The role of Pectobacterium brasiliense 1692 outer membrane vesicles on members of soft rot Enterobacteriaceae and Phytophthora parasitica

Abstract

Outer membrane vesicles (OMVs) are spherical nanoparticles formed from bulging and the subsequent fission of the outer membrane of Gram-negative bacteria. OMVs play a role in the secretion and transport of molecules in bacteria. They can transfer molecules from the inside to the outside of the cell, and as a result, they are regarded as a type zero secretion system. Furthermore, OMVs enclose multiple molecules such as enzymes (e.g., hydrolases), nucleic acids (e.g., DNAs and RNAs), and virulence factors. As a result, bacteria utilise OMVs in various microbial interactions, including competition. Potato (Solanum tuberosum) is one of the stable crop plants worldwide and has been under the threat of microbial infections. Pectobacterium brasiliense 1692 (Pbr1692), a soft rot bacterium, has resulted in a massive loss in the potato production industry and affected food security. Previously, it was reported that Pbr1692 cells could outcompete potato co-infectors using various mechanisms, namely, the secretion of antibiotic carbapenem, bacteriocins, and the type six secretion system. In this study, we investigated the role of Pbr1692 OMVs in microbial interactions, especially in competition. To achieve this, OMVs were isolated from Pbr1692 and confirmed through transmission electron microscopy and SDS-page. As anticipated, Pbr1692 OMVs exhibited antimicrobial activity against Dickeya dadantii. However, our data indicated that OMVs do not play a role in competition against Pectobacterium atrosepticum, P. carotovorum and P. parmentieri. Multiple studies have shown that bacterial OMVs participate in both intra and inter-kingdom interactions. Therefore, we investigated the role of Pbr1692 OMVs on plant pathogens from other kingdoms, specifically Phytophthora parasitica. Phytophthora parasitica is an oomycetes plant pathogen notorious for root and stem rot in economically important crops such as tobacco, potato, tomato, and peppers, as well as citrus plants. Since both P. parasitica and Pbr1692 are potato pathogens, we hypothesised that there is a likelihood that Pbr1692 could interact with Phytophthora parasitica. Therefore, we investigated the type of interaction that might occur between these two pathogens. From our analysis, it was evident that Pbr1692 cells do not inhibit the growth of P. parasitica INRA 310. Since OMVs reflect the biology of their parental cells, we were motivated to investigate whether Pbr1692 OMVs do not exhibit inhibition on P. parasitica. Despite the fact that P. parasitica INRA 310 zoospores were able to internalise Pbr1692 OMVs, they germinated in the same way as the untreated zoospores, implying that Pbr1692 OMVs do not inhibit P. parasitica INRA 310. Taken together, these viii results suggested that there might be an interaction between the two pathogens through the OMVs, but not competition. To shed some light on this, we conducted a phenotypic microarray analysis where Pbr1692 OMVs were shown to potentially assist P. parasitica INRA 310 zoospores in utilising some of the carbon sources tested. These preliminary results showed that zoospores co-inoculated with Pbr1692 OMVs showed improved growth in some chemicals, and these results were subsequently validated. Therefore, we could conclude that Pbr1692 OMVs participate in intra and inter-kingdom interactions. This study assists in understanding OMVs as a mechanism of interaction between plant pathogens. Also, it paves a way to target this mechanism to combat plant diseases caused by Pbr1692 or to look into Pbr1692 OMVs as antimicrobial agents in microbial communities.