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Systems biology of grape diseases
Dario Cantu, Assistant Professor!Viticulture and Enology Department, UC Davis!
GRCN July 10 2013 Davis, CA
Understanding grapevine diseases!
Planting density Moisture content Temperature Rain Wind Nutrient availability Other organisms
The host plant must be
susceptible to the pathogen
Environment
Disease The pathogen must be able to overcome
plant defenses
The environment must tip the balance in favor of the pathogen
Identification and characterization of novel sources of resistance!
Dissection of disease response molecular networks !
A systems biology approach!
Knowledge-based decisions to improve durability of resistance to diseases Integrating classical and systems biology approaches
Characterize mechanisms of pathogenicity and their evolution!
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density PHYLUM
AscomycotaBasidiomycota
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Basidiomycota (median=42.4Mb; N=427) Ascomycota (median=26.6Mb; N=1,106)
Fungal Genome Size Database. h5p://www.zbi.ee/fungal-‐genomesize.
Genomics of fungal pathogenicity!
“small” eukaryotic genomes = more accessible genomes and affordable re-sequencing
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density PHYLUM
AscomycotaBasidiomycota
Plant pathogens
Pathogen-specific focused annotation 6
A Fungal pathogen genome sequencing and assembly workflow
Isolation and taxonomic identification Shotgun sequencing
Illumina (HiSeq, MiSeq) PacBio RS
Quality control
Quality trimming/filtering Adapter removal Error correction K-mer analysis
De novo assembly
SOAPdenovo CLC Velvet SGA Mira …
Assembly QC
1. Contaminants
2. Assembly metric - Total length - N50 - L50 - Scaffold > 2Kb - Coverage
3. CEGMA
how many of the 248 Core eukaryotic genes are in the assembly?
Gene discovery
RNAseq
Repeat masker Maker (w/ snap) Augustus
[evidence-based and ab initio]
Quality control
Esca disease Phaeomoniella chlamydospora Phaeoacremonium spp. Togninia minima
Eutypa dieback Eutypa lata Eutypa leptolaca Diatrype prominens Diatrype stigma Diatrype whitemanensis Eutypella spp. …
Botryosphaeria canker Botryosphaeria spp. Neofusicoccum parvum
Grapevine trunk disease genomics
Esca disease Phaeomoniella chlamydospora Phaeoacremonium spp. Togninia minima
Eutypa dieback Eutypa lata Eutypa leptolaca Diatrype prominens Diatrype stigma Diatrype whitemanensis Eutypella spp. …
Botryosphaeria canker Botryosphaeria spp. Neofusicoccum parvum
Grapevine trunk disease genomics
Net income = Revenue – Expenses* *$60 per acre to remove diseased wood h%p://coststudies.ucdavis.edu/
Cankers are the main cause of grapevine dieback
Esca disease Phaeomoniella chlamydospora Phaeoacremonium spp. Togninia minima
Eutypa dieback Eutypa lata Eutypa leptolaca Diatrype prominens Diatrype stigma Diatrype whitemanensis Eutypella spp. …
Botryosphaeria canker Botryosphaeria spp. Neofusicoccum parvum
Grapevine trunk disease genomics
Net income = Revenue – Expenses* *$60 per acre to remove diseased wood h%p://coststudies.ucdavis.edu/
Cankers are the main cause of grapevine dieback
Esca disease Phaeomoniella chlamydospora Phaeoacremonium spp. Togninia minima
Eutypa dieback Eutypa lata Eutypa leptolaca Diatrype prominens Diatrype stigma Diatrype whitemanensis Eutypella spp. …
Botryosphaeria canker Botryosphaeria spp. Neofusicoccum parvum
Grapevine trunk disease genomics
Net income = Revenue – Expenses* *$60 per acre to remove diseased wood h%p://coststudies.ucdavis.edu/
Cankers are the main cause of grapevine dieback
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Eutypa lata (Eutypa dieback)
Togninia minima (Esca)
Neofusicoccum parvum (Bot canker)
Genome assembly Fungal purification
Grapevine trunk disease genomics
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Comparative analysis of the predicted secretome
signalP targetP TM-HMM
Predicted proteome
Secretome
pfam Blast2go CAZy
GH43: hemicellulase; GH61: monoxygenase
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Multiple hosts
Multiple geographical locations
Different virulence strengths
Sampling genetic diversity (whole genome resequencing)
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Multiple hosts
Multiple geographical locations
Different virulence strengths
Sampling genetic diversity (whole genome resequencing)
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0 50 100 150Mb
density PHYLUM
AscomycotaBasidiomycota
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“Difficult” genome: - Dikaryon (2n, ~5 heterokaryotic SNPs / Kb) - Large (expansion of intergenic repetitive regions). - DNA not from axenic cultures (contamination)
Puccinia striiformis f.sp. tritici genomics
Whole-genome shotgun sequencing!
Puccinia striiformis f.sp. tritici!
Two lanes of Illumina GAII -> 95% reads assembled in 29,178 contigs covering approximately ~90% of the PST genome (59x coverage; estimated size 80Mb) !
N50: 5.1Kb!
Illumina GAII!
Puccinia striiformis f.sp. tritici genomics
http://www.plosone.org/article/info%3Adoi/10.1371/journal.pone.0024230
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Puccinia striiformis f.sp. tritici genomics
Gene space quality control by pathway completeness
Assembly QC -> colinearity with Puccinia graminis f.sp. tritici (PGT) of longest contigs
Total length: 1.8 Gbp (~ 22X) Average length: 2400.11 nt Median length: 1,988 nt Longest sequence: 23,062 nt
Puccinia striiformis f.sp. tritici genomics
Error correction Illumina reads
PacBio
Hybrid assembly
Scaffolding
Assembled genomes Sequencing in progress DNA being collected
Whole Genome resequencing of 32 isolates from 12 different countries in 4 conFnents)
Illumina HiSeq2000
PacBio RS
Puccinia striiformis f.sp. tritici genomics
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Puccinia striiformis f.sp. tritici genomics
Dodds & Rathjen, 2010
Effectoromics
Effector candidate discovery 1. Genome analyses 2. Transcriptome profiling 3. Effector focused annotaQon
h%p://www.biomedcentral.com/1471-‐2164/14/270
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Haustoria transcripts
Puccinia striiformis f.sp. tritici genomics: 2. Transcriptome profiling
Haustoria isolation
Infected tissue
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Andy Walker (UCD), Kendra Baumgartner (USDA-ARS), Philippe Rolshausen (UCR), Caroline Roper (UCR), Andrew McElrone (USDA-ARS), Summaira Riaz (UCD), Ann Powell (UCD), Doug Gubler (UCD). Wheat rust:
Jorge Dubcovsky (UCD), Richard Michelmore (UCD), Cristobal Uauy (John Innes Centre), Sophien Kamoun (TSL), Diane Saunders (TSL), Xiaodong Wang (UCD), Xianming Chen (USDA-ARS).
Abraham Morales-Cruz
Aurelie Sastre
Barbara Blanco-Ulate
Eduardo Gutierrez Katherine
Harris Amrine Laura Jones
Our team
Collaborators
More info: h%p://cantulab.github.com