A gnotobiotic system reveals multifunctional effector roles in plant-fungal pathogen dynamics

Plants host diverse microbiota that influence physiological processes and can enhance resilience against invading pathogens that, in turn, evolved effector proteins to manipulate host microbiota in their favor. However, the complexity of microbial communities and their interactions complicates mechanistic research on processes governing microbiota assembly and function. Gnotobiotic systems are valuable tools to study plant microbiota by reducing complexity and enabling controlled microbiota reconstitution experiments. Despite their utility, no gnotobiotic systems have been established to investigate the role of antimicrobial effector proteins in the interactions between plants, their microbiota, and fungal pathogens. Here, we present a refined gnotobiotic system designed to study these interactions, establishing protocols for infections with the fungal pathogen Verticillium dahliae across multiple host plants under sterile conditions. We demonstrate that a synthetic microbial community (SynCom) derived from a culture collection generated for this study can be applied in this system where it interferes with fungal infections. Additionally, using our gnotobiotic system we reveal that specific antimicrobial effectors of V. dahliae, like Ave1L2, contribute to fungal virulence in a microbiota-dependent manner, whereas other antimicrobial effectors, such as Ave1, seem to possess functions beyond microbiota manipulation.