The evolutionary and functional paradox of cerato-platanins in the mycoparasitic fungus Trichoderma: high diversity, stabilizing selection, and a minor role in biotic interactions.

Cerato-platanins (CPs) form a family of fungal small-secreted cysteine-rich proteins (SSCPs) and are of particular interest not only because of their surface activity but also their abundant secretion by fungi. We performed an evolutionary analysis for 283 CPs from 157 fungal genomes with the focus on the opportunistic plant-beneficial and mycoparasitic fungus Trichoderma. Our results revealed the long evolutionary history of CPs in Dikarya fungi that have undergone several events of lateral gene transfer and gene duplication. Three genes were maintained in the core genome of Trichoderma, while some species have up to four CP-encoding genes. All Trichoderma CPs evolve under stabilizing natural selection pressure. The functional analysis of CPs in T. guizhouense and T. harzianum revealed that only EPL1 is active at all stages of the development but plays a minor role in interactions with other fungi and bacteria. The deletion of this gene results in increased colonization of tomato roots by Trichoderma spp. Similarly, the biochemical tests of the heterologously produced EPL1 by Pichia pastoris support the above claims. Based on the obtained results, we conclude that the function of CPs is probably linked to their surfactant properties and the ability to modify the hyphosphere of submerged mycelium and thus facilitate the nutritional versatility of fungi. The effector-like properties do not sufficiently describe the diversity and evolution of these proteins in fungi as they are also maintained, duplicated, or laterally transferred in the genomes of non-herbivore fungi. Importance Cerato-platanins (CPs) are the surface-active small proteins abundantly secreted by filamentous fungi. Consequently, immune systems of plants and other organisms recognize CPs and activate defence mechanisms. Some CPs are toxic to plants and act as virulence factors in plant-pathogenic fungi. Our analysis, however, demonstrates that the interactions with plants do not explain the origin and evolution of CPs in fungal Kingdom. We revealed the long evolutionary history of CPs with multiple cases of gene duplication and events of inter-fungal lateral gene transfers. In the mycoparasitic Trichoderma spp., CPs evolve under stabilizing natural selection and hamper the colonization of roots. We propose that the ability to modify the hydrophobicity of fungal hyphosphere is a key to unlock the evolutionary and functional paradox of these proteins.