Pinus-derived membrane vesicles disrupt pathogenic metabolism in fungi

Much of what we know about the biological impacts of vesicles (MVs) is derived from Arabidopsis thaliana. Our study focused on vesicles from species in the non-model plant group, Pinus (pine) (P. elliottii, P. radiata, and P. patula × Pinus tec (hybrid)). These plants have tougher tissues and strong, acicular-shaped leaves (needles). Herein, we first developed a protocol to guide effective collection of juice fluid from needles and roots in a clean and efficient manner. The effects of these vesicles were characterized in terms of the global nutrient profile of the pine pitch canker fungus, Fusarium circinatum, generated from growing fungal spores on ~400 substrates embedded across BioLog phenotypic microarray (PM) plates (PM1, PM2A: carbon sources; PM3B: nitrogen sources; PM9: osmolytes/pH; PM24C: chemicals). Our findings revealed that MVs, specifically needle-derived MVs (ndMVs) from P. elliottii, disrupt metabolite assimilation in several important pathways, including carbon and nitrogen metabolism. The PM data were also strongly correlated with observed phenotypic effects, including reduced viability and germination of spores in liquid media, as well as impaired filamentous growth on solid media. Importantly, these MV-induced phenotypic effects were reproducible in other filamentous pathogens (e.g., Botrytis cinerea, Chrysoporthe cubensis and F. graminearum) and during a glasshouse trial conducted with F. circinatum-infected P. elliottii seedlings, demonstrating the stable biological effects of ndMVs. Cumulatively, our results suggest that plant-derived vesicles can disrupt metabolism in pathogenic fungi and, therefore, serve as a cost-effective and sustainable source of novel plant protection molecules.