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Phytophthora Small RNA Database: Visualization of Small RNAs Using a Genome Viewer Deepthika Ennamuri Mentor: Dr. Niklaus J. Grnwald REU Summer 2011 Internship Horticultural Crops Research Laboratory

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USDA - National Institute of Food and Agriculture Grant $9 million grant Develop methods to manage Late Blight Disease http://www.csrees.usda.gov

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Phytophthora Genus of water molds Resemble fungi in growth habits More closely related to Brown Algae Resistant to most fungicides 100+ species have been discovered Some species can attack up to hundreds of different plant host species http://www.cdfa.ca.gov

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Phytophthora Cause root, fruit and stem rots on numerous species of plants First show symptoms of drought and starvation Affected plants become weaker and susceptible to attack by other pathogens or conditions http://www.nasa.gov/vision/earth/environment/invasive_agreement.html

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Phytophthora infestans Caused Irish potato famine Causes Late Blight Disease Destroys potato and tomato plants Costs $6.7 billion dollars annually Thrives in rainy and humid environments http://usablight.org/?q=node/18

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Phytophthora sojae Affects soybeans Costs $1-2 billion dollars annually Present in almost all soybean producing nations Can infect seeds, seedlings, and mature plants http://www.plantpath.wisc.edu/soyhealth/prr.htm

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Small RNAs Major role in regulating gene expression in eukaryotes Interfere with gene expression Degradation and repression of transcripts Small pieces of RNA bind to target mRNA and block its translation

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Significance Understand: The mechanisms these pathogens use to break down plants defenses Use P. sojae as a model to understand Phytophthora gene regulation Aim: Organize current small RNA information into a working online database

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Summer 2011 Project Develop a publicly accessible Phytophthora small RNA database Identify target genes miRNA sequence, gene ID, gene function Perl (programming language) Reannotate current P. sojae genome sequences MAKER annotation tool Rxlr and Crinkler genes gene families involved in infection

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Materials Perl A dynamic programming language MAKER Genome annotation tool Generic Genome Browser v2.4 (GBrowse) Genome viewing tool Ability to display different types of data

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GBrowse

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Identifying miRNAs Identify potential miRNA loci Capture FASTA sequence for the region

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Create foldback structure to determine if region contains miRNA

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Reannotating the P. sojae Genome Properly format P. sojae and P. infestans FASTA files Run FASTA files through MAKERMAKER outputs GFF3 files Format P. infestans and P.sojae GFF3 files Upload P. sojae and P. infestans small RNA GFF3 files to database

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Results http://phytophthora-smallrna-db.cgrb.oregonstate.edu/

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miRNA

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Artificial miRNA

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Acknowledgments Dr. Niklaus Grnwald Stephanie Bollmann Chris Sullivan Josh Cuperus Howard Hughes Medical Institute Dr. Kevin Ahern USDA NIFA US NSF - REU