Small RNA sequencing of Ustilago Maydis

The biogenesis of small non-coding RNAs is a molecular event that contributes to cellular functions. The basidiomycete fungus Ustilago maydis is a biotrophic pathogen parasitizing maize. A hallmark of its genome structure is an absence of RNAi machinery including Dicer and Argonaute proteins, which are responsible for the production of small RNAs in other organisms. However, it remains unclear whether U. maydis produces small RNAs during fungal growth. Here we found that U. maydis cells accumulate approximately 20-30 nucleotides of small RNA fragments during growth in the axenic culture condition. The RNA-seq analysis of these fragments identified that these small RNAs are originated from tRNAs and 5.8S ribosomal RNA. Interestingly, majority of their sequences are generated from tRNAs responsible for asparagine, glutamine and glycine, suggesting an bias of origin. The cleavage of tRNAs mainly occurs at the position near anticodon-stem-loop. U. maydis genome carries two genes UMAG_02611 and UMAG_03023 encoding RNase T2. The deletion mutants of two genes fail to accumulate tRNA-derived small RNAs. Furthermore, the mutants do not show the defect of fungal growth but shows small reduction of fungal virulence. Our results suggest that tRNA halves of U. maydis would contribute to the establishment of plant colonization.