Characterisation of the Extracellular Fraction in some economically relevant fungi

Botrytis cinerea, also known as “grey mould “, is a necrotrophic fungus part of the large fungal Ascomycota family. Over the years, B. cinerea has raised several concerns because of its wide host range and major impact on crops such as tomato, strawberry and grapevine. Despite much research, current management of B. cinerea is mostly based on chemical fungicides, raising environmental and health concerns. To explore innovative control strategies, this PhD project focused on the characterisation of the Extracellular Fraction (EF) of B. cinerea ungerminated macroconidia, because it represents the very first interface between the pathogen and its host at the beginning of the spring life cycle of the pathogen. The EF was defined as the entire repertoire of molecules, metabolites, extracellular vesicles (EVs) and proteins present outside the macroconidia, either associated with the cell wall surface (membrane-associated factors) or secreted via conventional and unconventional pathways, through active or passive mechanisms. A proteomic workflow was optimised to extract the EF and characterise the proteome profile (exoproteome and surfaceome) from ungerminated macroconidia of two B. cinerea strains with two different pathogenic backgrounds (B05.10, more aggressive, and T4, less aggressive). Experimental conditions were adjusted to preserve cell viability to avoid contamination from intracellular proteins released by lysis or cell death. Proteins were identified after trypsin digestion using nanoUPLC–MS/MS under conditions that do not allow EV rupture as reported in literature and bioinformatic analysis, such as functional classification, protein-protein interaction networks, effector classification, secretion and membrane-association prediction. Complementary TEM and nanoparticle tracking analysis confirmed that both strains release extracellular vesicles within one hour of incubation, making this the first report of EV production directly from macroconidia. Proteomic analysis revealed a common core of 80 proteins between the two strains. These proteins were clustered in different functional groups associated with protein folding, stress response, primary metabolism and intracellular trafficking. The presence of proteins involved in redox homeostasis, protein quality control and RNA-related processes highlights that macroconidia in an unstimulating environment, as a neutral buffer, appear to be metabolically active, releasing factors that may act as early virulence effectors. This core of proteins shared between the two strains highlighted an interesting functional conservation of these factors in the early steps of infection between B05.10 and T4. To investigate the functional role of the EF, detached leaf assays were performed in tomato (Solanum lycopersicum) and grapevine (Vitis vinifera). In tomato a pilot assay three concentrations of EF (25, 50 and 100 μg/mL) were tested. Results revealed a modulation of host defence genes such as PTI5, WRKY1 and PR1, indicating a plant immune-stimulating effect. Afterwards, in grapevine, pretreatment with EF prior to fungal inoculation resulted in a reduced development of necrotic lesions. This effect was supported by stereomicroscope observations performed at various time points (0, 6, 24, 30 and 54 hours post-infection) following 48 hours of immune stimulation. Gene expression analysis confirmed the induction of defence gene markers such as VvMYB14, VvSTS1 and VvJAR1, plus highlighted differences between infection with B05.10 and T4. Altogether, these findings support the role of the B. cinerea macroconidia exoproteome as an immune elicitor in plants and a probable virulence determinant. In conclusion, this work provides a first characterisation of the B. cinerea macroconidial exoproteome and surfaceome fraction from the EF, including likely EV-associated proteins with a bottom-up proteomic approach. The planta in-vivo assays results, instead, confirm that the entire EF acts as a priming stimulus that can enhance plant defence responses; parallel studies suggest that EF could probably prepare the macroconidial environment independently of its host to favour the subsequent infection. Considering the advances in understanding the early host-pathogen interaction mechanism, this research points towards potential applications of B. cinerea EF as a base for new eco-friendly crop protection strategy development. This work contributes to the search for sustainable alternatives to chemical fungicides in the fight against B. cinerea and other necrotrophic pathogens.