Systems biology of grape diseases - NAES · Systems biology of grape diseases Dario Cantu, Assistant Professor! Viticulture and Enology Department, UC Davis! GRCN July 10 2013 Davis,

Systems biology of grape diseases

Dario Cantu, Assistant Professor!Viticulture and Enology Department, UC Davis!

GRCN July 10 2013 Davis, CA

Nematodes

Bacteria

Viruses

Fungi

Grapevine diseases!

Oomyctes

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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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





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


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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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Stripe rust

Draft genome reference release

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Stripe rust

Draft genome reference release

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Cereal stripe rust genomics

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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!


http://www.plosone.org/article/info%3Adoi/10.1371/journal.pone.0024230

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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


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


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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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Puccinia striiformis f.sp. tritici genomics: 1. Genome analysis

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Haustoria transcripts

Puccinia striiformis f.sp. tritici genomics: 2. Transcriptome profiling

Haustoria isolation

Infected tissue

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Puccinia striiformis f.sp. tritici genomics: 3. Focused effector annotation

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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

Documents

Systems biology of grape diseases - NAES · Systems biology of grape diseases Dario Cantu, Assistant Professor! Viticulture and Enology Department, UC Davis! GRCN July 10 2013 Davis,