Project description:Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impacts EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and non-coding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally-relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and glial communities, and provide novel insights into the role of microglia-derived EVs in neuroinflammation.

Project description:It is of high importance to distinguish Tilletia caries and Tilletia laevis as causal agents of common bunt accurately from Tilletia controversa, the causal agent of dwarf bunt. All three of these wheat bunt diseases can lead to significant yield losses in crop production worldwide. But T. controversa is categorized as a quarantine pest in most areas of the world and must be discriminated from the T. caries / T. laevis complex. Usually, morphological characteristics of the teliospores are used to differentiate the three species. But due to natural hybridization and overlapping properties the discrimination is challenging. Germination behavior can also be considered for discrimination, but equivalent to their similar physiological and genetic traits the two agents of common bunt, T. caries and T. laevis could not be distinguished by this. It was suggested that the two species and maybe all three of those described Tilletia species might be conspecific. Up to now no molecular based method is available to differentiate the three species. Several studies have attempted the detection of the wheat bunt Tilletia species using PCR or other DNA-based methods. Other studies analyzed protein patterns with electrophoresis methods. But none of these approaches was able to distinguish between all of the three closely related Tilletia species. Several studies have shown that Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a useful tool to differentiate closely related fungal species. The aim of this study was to assess whether MALDI-TOF MS analysis is able to distinguish specimens of the three closely related pathogens T. caries, T. laevis, and T. controversa and may constitute an alternative method to the usually used morphology-based identification. Therefore MALDI-TOF MS was used to create subproteome fingerprints of the teliospores of 69 Tilletia specimens. These fingerprints were analyzed by comparing the mass spectra to each other by high-throughput multidimensional scaling (HiT-MDS ) together with hierarchical cluster analysis (HCA). The second approach was performed by discriminant analysis of principal components (DAPC). MALDI-TOF MS has proven to be a useful method for distinguishing between T. controversa and the two causal agents of common bunt, using our developed method of direct analysis of teliospores, but was unable to separate T. caries and T. laevis species. We conclude a potentially conspecific status of T. caries and T. laevis or even two morphotypes of one common species, causing identical disease symptoms and sharing the same germination requirements along with a related protein composition, shown in this study. Our developed MALDI-TOF MS method can be helpful in testing Tilletia bunt balls collected during field inspections, especially with regard to quarantine regulations or for breeding applications and may also be transferred to analyze further challenging sample material.

Dataset Information

Isolation and characterization of extracellular vesicles from Arabidopsis thaliana cell culture and investigation of the specificities of their biogenesis

Publications

Isolation and Characterization of Extracellular Vesicles from <i>Arabidopsis thaliana</i> Cell Culture and Investigation of the Specificities of Their Biogenesis.

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