Phytophthora Small
RNA Database:
Visualization of
Small RNAs Using a
Genome Viewer
Deepthika Ennamuri
Mentor: Dr. Niklaus J. Grünwald
REU Summer 2011 Internship
Horticultural Crops Research Laboratory
USDA - National Institute of Food and
Agriculture Grant
$9 million grant
 Develop methods
to manage Late
Blight Disease

http://www.csrees.usda.gov
Phytophthora
Genus of water molds
 Resemble fungi in
growth habits

◦ More closely related to
Brown Algae
◦ Resistant to most fungicides
http://www.cdfa.ca.gov
100+ species have been discovered
 Some species can attack up to
hundreds of different plant host species

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
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
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
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
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
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
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
GBrowse
Identifying miRNAs


Identify potential miRNA loci
Capture FASTA sequence for the region

Create foldback structure to determine if
region contains miRNA
Reannotating the P. sojae Genome
Properly format P. sojae and
P. infestans FASTA files
Run FASTA files through MAKER
MAKER outputs GFF3 files
Format P. infestans and P.sojae GFF3
files
Upload P. sojae and P. infestans small
RNA GFF3 files to database
Results

http://phytophthora-smallrna-db.cgrb.oregonstate.edu/
miRNA
Artificial miRNA
Acknowledgments

Dr. Niklaus Grünwald
◦ Stephanie Bollmann
◦ Chris Sullivan
◦ Josh Cuperus

Howard Hughes Medical Institute
◦ Dr. Kevin Ahern

USDA – NIFA

US NSF - REU