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Background Pseudozyma flocculosa is a highly efficient biocontrol agent (BCA) of powdery mildews whose mode of action remains elusive. It is known to secrete unique effectors during its interaction with powdery mildews but effectors have never been shown to be part of the arsenal of a BCA. Here, we characterize the role of the effector Pf2826 released by Pseudozyma flocculosa during its tripartite interaction with barley and the pathogen fungus Blumeria graminis f. sp. hordei. Results We utilized CRISPR-Cas9-based genome editing and confirmed that secreted P. flocculosa effector Pf2826 is required for full biocontrol activity. We monitored the localization of the effector Pf2826 with C-terminal mCherry tag and found it localized around the haustoria and on powdery mildew spores. His-tagged Pf2826 recombinant protein was expressed, purified, and used as bait in a pull-down assay from total proteins extracted during the tripartite interaction. Potential interactors were identified by LC–MS/MS analysis after removing unspecific interactions found in the negative controls. A two-way yeast two-hybrid assay validated that Pf2826 interacted with barley pathogenesis-related (PR) proteins HvPR1a and chitinase and with an effector protein from powdery mildew. Conclusions In contrast to the usual modes of action of competition, parasitism, and antibiosis ascribed to BCAs, this study shows that effector pf2826 plays a vital role in the biocontrol activity of P. flocculosa by interacting with plant PR proteins and a powdery mildew effector, altering the host–pathogen interaction.

Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous co-cultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.