Biophysical and proteomic analyses suggest functions ofPseudomonas syringaepvtomatoDC3000 extracellular vesicles in bacterial growth during plant infection

Abstract

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

The extracellular vesicle generation paradox: a bacterial point of view

“…Another recent study supports our findings as well as those from Bahar et al , showing that Pst vesicles lead to protection against bacterial pathogens and induce FRK1 expression in plants (preprint: Janda et al , 2021). Importantly, these results also show that Pst produces vesicles in planta that have similar biophysical properties to those isolated through traditional purification techniques (preprint: Janda et al , 2021).…”

“…Another recent study supports our findings as well as those from Bahar et al , showing that Pst vesicles lead to protection against bacterial pathogens and induce FRK1 expression in plants (preprint: Janda et al , 2021). Importantly, these results also show that Pst produces vesicles in planta that have similar biophysical properties to those isolated through traditional purification techniques (preprint: Janda et al , 2021). While current vesicle purification techniques limit the ability to test the function of vesicles produced in planta , these results provide critical evidence that vesicles are a physiologically relevant player in plant–microbe interactions.…”

“…Although a full biogenesis mechanism has yet to be revealed, OIMVs may form as a result of autolysin activity that transiently breaks down peptidoglycan to allow OIMV release (Kadurugamuwa & Beveridge, 1995; Pérez‐Cruz et al , 2013, 2015; Clarke, 2018). Naturally produced OIMVs have been observed in many different species including Shewanella vesiculosa , Neisseria gonorrhoeae , P. aeruginosa , Acinetobacter baumannii, V. shilonii , Pseudoalteromonas marina , Arhensia kielensis , and P. syringae , among others (Hagemann et al , 2014; Pérez‐Cruz et al , 2015; Li et al , 2016; preprint: Janda et al , 2021). In P. aeruginosa , vesicle production can also result from explosive cell lysis triggered by endolysins, yielding so‐called explosive OMVs (EOMVs) (Fig 3F) (Turnbull et al , 2016; Toyofuku et al , 2019).…”

“…T3SS effectors are well‐studied elicitors of potent plant immune responses that can result in local and systemic protection against invading pathogens (Vlot et al , 2009, 2021; Spoel & Dong, 2012; Conrath et al , 2015). T3SS effectors have also been found in vesicles isolated from plant bacteria, including Pst (Sidhu et al , 2008; Chowdhury & Jagannadham, 2013; preprint: Janda et al , 2021). Surprisingly, pre‐treatment with vesicles from these Pst mutants resulted in the same level of protection as vesicles from wild‐type Pst , revealing that vesicle‐mediated plant protection is T3SS‐independent (McMillan et al , 2021b).…”

“…Lin et al (2017) showed that P. aeruginosa secretes a T6SS effector, TseF, that binds PQS and helps deliver iron‐containing vesicles to the cell via FptA and OprF (Fig 2C). Vesicles from several other species including Francisella novicida , Bacillus subtilis , S. typhimurium , and P. syringae have also been implicated in nutrient acquisition (Dubey & Ben‐Yehuda, 2011; Galkina et al , 2011; McCaig et al , 2013; Sampath et al , 2018; preprint: Janda et al , 2021). Interestingly, some of these vesicles remain attached to the parent cell in a membrane tubule, perhaps to enable delivery of nutrients more efficiently to the cell (Dubey & Ben‐Yehuda, 2011; Galkina et al , 2011; McCaig et al , 2013; Sampath et al , 2018).…”