Dataset Information

First description of Botrytis cinerea Extracellular vesicles proteome: Unrevealing new tools for its infection process

ABSTRACT: Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several kinds of macromolecules implicated in cell communication. EVs have become an important topic in the study of pathogenic fungi, due to their relation with the fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EV cargo, because of plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, little is known about EVs of Botrytis cinerea, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the first isolation and characterization of B. cinerea EVs; and the description of their potential role during the infective process. All the experimental process was conducted in B. cinerea growing in mimimal salt medium supplemented with glucose as a constitutive stage, and deproteinized tomato cell walls (TCW) as virulence inductor. The isolation of EV was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EV fractions were visualised by TEM using negative staining. Proteomic analysis of EV cargo was addressed by LC-MS/MS. The used methodology had allowed, for the first time, the correct isolation of B. cinerea EVs, and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in the morphology under both assayed conditions. GO analysis of EV proteins showed an enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed difference in EV function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, between others. This is the first evidence and characterization of B. cinerea EVs production, showing that EVs are essential in the infection process of B. cinerea and share crucial functions with the conventional secretion pathway.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Botryotinia Fuckeliana (noble Rot Fungus) (botrytis Cinerea)

TISSUE(S): Plant Cell

SUBMITTER: Sara Christina Stolze

LAB HEAD: Hirofumi Nakagami

PROVIDER: PXD040614 | Pride | 2023-10-24

REPOSITORIES: Pride

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Publications

The Adaptation of <i>Botrytis cinerea</i> Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Escobar-Niño Almudena A Harzen Anne A Stolze Sara C SC Nakagami Hirofumi H Fernández-Acero Francisco J FJ

Journal of fungi (Basel, Switzerland) 20230824 9

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of <i>Botrytis cine ...[more]

PMID: 37754980

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Project description:Introduction: Cell-derived extracellular vesicles ([EVs], i.e., exosomes and microparticles) have been reported to mediate the cardioprotective effects of stem cells. However, a head-to-head comparison of their effects with those of the cells from which they derive has not been reported. Hypothesis: The effects of the EV content of human embryonic stem cell (hESC)-derived cardiac progenitors may be equivalent to those of the parent cells. Methods: Two series of immunodeficient (nude) mice underwent permanent coronary artery ligation. Three weeks later, those with an echocardiographically-determined LV ejection fraction â¤ 50% were randomly allocated to receive transcutaneous echo-guided peri-infarct injections of alpha-MEM media (controls), hESC-derived SSEA-1+ cardiac progenitors (500000 cells in 30 Î¼L), or total EV secreted by those 500000 cells in 48 hours in an equivalent volume. The second series also included sham-operated animals for which the needle was inserted into the myocardium in three places without injection. EVs were collected from the cell-secreted medium by ultracentrifugation and characterized by flow cytometry, and nanosight NTS. Outcomes were assessed after 6 weeks by echocardiography, taking LV end-systolic volume (ESV) as the primary end point. Hearts were processed for the assessment of fibrosis and angiogenesis. Total RNA was extracted from carefully selected animals for gene expression analysis by Affymetrix chip. Only animals with similar VTS at baseline were used for this analysis. All data were collected and analyzed blindly. Results: Cardiac progenitors were successfully generated by exposure of the pluripotent ESC to bone morphogenetic protein-2, purified by anti-CD15 immunomagnetic sorting and then cultured on vitronectin for 48 hours, after which cells or EVs derived from the same cell batch were injected. After 6 weeks in the first series, LVESV [mÂ±SEM] in controls did not significantly differ from the pre-injection value (difference: -2.64 Â± 1.54 ÂµL, p=0.12, n= 12). Conversely, in the cell-treated group, LVESV significantly decreased by -4.20 Â± 0.96 ÂµL (p=0.0007, n=16). A similar decrease was seen in the EV-treated group: -5.73 Â± 1.21 ÂµL (p=0.0003, n=15). Similar patterns were seen for LV end-diastolic volumes: controls (-2.46 Â± 1.19 ÂµL, p=NS), cells (-4.48 Â± 1.47 ÂµL, p=0.009), EV (-4.29 Â± 1.31 ÂµL, p=0.005). For the second series, the VTD of cell- and EV-treated animals remained stable (cell: -1.85 Â± 3.27 ÂµL, p=NS, n= 4; EV: +0.05 Â± 1.53 ÂµL, p=NS, n = 4), whereas sham- and control-treated animals deteriorated significantly (sham: +2.20 Â± 0.35 ÂµL, p = 0.003, n = 5; control: +3.27 Â± 0.87 ÂµL, p = 0.03, n = 4). The trend held for VTS, though differences were not significant. Analysis of gene expression data revealed interesting pathways that were more highly expressed in cell- and EV-treated animals than in controls and sham-operated animals. Conclusions: These data support the hypothesis that EVs may be critical mediators of the paracrine effects of cell therapy, and for the sake of streamlining translational processes, deserve to be considered as potential alternatives to stem cell transplantation. Gene expression levels were compared between mouse hearts.

Dataset Information

First description of Botrytis cinerea Extracellular vesicles proteome: Unrevealing new tools for its infection process

Publications

The Adaptation of <i>Botrytis cinerea</i> Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Similar Datasets

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