Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Donaldson ME

PROVIDER: S-EPMC5414199 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Donaldson Michael E ME Ostrowski Lauren A LA Goulet Kristi M KM Saville Barry J BJ

BMC genomics 20170502 1

<h4>Background</h4>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 an ...[more]

PMID: 28464849

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Project description:BackgroundSerial Analysis of Gene Expression (SAGE) is a powerful tool for genome-wide transcription studies. Unlike microarrays, it has the ability to detect novel forms of RNA such as alternatively spliced and antisense transcripts, without the need for prior knowledge of their existence. One limitation of using SAGE on an organism with a complex genome and lacking detailed sequence information, such as the hexaploid bread wheat Triticum aestivum, is accurate annotation of the tags generated. Without accurate annotation it is impossible to fully understand the dynamic processes involved in such complex polyploid organisms. Hence we have developed and utilised novel procedures to characterise, in detail, SAGE tags generated from the whole grain transcriptome of hexaploid wheat.ResultsExamination of 71,930 Long SAGE tags generated from six libraries derived from two wheat genotypes grown under two different conditions suggested that SAGE is a reliable and reproducible technique for use in studying the hexaploid wheat transcriptome. However, our results also showed that in poorly annotated and/or poorly sequenced genomes, such as hexaploid wheat, considerably more information can be extracted from SAGE data by carrying out a systematic analysis of both perfect and "fuzzy" (partially matched) tags. This detailed analysis of the SAGE data shows first that while there is evidence of alternative polyadenylation this appears to occur exclusively within the 3' untranslated regions. Secondly, we found no strong evidence for widespread alternative splicing in the developing wheat grain transcriptome. However, analysis of our SAGE data shows that antisense transcripts are probably widespread within the transcriptome and appear to be derived from numerous locations within the genome. Examination of antisense transcripts showing sequence similarity to the Puroindoline a and Puroindoline b genes suggests that such antisense transcripts might have a role in the regulation of gene expression.ConclusionOur results indicate that the detailed analysis of transcriptome data, such as SAGE tags, is essential to understand fully the factors that regulate gene expression and that such analysis of the wheat grain transcriptome reveals that antisense transcripts maybe widespread and hence probably play a significant role in the regulation of gene expression during grain development.

Project description:The first taxonomic treatment of the smut fungi in Greenland is provided. A total of 43 species in 11 genera are treated and illustrated by photographs of sori, microphotographs of spores in LM and SEM, and distribution maps. Two species, Anthracoidea pseudofoetidae and Urocystis tothii, are recorded as new from North America. Thirteen species, Anthracoidea altera, A. capillaris, A. limosa, A. liroi, A. pseudofoetidae, A. scirpoideae, A. turfosa, Microbotryum lagerheimii, M. stellariae, Schizonella elynae, Stegocintractia luzulae, Urocystis fischeri, and U. tothii, are reported for the first time from Greenland. Three new fungus-host combinations, Anthracoidea capillaris on Carex boecheriana, Anthracoidea pseudofoetidae on Carex maritima, and Urocystis tothii on Juncus biglumis, are given. Five plant species are reported as new hosts of smut fungi in Greenland, namely, Carex nigra for Anthracoidea heterospora, C. canescens for Anthracoidea karii, C. fuliginosa subsp. misandra for Anthracoidea misandrae, C. maritima for Orphanomyces arcticus, and C. fuliginosa subsp. misandra for Schizonella melanogramma. Three species, Microbotryum violaceum s. str. (recorded as 'Ustilago violacea'), Urocystis anemones, and U. junci, which were previously reported from Greenland, are considered wrongly identified. Additional distribution records are given for 12 species from Greenland: Anthracoidea bigelowii, A. caricis, A. elynae, A. lindebergiae, A. misandrae, A. nardinae, A. rupestris, A. scirpi, Schizonella melanogramma, Stegocintractia hyperborea, Urocystis agropyri, and U. sorosporioides. The most numerous distribution groups are the following: circumpolar-alpine and Arctic-alpine species - 14; circumboreal-polar species - 10; and circumpolar and Arctic species - 6. The most widely distributed smut fungi in Greenland were Anthracoidea bigelowii, A. elynae, Microbotryum bistortarum, and M. vinosum. Most species were found in the High Arctic zone (29 species), while from the Low Arctic zone and the Subarctic zone, 26 and 19 species were known, respectively. Ten species, Anthracoidea bigelowii, A. capillaris, A. elynae, Microbotryum bistortarum, M. koenigiae, M. pustulatum, M. silenes-acaulis, M. vinosum, Schizonella elynae, and Urocystis sorosporioides, were recorded from all three zones. Only plants belonging to six families, Cyperaceae, Poaceae, Juncaceae, Ranunculaceae, Caryophyllaceae, and Polygonaceae, out of a total of 55 in the flora of Greenland, hosted smut fungi. Cyperaceae was the plant family with most host species (23). Carex was the genus with the highest number of host species (22). The total number of the host plants (45 species) was 8.5 % out of a total of 532 vascular plants in the flora of Greenland. A new combination in Carex, C. macroprophylla subsp. subfilifolia, is proposed for Kobresia filifolia subsp. subfilifolia.

Project description:Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets.To gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3'-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes.Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3' untranslated region length of 523 bp.Our strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3' UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum.

Dataset Information

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

Background

Results

Conclusions

Publications

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

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