Transcriptomic insights into the tritrophic plant-pathogen-mycoparasite interaction reveal coordinated reprogramming fungal secretomes and plant amino acid metabolism.

O_LIThe tomato-Cladosporium fulvum (syn. Fulvia fulva) pathosystem has served as a model for the gene-for-gene concept of effectors and resistance proteins, but this binary framework does not include the potential influence of other microbial participants. Here we describe the dramatic changes in gene expression of all members of the tritrophic interaction among tomato, C. fulvum, and mycoparasitic fungus Hansfordia pulvinata. C_LIO_LITranscriptomic analyses of the mycoparasite H. pulvinata during parasitism of C. fulvum on tomato on planta and in vitro revealed a dramatic upregulation of genes encoding small secreted proteins during mycoparasitism, notably, a Nep1-like protein (HpNlp1) lacked typical necrosis-inducing activity but induced the accumulation of antifungal compounds inhibiting spore germination of C. fulvum. C_LIO_LISimilarly, in C. fulvum parasitized by H. pulvinata, effector genes were highly expressed. Strikingly, effector protein Ecp2 was found to share structural similarity with pathogen killer toxin 4 proteins and had broad-spectrum antifungal activity, indicating a dual function in fungal competition and Cf-ECP2-mediated plant resistance. C_LIO_LIIn tomato plants infected by C. fulvum parasitized by H. pulvinata, primary metabolism and defense-related genes were exclusively activated. These results suggest that in the tritrophic interaction, the mycoparasite simultaneously suppressed the pathogen and induced plant resistance. This study uncovers a multilayered molecular network in which the mycoparasite coordinates pathogen suppression and plant defense within the tritrophic interaction. C_LI