Isolation and Characterization of Extracellular Vesicles From <i>Ascochyta rabiei</i> , a Necrotrophic Fungal Chickpea Pathogen

Extracellular vesicles produced by pathogenic organisms play a role in host recognition and invasion across kingdoms, including fungi interacting with their plant hosts. Little is known about EVs from phytopathogenic fungi compared to human fungal pathogens. Ascochyta rabiei is an endemic and widespread necrotrophic ascomycete fungus that causes a significant impact on chickpea production. The presence of EVs generated by A. rabiei during the invasion of chickpea has not previously been reported. Here, A. rabiei EVs were identified and isolated from liquid fungal broth cultures, both with and without host, using optimized ultracentrifugation and filtration techniques. Nanoparticle tracking analysis, transmission electron microscopy, and mass spectrometry revealed 40-200 nm cup-shaped, double-membraned structures. Protein cargo analysis showed significant variations depending on host presence, affecting EV production, size, and composition. The cargo included proteins involved in vesicle transport, basic cellular and oxidation/reduction-related processes, as well as pathogenesis. Some were upregulated in the presence of the host, likely facilitating immune modulation, plant defence alteration, or pathogen establishment. These findings highlight the presence and functional importance of A. rabiei EVs in the interaction with and invasion of chickpea. Future research should concentrate on characterizing EVs under various growth circumstances and confirming fungal-specific EV markers. SUMMARY: This study presents the first characterization of extracellular vesicles (EVs) produced by Ascochyta rabiei, a major phytopathogenic fungus affecting chickpea. An optimized ultracentrifugation and filtration-based isolation method enabled efficient EV recovery without reliance on specialized equipment. Comprehensive proteomic analysis revealed that EV production, size distribution, and protein composition were modulated by the presence of the host. Identified EV-associated proteins were linked to virulence, oxidative stress response, cell wall modification, and effector functions, suggesting a role in host-pathogen interactions. These findings advance the current understanding of fungal EVs in plant infection processes and establish a foundation for future studies investigating their functional significance in pathogenesis.