Fusarium graminearum KP4-like proteins possess root growth-inhibiting activity against wheat and potentially contribute to fungal virulence in seedling rot.

The virally encoded KP4 killer toxin protein was first identified from Ustilago maydis (Um), and its homologues are present in diverse fungi and in one species of moss. No KP4-like (KP4L) proteins have been functionally characterized. Here, we report the identification and functional analysis of four KP4L proteins from Fusarium graminearum (Fg), the primary causal pathogen of Fusarium head blight (FHB), which is also known to associate with seedling rot of wheat. The four FgKP4L proteins (FgKP4L-1, -2, -3 and -4) are encoded by small open reading frames (378-825 bp) located on chromosome 1 with the FgKP4L-1, -2 and -3 genes clustering together. Sequence analysis indicated that FgKP4L proteins have conserved domains predicted to form a three-dimensional alpha/beta-sandwich structure as first reported for UmKP4, with FgKP4L-4 featuring double Kp4 domains. Further analyses revealed that the FgKP4L genes are expressed in vitro under certain stress conditions, and all up-regulated during FHB and/or seedling rot development, the recombinant FgKP4L-2 protein does not induce cell death in wheat leaves or spikelets, but inhibits root growth of young seedlings, and the elimination of the FgKP4L-1/-2/-3 gene cluster from the fungal genome results in reduced virulence in seedling rot but not in FHB. Database searches revealed KP4L proteins from ∼80 fungal species with more than half from human/animal pathogens. Phylogenetic analysis suggested that UmKP4 and the moss KP4L proteins are closely related to those from a zygromycete and Aspergillus, respectively, implying cross-kingdom horizontal gene transfer.