Functional diversification of orthologous effector shapes pathogenic lifestyle in smut fungi

The smut fungi Ustilago maydis and Sporisorium reilianum both parasitize maize. U. maydis causes tumor formation and anthocyanin induction on all above ground organs of the maize plant, while S. reilianum causes disease symptoms only in the floral organs without tumor formation and anthocyanin induction. These two pathogens share highly similar genome structures. It is yet unclear what drives their diversity in pathogenic lifestyles. Here we show that the functional diversification of orthologous effector proteins contributes to the distinct pathogenic lifestyles. The secreted Tin2 effector of U. maydis contributes to virulence and is responsible for anthocyanin induction in maize, and this is achieved by stabilizing the maize kinase ZmTTK1. The genome of S. reilianum encodes a Tin2 effector orthologue at a genomic location syntenic to U. maydis tin2. When U. maydis tin2 is expressed in S. reilianum, anthocyanin is induced in infected leaf tissue. On the other hand, tin2 from S. reilianum (Srtin2) is unable to induce anthocyanin induction in the U. maydis tin2 mutant. We demonstrate that Srtin2 in S. reilianum is also required for virulence. SrTin2 fails to interact with ZmTTK1 but interacts with ZmTTK2 and ZmTTK3, two paralogues of ZmTTK1. Furthermore, recombinant SrTin2 protein negatively affects the kinase activity of ZmTTK2 and ZmTTK3 in vitro. A resurrected ancestral Tin2 protein does not complement anthocyanin induction of the U. maydis tin2 mutant. This suggests that the U. maydis Tin2 effector has evolved unique features, which are not shared by Tin2 effectors of other smut fungi.