Bacterial Extracellular Vesicles and the Plant Immune Response

Abstract

Cells from all levels of life secrete vesicles, which are nanoscale proteoliposomes packaged with a variety of proteins, lipids, and small molecule cargo. Depending on their origin, these extracellular vesicles are termed exosomes, microvesicles, exomeres, and membrane vesicles, to list a few. Vesicles released from Gram-negative bacteria bud from the outer membrane and are, therefore, referred to as outer membrane vesicles (OMVs). In mammalian systems, OMVs facilitate bacterial survival by alleviating membrane stress, serving as a decoy for bacteriophage and antibiotics, and providing a fast membrane remodeling mechanism. OMVs also contribute to virulence by delivering toxins and other soluble and insoluble cargo to the host cell. The role OMVs play in plant systems remains unknown.Previous studies revealed that plant pathogenic bacterial vesicles contain virulence factors, type III secretion system effectors, plant cell wall-degrading enzymes, and more, suggesting that vesicles may play similar roles to those from mammalian pathogens in host-pathogen interactions. Further, OMVs elicit several markers for pathogen-associated molecular pattern triggered immunity in plants. These responses include increased transcription of defense markers such as FRK1 and production of reactive oxygen species. Building on these findings, here we show that OMVs from the plant pathogen Pseudomonas syringae and the plant beneficial Pseudomonas fluorescens elicit plant immune responses in Arabidopsis thaliana that protect against future pathogen challenge. Intriguingly, protection is independent of salicylic acid plant defense pathways and bacterial type III secretion. OMVs also inhibit seedling growth, another indication of plant immune activation. Our initial biochemical studies suggested that the immunogenic OMV cargo was larger than 10 kDa and differed between the pathogen and beneficial species despite similar plant immunity outcomes. Interestingly, protective OMV-mediated responses were protein-independent, while the seedling growth inhibition phenotype was entirely protein dependent. Proteomics analysis confirmed that OMV protein cargo differed between P. syringae and P. fluorescens. While media culture conditions did not dramatically impact the immunogenic activity of isolated OMVs from either species, proteomics analysis revealed a significant shift in P. syringae OMV cargo between complete and minimal media conditions. P. fluorescens OMV cargo was largely the same in the two media conditions, with no significantly enriched proteins in minimal or complete media. Further analysis of the proteins enriched in the P. syringae minimal OMV condition identified one set of proteins with the same baseline abundance in P. syringae and P. fluorescens complete OMVs and another set with a lower baseline abundance compared to P. fluorescens OMVs. These two subsets could contribute to virulence and stress tolerance, respectively. Enrichment analysis uncovered particularly interesting protein categories in the subset with the same baseline abundance. Of interest, several lipoprotein and lipid binding categories were enriched, and proteins involved in synthesis of the phytotoxin coronatine were also enriched in this same-baseline subset. These results support our hypothesis that proteins enriched in P. syringae minimal OMVs with the same baseline abundance in P. fluorescens complete OMVs may contribute to OMV-mediated bacterial virulence in plants. Our findings also suggest that our forthcoming OMV metabolomic analyses may reveal non-proteinaceous cargo that is critical for OMV-mediated plant immune activation.The work presented here lays the groundwork for future exploration of OMV-plant interactions and adds a new layer of complexity to plant-bacteria interactions. Further, these results reveal that OMVs elicit complex plant immune responses that would be difficult for pathogens to adapt to and overcome, supporting a role for bacterial OMVs in agricultural applications to promote durable resistance and revealing a new potential avenue for disease prevention and management.