Transcript
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
Coevolutionary struggle between plants and pathogens
pathogenplantSUSCEPTIBLE
Coevolutionary struggle between plants and pathogens
RESISTANTpathogen
plant
Coevolutionary struggle between plants and pathogens
pathogenplantSUSCEPTIBLE
Coevolutionary struggle between plants and pathogens
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
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
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
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
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
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
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