Exploring links between antisense RNAs and pathogenesis in Ustilago maydis through transcript and gene characterization.

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.

Transcriptome analysis of smut fungi reveals widespread intergenic transcription and conserved antisense transcript expression.

BACKGROUND: Biotrophic fungal plant pathogens cause billions of dollars in losses to North American crops annually. The model for functional investigation of these fungi is Ustilago maydis. Its 20.5 Mb annotated genome sequence has been an excellent resource for investigating biotrophic plant pathogenesis. Expressed-sequence tag libraries and microarray hybridizations have provided insight regarding the type of transcripts produced by U. maydis but these analyses were not comprehensive and there were insufficient data for transcriptome comparison to other smut fungi. To improve transcriptome annotation and enable comparative analyses, comprehensive strand-specific RNA-seq was performed on cell-types of three related smut species: U. maydis (common smut of corn), Ustilago hordei (covered smut of barley), and Sporisorium reilianum (head smut of corn). RESULTS: In total, >1 billion paired-end sequence reads were obtained from haploid cell, dikaryon and teliospore RNA of U. maydis, haploid cell RNA of U. hordei, and haploid and dikaryon cell RNA of S. reilianum. The sequences were assembled into transfrags using Trinity, and updated gene models were created using PASA and categorized with Cufflinks Cuffcompare. Representative genes that were predicted for the first time with these RNA-seq analyses and genes with novel annotation features were independently assessed by reverse transcriptase PCR. The analyses indicate hundreds more predicted proteins, relative to the previous genome annotation, could be produced by U. maydis from altered transcript forms, and that the number of non-coding RNAs produced, including transcribed intergenic sequences and natural antisense transcripts, approximately equals the number of mRNAs. This high representation of non-coding RNAs appears to be a conserved feature of the smut fungi regardless of whether they have RNA interference machinery. Approximately 50% of the identified NATs were conserved among the smut fungi. CONCLUSIONS: Overall, these analyses revealed: 1) smut genomes encode a number of transcriptional units that is twice the number of annotated protein-coding genes, 2) a small number of intergenic transcripts may encode proteins with characteristics of fungal effectors, 3) the vast majority of intergenic and antisense transcripts do not contain ORFs, 4) a large proportion of the identified antisense transcripts were detected at orthologous loci among the smut fungi, and 5) there is an enrichment of functional categories among orthologous loci that suggests antisense RNAs could have a genome-wide, non-RNAi-mediated, influence on gene expression in smut fungi.