Biotrophic basidiomycete plant pathogens cause billions of dollars in losses to cereal crops annually. The model for this group of fungi is the corn smut pathogen Ustilago maydis. Annotation of its genome identified antisense RNAs (asRNAs) complementary to over half of the coded mRNAs, some of which are present at high levels in teliospores but detected at very low levels or not at all in other cell types, suggesting they have a function in the teliospore or during teliospore formation. Expression of three such asRNAs (as-UMAG_02150, ncRNA1, and as-UMAG_02151) is controlled by two adjacent genomic regions. Deletion of these regions increased transcript levels of all three asRNAs and attenuated pathogenesis. This study investigated the reason for this marked reduction in pathogenesis by: (1) using deletion analyses to assess the involvement of genes, complementary to the asRNAs, in pathogenesis; (2) determining that one of the linked genes encodes a putative xylitol dehydrogenase; and (3) identifying and functionally characterizing asRNAs that could influence expression of protein-coding genes. The results presented suggest that the influence of the asRNAs on pathogenesis occurs through their action at unlinked loci.,Copyright ? 2019 The Authors. Published by Elsevier Inc. All rights reserved.