O4 Cooke

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O4 Cooke

  1. 1. A clearing picture of Phytophthora evolution: from the wide-angle to the zoom lens for optimal phylogenetic focus David Cooke, SCRI
  2. 2. • Introduction – aims of paper • Early evolution of oomycetes • Lessons learned from genomics • Current status of Phytophthora phylogeny • Tools and drivers: fine-scale population shifts • Conclusions • Acknowledgements
  3. 3. Genomics in the oomycetes P. infestans P. ramorum P. sojae H. parasitica P. capsici P. andina P. mirabilis P. phaseoli Albugo Pythium? Saprolegnia? Aphanomyces High throughput sequencing technologies providing many new opportunities
  4. 4. Eukaryote tree of life Lane & Archibald, TREE 2008
  5. 5. Contributions of genomics Tyler et al., 2006 Science P. ramorum and P. sojae genomes Found 855 Phytophthora genes with similarity to red algae or cyanobacteria Whisson et al 2007 Nature Shared mechanisms of effector transport into host cells – oomycete and Apicomplexans Lane & Archibald, TREE 2008 RxLR class of effectors considered key to pathogenicity: a reduced repertoire noted in Hy. parasitica genome (Warwick HRI). Also an absence of genes involved in zoospore formation…
  6. 6. Long history of parasitism in oomycetes Gordon Beakes – Chapter in “Oomycete Genetics and Genomics: Diversity, Plant and Animal Interactions, and Toolbox” (in press, 2008) Long history of parasitism in oomycetes – diverse and elegant range of infection devices & abundance of marine species. Tree of Life upgrade planned
  7. 7. Basal oomycetes rDNA 18S tree The Development, Ultrastructural Cytology, and Molecular Phylogeny of the Basal Oomycete Eurychasma dicksonii … Satoshi Sekimoto, Gordon W. Beakes, Claire M.M. Gachon, Dieter G. Müller, Frithjof C. Küpper, Daiske Honda Protist, 2008 Parasites of plants across oomycete lineages
  8. 8. Cooke et al 2000 c.f. Blair et al 2007 • Multigene approach more robust • Clades shared • Basal branch structure similar
  9. 9. Pquininea Pmacrochl Prichard EU301150Psp.TIB20079 Pfallax Pcaptiosa EU644726Psp.BFA EU196368Psp.167008 EU196369Psp.167011 Pgallica Pboehmeriae DQ066921Psp.Drimys winteri DQ002008Psp.cc2124 Pkernoviae 9 & 10 ITS Tree 2008 Ppolonica Pinsol cooke EF590254Pcuyabensis EF680320Pnapoensis EU419612Psp.6216900 EU419615Psp.8021391 EU334634Pirrigata23J7 EU644721Psp.BF3 EU644715Psp.BF4 EU644714Psp.BFK EF680321Pthermophilum EU644725Psp.BF1 EU583798Phydropathica SCRP237 Rf6 EU164426.1Pzentmyerii EF590256Plagoariana EU644720Psp.BFR EU644722Psp.BF36 Rf17 • Scan of GenBank Taxonomy Pbrassicae Pfoliorum Pramorum Pprimulae Pporri Phibernalis browser for ITS seq. Plateralis Pmedicaginis Ptrifolii Psansomea Psyringae P. austrocedrae Pdrechsleri 8 • 146 seq but some redundancy Pkelmania Pcryptogea Perythroseptica P. fragaefolia Psojae Pniederhauserii Ppistaciae Pmelonis • Clade 8 position of P. 7 Pcajani Pvignae Pcinnamomi Pfvr Pcinnparvi Puliginosa Peuropaea cryptogea & drechsleri Palnimult Pcambivora Pquercina Pilicis Ppsychrophila Ppseudosyringae Pnemorosa Pheveae 3 • Clade 7 P. fragaefolia on deep Pkatsurae EF067922Psp.NZICMP 5 P. novaeguinee Ppseudotsugae Pcact Pidaei root Phedraiandra Pnicotianae Ptentaculata Pclandestina Piranica Pinfestans & andina Pipomoeae Pmirabilis 1 • Clade 5 P. novaeguinee and a Pphaseoli P. frigida EU644723Psp.BLW71 Pmultivesiculata EU301125Psp.TIB20072 strain Pbisheria NZ isolate Pcolocasiae Pbotryosa EU419614Psp.204B Pcitrophthora EU419613Psp.1211 EF523387Psiskiyouensis Ptropicalis Pcapsici Pglovera DQ821185Psp.BR514 2 • Clade 2 P. frigida in basal EU301132Psp.TIB20074 Pcitricola Pinflata EU877749Psp.59 position EU877753.1Psp.63 EU877755Psp.65 EU877754Psp.64 Pmegakarya Pelitch vogl EU419617Psp.1931 • Clade 4 ‘tropical and P. alticola P. quercetorum EU301118Psp.TIB20071 strain Psp banksia Ppalmivora Parecae 4 australasian flavour?’ Paspara EF590257Psulawesiensis EU593265Psp.IMI329669 EU301170Psp.TIB20076 Pinundata Phumicola EU000108Psp.77231 EU594601Psp.1FFL2008 EF153672Psp.P12745 • Clade 6 dramatic expansion & Pmegasperma EU301174Psp.TIB20077 DQ396412Psp.AG34UASWS0200 EU000143Psp.AG43 appearance of aerial habit 6 EU106591Psp.92209C EU594600Psp.2FFL2008 EU106590Psp.92207 EU301176Psp.TIB20078 EU869199PaustralisVHS EU301158.1Psp.TIB20073 strai EF152514Psp.GD2f EF522140Psylvatica EU725809Ppinifolia EU594598Phungarica EU594599Psp.4FFL2008 Pgonapodyides EU419616Psp.B6 EU000124Psp.91112 AY787023Psp.AG52 0.1
  10. 10. Expansion of clade 9 & 10 P. quininea P. macrochlamydospora P. richardiae EU301150Psp.TIB20079 • Discrete lineage from P. fallax P. captiosa 9 majority of genus EU644726Psp.BFA EU196368Psp.167008 EU196369Psp.167011 • No obvious common P. gallica 10 morphological/behaviour P. boehmeriae al traits P. kernoviae P. polonica • Distinct geographical P. insolita P. cuyabensis origin but blurred by P. napoensis EU419612Psp.6216900 migration? EU419615Psp.8021391 P. irrigata EU644721Psp.BF3 EU644715Psp.BF4 EU644714Psp.BFK P. thermophilum EU644725Psp.BF1 P. hydropathica 9 P. parsiana P. zentmyerii P. lagoariana EU644720Psp.BFR EU644722Psp.BF36 Rf17
  11. 11. Status of Phytophthora phylogenetics? • Move from single gene to multigene phylogenies • ITS remains good starting point • Number of species increasing but main structure unchanged • Expansion of clades 9 & 10 most marked • Ever expanding range of taxa in clade 6 • Monitoring methods by Silvia Scibetta – powerful tool • Still issues with GenBank – reference collection better in independent source (Phytophthora database)
  12. 12. Oomycete pathogen population analysis • Mutation • Neutral markers • Recombination • Natural selection AFLPs • Gene flow SSRs • Random genetic drift P. infestans, P. ramorum, Pl. viticola • Migration Accelerated discovery examined by Schena and Cooke (BMC Genomics submitted) Sequencing Coalescent analysis • Functional markers Carbone web page Host range Virulence Aggressiveness & Fitness Fungicide resistance Sequencing genes under strong selection effector genes e.g. RxLRs P. infestans, Hy. parasitica
  13. 13. Simple Sequence Repeats – P. infestans Marker G11 Isolate 1 Isolate 2 ABI 3730 capillary sequencer Isolate 3 Isolate 4 Multiplex PCR – protocols on www.eucablight.org
  14. 14. Changes in British P. infestans population • Eucablight Scotland database - powerful • Increase in A2 mating type seen in GB • What about genotypes? England
  15. 15. Derived data plotted on web • Differences in allele frequency between countries • Expansion to South and Central America complete • Further expansion under discussion
  16. 16. Great Britain SSR genotypes 1982-2007 GB genotypes 100% 90% 3 80% 10 70% 13 71% 60% Fre que nc y US1 old A2 50% 7,8 40% misc. 6 30% 2 20% A1 10% 4 5 10% 12 0% 1982 1995 1996 1997/8 2003 2004 2005 2006 2007 Isolate no. 34 85 188 264 290 581 73 899 1452 • Major change in 2006 and 2007 • Some genotypes very durable, others not. Why?
  17. 17. GB genotype frequency within 2006 season 100% 90% 80% 70% 60% 50% 40% 30% 20% Number of 10% outbreaks 0% May(1-31) June(1-15) June(16-30) July(1-15) July(16-31) Aug(1-15) Aug(16-31) Sep(1-30) 5 44 27 20 13 10 18 18 31 249 152 116 82 60 112 87 Number of isolates
  18. 18. GB genotype frequency within 2007 season 100% 90% 80% 70% 60% What drives this change? 50% 40% 30% 20% 10% Number of outbreaks 0% May(1-15) May(16-31) June(1-15) June(16-30) July(1-15) July(16-31) Aug(1-15) 4 4 22 80 139 45 14 29 23 144 387 574 220 52 Number of isolates
  19. 19. Lesion area • • 6_ A1 _4 0 2 4 6 8 10 12 14 16 10 0A N 13 L0 _A 62 2_ 69 6_ 388 A1 4 _3 B 92 0A N 13 L0 _A 42 2_ 46 13 39 _A 64 2_ A 17 39 _A 2 2_ 8A 43 7_ 88 A1 D _4 7_ 16 A1 8C 10 _41 _A 6 2_ 8B 13 44 _A 40 C 10 2_4 _A 13 2_ 2 39 B 36 C S 2 E0 30 S 58 E 2_ 030 A1 8 _3 7 3b 88 _A 8A 2_ 42 2_ 44 A1 E _4 06 LD 8B 15 1 P 8a M _A P6 1_ 22 42 3_ 56 A2 B _4 01 2F M P On average, genotype 13 isolates result in 61 8_ 8 larger lesions than other genotypes at 13oC 2a Genotype 6 largest lesions of A1 genotypes _A 1_ C4 42 32 E C Lesion area (mean of all varieties) 2 13 oC
  20. 20. Days 13 _A 0 1 2 3 4 5 6 7 8 2_ • • 38 84 N B L0 4 24 N 6 L0 13oC 6_ 6 26 A1 9 _ 41 13 00 _A A 2_ 13 39 _A 64 2_ A 17 41 _A 32 2_ B 13 43 _A 88 D 2_ 39 6_ 28 A1 A _3 3b 92 _A 0A 2_ 10 42 _A 44 2_ E 44 2_ 4 A1 0C 10 _4 _A 06 2_ 8B 39 2_ 36 A1 C2 _3 7_ 88 A1 8A _4 16 8 LD C 3 _ 15 1 A2 P _4 7_ 01 A1 2F _4 16 8 SE B sooner than other genotypes at 13oC 03 05 8 M P6 22 Genotype 6 shortest LP of A1 genotypes 8_ SE 2a 03 _A 08 1_ 7 On average, genotype 13 isolates sporulate 8a 42 Latent period _A 32E 1_ 42 56 B M P6 18
  21. 21. Field trial results 100.00% Percentage of each genotype 90.00% 80.00% 70.00% x 60.00% 7 50.00% 2 40.00% 8 6 30.00% 13 20.00% 10.00% 0.00% D1 D2 D3 D4 D5 D6 No. lesions 67 67 99 100 17 20 • All isolates were pathogenic in lab test at D0 • Domination of genotype 13 clear • Four other genotypes (1 alien) rare
  22. 22. Virulence of genotype 8- A1 R1 R2 R3 R4 R5 R6 R7 R8 R10 R11 BPC_06_4256B = 8_A1 1,4,7,10,11 Less complex and less aggressive (note 7,10&11)
  23. 23. Virulence of genotype 13 - A2 R1 R2 R3 R4 R5 R6 R7 R8 R10 R11 BPC_06_3928A = 13_A2 1,2,3,4,5,6,7,9,10,11 Complex virulence and aggressive Resistant to metalaxyl
  24. 24. Structured screening of RxLR diversity • Study of evolutionary forces on key pathogen traits 3 D Panel of 30 GB isolates 3_A2 2 B • Avr 3a 10_A2 • 3 SNPs (3 replacement changes) • 23 EM homozygous (virulent) • 6 EM/KI heterozygous (avirulent) 13_A2 • 1 KI homozygous (avirulent) 4 A • avirulent strains confined to a specific lineage which explains decline of this avirulence • R3a not deployed widely (3.5% area) – low selection pressure implies no ‘cost’ to virulence for avr3a • PEX 161 2 C 1_A1 • 9 SNPs (1 outside ORF, 2 silent, 6 replacement) 2 A • 5 haplotypes 6_A1 • 7 TTGAATCGC A 3 D • 2 YTGMWWYRY B 2_A1 • 2 YWRMWWYRY C 6 D • 14 YWRAAWYRY D 7&8_A1 • 1 CAAAAATAT E • Haplotypes correspond to SSR lineages 7/1A/B/D/E rare
  25. 25. Conclusions • Genomics High throughput, affordable (£5K per 100 Mb) DNA sequencing offering insights into mechanisms of pathogenicity, host range and ultimately host resistance responses. Post-genomics is going to be a ‘long haul’. • Phylogenetics Exciting prospects for environmental monitoring of Phytophthora diversity in support of conventional approaches. Evaluating risks. Exploring what happens in ‘balanced’ natural ecosystems as an aid to understanding invasive species. Phylogeography to improve our understanding of origins of species. Phylogenomics to understand mechanisms conserved across genus. • Population analysis Tracking populations – co-ordinated responses needed – tracking using standardised tools – sharing ‘intelligence’ on local problems. Predicting risk? How does population structure influence management? Examining selection pressures on key genes.
  26. 26. Acknowledgments • Eucablight team Jens Hansen, Poul Lassen & Alison Lees • SCRI Team: Alison Lees, Naomi Williams and Louise Sullivan, • Italian team: Silvia Scibetta, Leonardo Schena, Santina Cacciola and others • BPC team: David Shaw, Ruairidh Bain, Nick Bradshaw, Moray Taylor • Many, many people who have contributed inspiration, friendship & cultures • Potato Council & RERAD for funding

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