Outer membrane vesicles (OMVs) secreted by Paraburkholderia phytofirmans PsJN: characterization of associated RNA content and interaction with plant cells

Abstract

Plant growth-promoting bacteria (PGPB) support plant development, increase productivity, and improve resilience to stress. These benefits arise from complex interactions between species, involving the exchange of various signaling and effector molecules. Extracellular vesicles (EVs) are an important means of signaling between cells, organisms, and even across biological kingdoms. Among EVs, outer membrane vesicles (OMVs)—produced by Gram-negative bacteria—can elicit both immune-activating and immune-suppressing responses in plants. However, direct interaction between OMVs and plant cell membranes has been shown for only a single phytopathogenic bacterial strain. Even less is known about the role of OMVs in symbiotic relationships between plants and beneficial bacteria, with existing data mostly limited to rhizobia-legume systems. To explore OMV-mediated interactions between non-rhizobial plant-beneficial bacteria and plant cells, we isolated and characterized OMVs from Paraburkholderia phytofirmans PsJN—a wellknown plant growth-promoting and stress-mitigating strain effective across various plant species. Using OMVs labeled with the fluorescent lipid-binding dye Vybrant™ DiD and visualization via confocal laser scanning microscopy, we demonstrated that PsJN-OMVs directly interact with the root hairs and epidermal cells of Arabidopsis thaliana and tomato. Using the membrane fusion probe octadecyl-rhodamine B chloride (R18), we confirmed that PsJN-OMVs can fuse with plant cell membranes. We examined the RNA content of PsJN-OMVs. The OMV-associated RNA, which was mostly sized up to 200 nt, was sequenced using the DNBSEQ Sequencing Platform. Prior to library synthesis, samples were separated into two subsamples: one up to 40 nt and the other from 40 to 200 nt. These were then sequenced using the SE50 and PE100 approaches, respectively. The same procedure was used for small RNA isolated from PsJN cells. Comparison of RNA sequences from PsJN OMVs and cells showed substantial differences in their size distribution, biotypes, and genome regions of origin. The results indicate that the packaging of RNA into PsJN-OMVs is not random, but involves the selective enrichment of certain RNA species in OMVs, while others are predominantly retained within the cells. Previous studies suggested that EVs may both remove unnecessary cellular RNAs and transfer RNAs to other cells, as signaling functions. The molecular mechanisms for selective loading of eukaryotic EVs are only partially described, while those responsible for bacterial vesicles are poorly understood. The data on RNA associated with OMVs secreted by plant beneficial bacteria are even more scarce. Our results make a significant step toward understanding the role of OMVs in the initial phases of root colonization and symbiosis establishment. Advancing knowledge in this field may lead to the development of innovative, environmentally friendly strategies to address agricultural challenges and promote sustainable crop production.Book of abstract: Arturo Falaschi Conference “Cell-cell communication in bacteria: new insights and future trends”, 28-30 October 2025 | Trieste, ITAL