Extracellular Vesicles of the Plant Pathogen Botrytis cinerea

Abstract

Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.

Keywords: EV; cell–cell communication; electron/confocal microscopy; fungus; proteomics; secretion; tomography.

Figure 1

Hyphae of B. cinerea produce EVs in vitro and in planta. (A–C) TEM of B. cinerea hyphae grown in vitro. Single (black arrow) and groups (black star) of extracellular vesicles (BcEVs) are visible between the plasma membrane (PM) and the fungal cell wall (FCW). Multi-vesicular bodies are indicated (MVB). Images are representative of hyphae collected from either solid or liquid cultures. (D) Electron tomography of a MVB adjacent to the plasma membrane (hypothesized pre-fusion stage) and containing ovoid (blue) and tubular (green) BcEVs. (E) TEM images of B. cinerea hyphae (Hy) located on the external aerial side of bean epidermis cells (Ext) and inside plant cell cytoplasm (PC). The plant cell wall is indicated (PCW, made with parallel lines), covered by the cuticle in the outer-periclinal sides of the two epidermis cells. At higher magnification, single (black arrow) and groups (black star) of BcEVs are visible between the fungal PM and the FCW (more translucent material) as well as MVB-like structures (MVB-L). Scale bar: 200 nm unless indicated.

Figure 2

Isolation of EVs from solid cultures of B. cinerea. (A) EVs were isolated using differential centrifugation and further separation by density (iodixanol) gradient ultracentrifugation. The fractions (F1 to F6) were analyzed through measurements of their densities (9 biological replicates), protein concentrations (28 biological replicates, F5: 19.6 $\pm$ 4.7 µg/4.6 $\pm$ 0.1 g dry mycelium) and labelling with fluorescent CFSE (6 biological replicates). Protein and fluorescence quantifications were performed after washing of the iodixanol and collection of the biological material by centrifugation. Results are expressed as mean values $\pm$ SEM. (B) TEM of fraction F5, showing vesicles of different sizes and densities. (C) Dynamics light scattering of fraction F5, showing particles mostly (>95%) in the range of 90 to 500 nm in size (8 biological replicates).

Figure 3

Diversity of the 271 membrane and GPI-anchored proteins identified in BcEVs. (A) Top 10 enriched biological processes (dark blue, p-value < ${10}^{-8}$; blue, p-value < ${10}^{-7}$; white, ${10}^{-6}$ < p-value < ${10}^{-3}$). (B) Predicted sub-cellular compartments. (C) Predicted enzymes. (D) Classification of the 206 proteins with a predicted function into 8 categories and 24 sub-categories. The proteins with unknown functions are not represented. The number of proteins inside each pie chart is indicated in white or black.

Figure 4

Diversity of the 402 soluble proteins identified in BcEVs. (A) Top 10 enriched biological processes (dark blue, p-value <${10}^{-8}$; blue, p-value <${10}^{-7}$; light blue, p-value <${10}^{-6}$). (B) Predicted sub-cellular compartments. (C) Predicted enzymes. (D) Classification of 351 proteins with a predicted function into 12 categories and 27 sub-categories. The proteins with unknown functions are not represented. The number of proteins inside each pie chart is indicated in white or black.

Figure 5

BcEVs stain onion cells. BcEVs labelled with CFSE were deposited onto onion epidermal cells, incubated for 3 h and washed away before imaging by confocal microscopy. (A) Low magnification of the CFSE labelling of the epidermal cells (left) and higher magnification showing the labelling of the plasma membrane (center). (B) Subcortical nuclear zones of adjacent epidermal cells showing intracellular CFSE labelling. The absence of fluorescence was verified in the epidermal cells exposed to the PBS control sample (see Section 2.3). Scale bar: 20 µm.

Figure 6

BcEVs stain cells of B. cinerea and F. graminearum, but not that of the yeast S. cerevisiae. (A) Confocal microscopy of germinated B. cinerea conidia (B. c) incubated for 1 h and 4 h with CFSE-BcEVs. The absence of green fluorescence (CFSE) was verified in hyphae exposed to the PBS control sample (control). (B) Yeast cells (S. cerevisiae) collected in exponential phase and incubated for 1 h with CFSE-BcEVs. Co-staining with aniline blue (glucan marker) is shown. (C) Germinated conidia of F. graminearum (F. g) incubated for 1h with CFSE-BcEVs. Fluorescent BcEVs aggregates are visible in B and C (strong green fluorescent signal outside the fungal cells). Scale bar: 5 µm.

Figure 7

BcEVs can improve the growth of B. cinerea. (A) Growth curves of B. cinerea in liquid medium supplemented with CFSE-BcEVs or with PBS control sample. (B) Growth curves of F. graminearum grown under the same conditions used for B. cinerea in (A). Six biological replicates were performed. Data are expressed as mean values $\pm$ SEM (Student’s t-test p-value * <0.05; ** <0.01; *** <0.001).

Figure 8

Schematic representation of BcEVs. The diverse functions highlighted by the BcEVs proteomic analysis are indicated, with orange ovals positioned where membrane proteins have been identified. EV marker proteins are indicated in pink.