Functional analysis of secreted long non-coding RNAs contributing to the virulence of the corn smut fungus Ustilago maydis

Plant pathogenic fungi secrete and deliver effector molecules into plant cells to establish parasitism. To date, most studies have focused primarily on proteinaceous molecules. Here, we report that the corn smut fungus, Ustilago maydis, secretes hundreds of long non-coding RNAs (lncRNAs) via extracellular vesicles. These lncRNAs are transcribed from intergenic regions of the genome and are found to be highly expressed. Many of them appear to be non-polyadenylated. The expression of some lncRNAs is specifically upregulated during plant infection. Among them, the deletion mutant of lncRNA_5109 exhibited the most significant reduction in pathogenicity. The deletion mutant complemented with intergenic region including lncRNA_5109 showed transcript accumulation and restored pathogenicity. To clarify the function of lncRNA_5109, we conducted a pull-down assay using in vitro-transcribed RNA and cell lysate extracted from infected plant leaves. Through proteomic analysis of the precipitates, we identified maize LSm4, which may play a role in pre-mRNA splicing, as the most significant interaction partner of lncRNA_5109. Furthermore, the interaction was verified through a pull-down assay using recombinant LSm4 protein and lncRNA_5109. Alternative splicing analysis revealed that exon skipping occurred in many plant genes infected with wild type compared to the deletion mutant. These results suggest that U. maydis utilizes lncRNA to modulate host mRNA metabolism to promote virulence.