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Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis

March 2015 , Volume 28 , Number  3
Pages  298 - 309

Alyssa Burkhardt,1 Alex Buchanan,2 Jason S. Cumbie,2,3 Elizabeth A. Savory,2 Jeff H. Chang,2,3,4 and Brad Day1,5,6

1Graduate Program in Cell and Molecular Biology, Michigan State University, East Lansing, MI 48824, U.S.A.; 2Department of Botany and Plant Pathology, 3Molecular and Cellular Biology Program, and 4Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, U.S.A.; 5Graduate Program in Genetics, and 6Department of Plant, Soil and Microbial Sciences, Michigan State University

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Accepted 30 October 2014.

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5′- and alternative 3′-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.

© 2015 The American Phytopathological Society