While human extracellular vesicles (EVs) have attracted a big deal of interest and have been extensively characterized over the last years, plant-derived EVs and nanovesicles have earned less attention and have remained poorly investigated. Although a series of investigations already revealed promising beneficial health effects and drug delivery properties, adequate (pre)clinical studies are rare. This fact might be caused by a lack of sources with appropriate qualities. Our study introduces plant cell suspension culture as a new and well controllable source for plant EVs. Plant cells, cultured in vitro, release EVs into the growth medium which could be harvested for pharmaceutical applications. In this investigation we characterized EVs and nanovesicles from distinct sources. Our findings regarding secondary metabolites indicate that these might not be packaged into EVs in an active manner but enriched in the membrane when lipophilic enough, since apparently lipophilic compounds were associated with nanovesicles while more hydrophilic structures were not consistently found. In addition, protein identification revealed a possible explanation for the mechanism of EV cell wall passage in plants, since cell wall hydrolases like 1,3-β-glucosidases, pectinesterases, polygalacturonases, β-galactosidases and β-xylosidase/α-L-arabinofuranosidase 2-like are present in plant EVs and nanovesicles which might facilitate cell wall transition. Further on, the identified proteins indicate that plant cells secrete EVs using similar mechanisms as animal cells to release exosomes and microvesicles.

Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H₂O₂ concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H₂O₂ results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H₂O₂ at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H₂O₂ induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypic resistance against H₂O₂ alters the evolution of resistance against oxygen stress. Experimental evolution of H₂O₂ resistance revealed faster evolution and higher levels of resistance in primed cells. Several mutations were found to be associated with resistance in evolved populations affecting different loci but, counterintuitively, none of them was directly associated with scavenging systems. Our results have important implications for host colonisation and infections where microbes often encounter reactive oxygen species in gradients. Throughout evolution, bacteria were exposed to reactive oxygen species and evolved the ability to scavenge toxic oxygen radicals. Furthermore, multicellular organisms evolved the ability to produce such oxygen species directed against pathogens. Recent studies also suggest that ROS such as H₂O₂ play an important role during host gut colonisation by its microbiota. Traditionally, experiments with different antimicrobials have been carried out using fixed concentrations while in nature, including in intra-host environments, microbes are more likely to experience this type of stress in steps or gradients. Here we show that bacteria treated with sub-lethal concentrations of H₂O₂ (priming) survive far better than non-treated cells when they subsequently encounter a higher concentration. We also found that the 'priming' response has a protective role from lethal mutagenesis. This protection is provided by long-lived proteins that, upon priming, remain at a high level for several generations as determined by time-lapse LC-mass spectrometry. Bacteria that were primed evolved H₂O₂ resistance faster and to a higher level. Moreover, mutations that increase resistance to H₂O₂, as determined by whole-genome sequencing (WGS), do not occur in known scavenger encoding genes but in loci coding for other functions, at least in E. coli.

Insects show long-lasting antimicrobial immune responses that follow the initial fast-acting cellular processes. These immune responses are discussed to provide a form of phrophylaxis and/or to serve as a safety measure against persisting infections. The duration and components of such long-lasting responses have rarely been studied in detail, a necessary prerequisite to understand their adaptive value. Here, we present a 21 day proteomic time course of the mealworm beetle Tenebrio molitor immune-challenged with heat-killed Staphylococcus aureus . The most upregulated peptides are antimicrobial peptides (AMPs), many of which are still highly abundant 21 days after infection. The identified AMPs included toll and imd-mediated AMPs, a significant number of which have no known function against S. aureus or other Gram-positive bacteria. The proteome reflects the selective arena for bacterial infections. The results also corroborate the notion of synergistic interactions in vivo that are difficult to model in vitro . This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.

BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex.

Known elicitors of plant defenses against eggs of herbivorous insects are low‐molecular‐weight organic compounds associated with the eggs. However, previous studies provided evidence that also proteinaceous compounds present in secretion associated with eggs of the herbivorous sawfly Diprion pini can elicit defensive responses in Pinus sylvestris. Pine responses induced by the proteinaceous secretion are known to result in enhanced emission of (E)‐β‐farnesene, which attracts egg parasitoids killing the eggs. Here, we aimed to identify the defense‐eliciting protein and elucidate its function. After isolating the defense‐eliciting protein from D. pini egg‐associated secretion by ultrafiltration and gel electrophoresis, we identified it by MALDI‐TOF mass spectrometry as an annexin‐like protein, which we named ‘diprionin’. Further GC‐MS analyses showed that pine needles treated with heterologously expressed diprionin released enhanced quantities of (E)‐β‐farnesene. Our bioassays confirmed attractiveness of diprionin‐treated pine to egg parasitoids. Expression of several pine candidate genes involved in terpene biosynthesis and regulation of ROS homeostasis was similarly affected by diprionin and natural sawfly egg deposition. However, the two treatments had different effects on expression of pathogenesis‐related genes (PR1, PR5). Diprionin is the first egg‐associated proteinaceous elicitor of indirect plant defense against insect eggs described so far.

Feae’s viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.