Tyler impact next gen fri 0900

Evolution and function of the P. sojae effectorome:

What we’ve learned from next generation sequencing

Evolution and function of the P. sojae effectorome:

What we’ve learned from next generation sequencing

Brett TylerVirginia Bioinformatics Institute

Virginia Tech

Brett TylerVirginia Bioinformatics Institute

Virginia Tech

Coevolutionary struggle between plants and pathogens

pathogenplantSUSCEPTIBLE

pathogenplantRESISTANT




RESISTANTpathogen

plant




Plant defenses and effectorsEffectors suppress PAMP-triggered immunity (PTI) and Effector-

triggered immunity (ETI)

Extracellulareffector

intracellulareffector

R

Rimmunity

PRR

Pathogen-associated molecular pattern (PAMP)

Pattern-recognition receptor (PRR)

PTI

R

R

ETI

Some effectors suppress ETI

Some effectors suppress ETI

Some effectors suppress PTI

Some effectors suppress PTI

Effector – R gene Coevolutionary struggle H

ein et al Molecular Plant Path (2009)

Loss of Avr gene transcription

• Loss of transcription allows later reactivation of the gene

• May involve rapid epigenetic changes

P. sojae Avr1b

P. sojae Avr3a

Loss of Avr gene function by mutation

P. infestans Avr4(van Poppel et al., 2008)

Additional examples: C. fulvum Avr4

New functional variants• P. infestans Avr3a

• Avr3a is essential for infection – silencing reduces infection (Bos et al., 2010)– R genes are being sought against EM allele

Avrvir

Avrvir

Avrvir

New functional variants

Hyaloperonospora arabidopsidis ATR13 P. sojae Avr1b

• Positive selection occurs when a mutation that causes an amino acid change is favored over one that does not

• Synonymous mutations are frequently lost by genetic drift

• Non-synonymous mutations are preserved by selection

• Ability to grow on a plant containing a resistance gene is an example of positive selection

• Measured by a statistical test comparing the frequencies of synonymous mutations (dS) to that of non-synonymous ones (dN), adjusted for the frequency of codons available for each type of substitution.

• ATR1 and Avr1b display dN/dS >> 1

• dN/dS ratio is a valuable tool for finding pathogen genes involved in co-evolutionary conflict with host

Positive (divergent) selection

Huge superfamily of Avr-like genes with accelerated divergence

P. sojae ~400P. ramorum ~370P. infestans ~550H. arabidopsidis ~130

Signal Peptide RXLR dEER

27aa10-67aa

14aa7-55aa

126aa28-835aa

medianrange

Includes all 15 cloned oomycete avirulence genesPsAvr1a, PsAvr1b, PsAvr1k, PsAvr3a,PsAvr3c, PsAvr4/6, PsAvr5PiAvr1, PiAvr2,PiAvr3a, PiAvr4, PiAvrPlb1/ipiO1, PiAvrPlb2, HpAtr1, HpAtr13

Jiang et al (2008) PNAS 105(12), 4874-4879

Sequencing of P. sojae genomes

• P6497 reference strain. Genotype I

– 2002-2004 DOE JGI

– Sanger sequencing with Megabase

– 9X draft assembly

• P6497 finishing

– 2007-2010 Hudson Alpha Institute

– Gap filling; BAC finishing; genetic mapping

• Resequencing. 2009 VBI CLF

– P7076 (genotype II)

– P7074 (genotype III)

– P7064 (genotype IV)

– 454 Titanium sequencing. $25,000 each

– 9x coverage. Newbler assembly

Sequencing the variation in P. sojae

Forster et al.(1994) MPMI 7, 780-791

Distribution of variation in the effectorome

Characterization of P. sojae transcriptomes

• 3000 Sanger ESTs 1998

– Mycelia, zoospores, infected hypocotyls

– Qutob, D.et al (2000) Plant Phys. 123, 243-253.

• 27,000 Sanger ESTs 2002

– Mycelia (various treatments), zoospores, infected hypocotyls

– Torto-Alalibo et al (2007). Mol. Plant-Microbe Interact. 20, 781-793.

• Affymetrix microarrays 2006

– 15,800 probe sets (along with soybean)

– About half the effector genes are missing

– Various treatments including infection

• ABI Solid™ sequence tags 2009

– 50 million from mycelia

– 200 million from infected soybean (~40m from P. sojae)

Effector Gene Expression ProgramEffector Gene Expression ProgramAffymetrix GeneChip data quantile-normalized to an external reference

E

E

Expression Patterns of Elicitors and SuppressorsExpression Patterns of Elicitors and Suppressorslo

g2

exp

ress

ion

leve

ls

0 3 6 12

hours post-inoculation (biotrophic phase)

elicitorssuppressorselicitorssuppressors

suppression

Early expressed effectors suppress plant response to later effectors

1.GFP+GFP+INF1

2.Avh172+GFP+INF1

3.GFP+Avh238+INF1

4.GFP+Avh238+INF1

5.Avh172+Avh238+INF1

6.GFP+Avh238

7.Avh172+Avh238

Avh172

1

645

2 37

Cooperation among effectors

Avh238 INF1

Yuanchao Wang Nanjing Agricultural University

immediate-early early PAMP

1.GFP+GFP+INF1

2.Avh172+GFP+INF1

3.GFP+Avh238+INF1

4.GFP+Avh238+INF1


6.GFP+Avh238

7.Avh172+Avh238

Avh172

1

645

2 37


Avh238 INF1



1.GFP+GFP+INF1

2.Avh172+GFP+INF1

3.GFP+Avh238+INF1

4.GFP+Avh238+INF1


6.GFP+Avh238

7.Avh172+Avh238

Avh172

1

645

2 37


Avh238 INF1



1.GFP+GFP+INF1

2.Avh172+GFP+INF1

3.GFP+Avh238+INF1

4.GFP+Avh238+INF1


6.GFP+Avh238

7.Avh172+Avh238

Avh172

1

645

2 37


Avh238 INF1



1.GFP+GFP+INF1

2.Avh172+GFP+INF1

3.GFP+Avh238+INF1

4.GFP+Avh238+INF1


6.GFP+Avh238

7.Avh172+Avh238

Avh172

1

645

2 37


Avh238 INF1



Avh172 and Avh238 are essential for infection

Stable and transient silencing of effector genes in P. sojae

RNA seq reveals that disproportionately few genes contribute most transcripts

P. sojae RXLR effector genes ranked by expression during infection

MutationsPer gene

> 105-101-40

Expr

essi

on d

urin

g in

fecti

on (l

og2R

PKM

)

Expression in mycelia (log2RPKM)

Avh172

Avh238

Expression and variation of the P. sojae effectorome

Hypothesis: balancing selection results in expansion and divergence of effector gene family

• Gene duplication increases virulence and is selected for

• Gene duplication increases selection pressure from R genes

– Gene divergence or loss is selected for

• Genes damaged by mutation are replaced as functional genes are duplicated

– Gene birth and death model

Jiang et al (2008) PNAS 105(12), 4874-4879

Evolution of RXLR effector gene family

Seasons

400

350

300

250

200

150

100

50

00 5000 10000 15000 20000

Eff

ecto

r G

enes

Evolution of effector gene numberEvolution of effector gene number

Computer modelingComputer modeling

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 50 100 150 200

Series2

Predictions: a few genes contribute most to virulence many gene duplications and pseudogenes

Individual contributions

cumulative contribtions

Contributions of genes to virulence in model

50%

90%

Effector genes ranked by virulence contribution

BIRTH

DEATH

Viru

lenc

e C

ontr

ibut

ion

Predictions: a few genes contribute most to virulence

P. sojae RXLR effector genes ranked by expression during infection

Avh172

Avh238

Many effector genes are drifting under neutral selection

Duplications and pseudogenes in the Avr3a/5 region

Avh assembly 1.1 new Arachne assembly

339 scaffold_42:379748-380908 (1160) 8:370427-371587231 scaffold_42:404520-405257 8:395936-39520235 scaffold_42:408109-408633 8:398788-399090108 scaffold_42:432464-432612 8:423142-423393pseudo 8:428281-42847037 scaffold_80:333293-333700 8:572644-572237Avr3a/92a2 scaffold_80:316435-316103 not in new assemblyAvr3a/92a1 scaffold_80:300039-300374 8:590004-59021336 scaffold_80:294754-294951 8:595427-59565138 scaffold_80:268360-268782 8:621596-62201871 scaffold_80:167143-167330 8:723048-72327588 scaffold_80:136261-136524 8:758270-7580857b1 scaffold_80:26979-27344 8:862396-8620677c scaffold_80:15473-15802 8:876036-8763657a scaffold_31:704559-704233 8:894007-894333new scaffold_31:703472-703787 8:895094-894915pseudo321 scaffold_31:603816-604153 8:996448-996126320 scaffold_31:593690-594190 8:1006574-1006359pseudo?387 scaffold_31:579565-579997 8:1020802-102105321 scaffold_31:573401-573910 8:1026863-1026669319 scaffold_31:558714-558369 8:1041678-1041935new missing from old assembly 8:1045964-1045779pseudo scaffold_31:548437-548586 8:1052534-105238514 scaffold_31:480665-481244 8:1119642-111930168 scaffold_31:467979-468151 8:1132322-1131981pseudo scaffold_31:464136-464446 8:1136164-1135815new scaffold_31:459828-459655… 8:1140472-1140666pseudo scaffold_31:449859-449122 8:1150441-1151178318 scaffold_31:446373-445942 8:1153927-115435896 scaffold_31:373894-374105 8:1226195-1226413

7b2 scaffold_1152:3295-3627 not in new assembly

Predictions: many gene duplications and pseudogenes

Genomics of oomycete effectors

• Co-evolutionary conflict between pathogens and their hosts drives rapid change in effector genes

• Effector gene repertoires can change through gene deletion, transcriptional inactivation and rapid mutation

• A small number of effector genes contributes disproportionately large number of transcripts

• Birth-and-death evolution may shape the oomycete RXLR effector repertoire

Summary

Documents

Tyler impact next gen fri 0900