P-088 - Characterization of an Early-Expressed Ustilago maydis Effector Family with Copy-Number Variation in Smut Fungi

Ustilago maydis is a biotrophic smut fungus that infects aerial organs of maize. The fungus manipulates host immunity by the secretions of many effector proteins. We identified three putative effector gene paralogues (Rhi1/Rhi2 and Rhi3). Rhi1 and Rhi2 seem to have undergone evolutionary recent gene duplication and share identical coding sequences with a single synonymous SNP. Rhi3 exhibits sequence divergence yet retains conserved domains with Rhi1/Rhi2. Expression analysis indicates high expression during initial infection, which is then suppressed from two days post fungal inoculation on maize. Comparative genomic analysis across smut fungi revealed copy-number variation, with most species harboring at least one homologue, yet U. maydis harbors three copies – the most of any species assessed. This indicates a potential evolutionary adaptation in U. maydis. Several conserved motifs and domains, including a putative nuclear localization signal, were also identified. To functionally characterize Rhi1/Rhi2/Rhi3, expression was induced in Arabidopsis seedlings which revealed a significant increase in root hair growth, indicating a potential role in plant development modulation. We then sought to determine their subcellular localization in Nicotinia benthamiana, with early results supporting nuclear localisation. Future experiments will seek to assess their role in fungal virulence and assess any distinct roles during early infection.