs) for small regulatory noncoding RNAs (sRNAs) has recently been demonstrated. Nevertheless, to perform their function, these sRNAs usually need a protein cofactor called Hfq. In this work we show, by using a combination of infrared and circular dichroism spectroscopies, that Hfq, after interacting with the inner membrane, can be translocated into the periplasm, and then be exported in OMVs, with the possibility to be bound to sRNAs. Moreover, we provide evidence that Hfq interacts with and is inserted into OMV membranes, suggesting a role for this protein in the release of sRNA outside the vesicle. These findings provide clues to the mechanism of host-bacteria interactions which may not be defined solely by protein-protein and protein-outer membrane contacts, but also by the exchange of RNAs, and in particular sRNAs.