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O27 Martin

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Information about O27 Martin

Published on November 24, 2008

Author: bongsoopark

Source: slideshare.net

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2008 Phytophthora Workshop
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Advances in systems for identification and diagnosis of Phytophthora, Pythium and related genera Frank Martin USDA-ARS, Salinas, CA

Identification of Isolates • Challenges of morphological identification – Level of expertise needed – Not all isolates produce necessary structures – Overlap of morphological features – Convergent evolution – Time necessary

Molecular Identification • Generally takes less time • Less subjective for identification • Can sometimes differentiate isolates below the species level.

Desired Marker Characteristics • Look for a single region that is conserved within a species but variable between species. • Have conserved sequences flanking variable region • Amplicon size suitable for real-time PCR • High copy number

Molecular Loci Used for Species Identification • Nuclear – rDNA – β-tubulin – Elicitin, cellulose binding elicitor lectin – Translation elongation factor 1 α – Ypt1 gene – Elicitin gene par1, putative storage protein Lpv – 60S Ribosomal protein L10, enolase, heat shock protein 90, TigA gene fusion protein

Molecular Loci Used for Species Identification - Nuclear • Nuclear – Multiple copy • rDNA – ITS region most commonly used for – sequence based ID (good representation in GenBank) – As source of sequences for designing species-specific markers – “Single” copy • Translation elongation factor 1 alpha – phylogeny – Kroon et al. 2004, Blair et al. 2008 • ß -tubulin – phylogeny and molecular diagnostics – Kroon et al. 2004, Blair et al. 2008, Bilodeau et al. 2007 • Elicitin, cellulose binding elicitor lectin – molecular diagnostics – Bilodeau et al. 2007a, b • Ypt1 gene – molecular diagnostics – Schena et al. 2006, 2007 • Elicitin gene par1, putative storage protein Lpv – Kong et al. 2003a, b • 60S Ribosomal protein L10, enolase, heat shock protein 90, TigA gene fusion protein – phylogeny – Blair et al. 2008

rDNA Organization 18 S 5.8 S 28 S IGS ITS 1 ITS 2 5S Pythium Filamentous Sporangia For Pythium species with spherical sporangia/hyphal swellings the 5 S rDNA is dispersed as an array in other regions of the genome •Spacer regions between copies useful for species-specific markers Cistron present in multiple copies in head to tail array

Ypt1 Gene Species-Specific Diagnostic Markers Genus–specific primers and 15 species-specific Phytophthora Phytophthora genus-specific genus-specific From Schena et al. 2007

Molecular Loci Used for Species Identification - Mitochondrial Mitochondrial – multiple copy – cox1 - phylogeny and molecular diagnostics • Kroon et al. 2004a, b, Levesque et al. (bar code, personal comm.) – cox2 – phylogeny • Martin et al. 2000, 2003a, b, Hudspeth et al. 2000, Kageyama et al. 2005, Villa et al. 2006 – cox1 and cox2 spacer -molecular diagnostics • Martin et al. 2004, Tooley et al. 2006 – nad1 – phylogeny • Kroon et al. 2004 – nad5 – phylogeny • Ivors et al. 2004

Nuclear vs Mitochondrial Markers • Mitochondria are uniparentally inherited from maternal parent • Copy number may change depending on physiological status of the pathogen, so may not be best for quantification

Copy Number vs Sensitivity • Multiple copy vs “single” copy – Similar Ct in real-time PCR for P. ramorum using ITS and elicitin markers, • The Ct for both these loci averaged 3.7 lower than β tubulin – Bilodeau et al. 2007, unpublished • Consistency for rDNA copy number – In Pythium, rDNA hybridizes to different number of chromosomal bands in PFGE • different hybridization intensity relative to other “single” copy probes as well. – Different real-time PCR Ct observed for various isolates of P. infestans when normalized to Ct of “single” copy loci (Z. Atallah, personal comm.)

Techniques Used for Molecular Identification • Techniques used are dependent on the type of analysis that is needed – Identification of isolates to species level that have been cultured – Identification of isolates from field samples – Identification of a particular species of regulatory importance from field samples – Identification of subpopulations within a species

Molecular Techniques for Isolate Identification • DNA sequencing – Specific genes for ID and phylogenetic analysis • Pythium – Nuclear – ITS, large ribosomal subunit, β tubulin, – Mitochondrial – cox1, cox 2 • Phytophthora – Nuclear – ITS, β tubulin, translation elongation factor 1 α, elicitin, 60S Ribosomal protein L10, enolase, heat shock protein 90, TigA gene fusion protein, Ypt1 – Mitochondrial – cox1, cox2, nad1, nad5 – Molecular tool box for identification and characterization of Phytophthora spp. • 4 mtDNA intergenic regions, a portion of the rDNA-IGS, a portion of Ypt1 (a ras related protein). – Schena and Cooke 2006

Molecular Techniques for Isolate Identification • Micro/macro arrays – Identification of isolates to species level • Reverse dot blot – Levesque et al. 1998 • Reviewed in Lievens and Thomma 2005 – Use single nucleotide polymorphisms (SNPs) on array to identify subpopulations

Molecular Techniques for Isolate Identification • Single Strand Conformational Polymorphism – SSCP of ITS sequences - Both Pythium and Phytophthora spp. • C. Hong’s lab at VPI (2003 – 2005) • Automated sequencer for Phytophthora ID – Tom Kubisiak, USDA Forest Service, MS (unpublished) – SSCP with cox spacer region for Phytophthora spp. • E. Hansen (unpublished)

PCR-RFLP for Isolate Identification • RFLP analysis of PCR amplified fragments – ITS region of the rDNA • Phytophthora – David Cooke (PhytID) • Pythium – Chen et al. 1992, Wang and White 1997 – MtDNA • cox 1 and 2 gene cluster – Phytophthora - Martin and Tooley 2004 – Pythium – Martin (unpublished) • Spacer between cox 1 and 2 genes – Phytophthora - Martin (unpublished)

Martin and Tooley Phytopathology 2004

RFLP Analysis for ID of Pythium spp. • Similar in approach to Phytophthora RFLP analysis – Different primers used – Amplicon a little more than half the size of the Phytophthora amplicon • Tested on over 160 isolates representing 40+ species – Clearly delineated species – Limited intraspecific variation

Alu1 100 bp ladder P. catenulatum P. heterothallicum P. myriotylum P. sylvaticum P. torulosum P. ultimum – Korea P. ultimum – New Zealand P. ultimum – Maryland P. ultimum – New York P. ultimum – HS – Iowa P. aphanidermatum P. myriotylum P. spinosum P. splendens P. sylvaticum P. ultimum

Phytophthora genus-specific Amplification cox 2 spacer cox 1 Phy-8b Phy-10b Phytophthora Approximately 450-500 bp Primers amplify Phytophthora, but not the Pythium and plant species tested •Analysis can be done directly on amplifications from infected tissue

25 bp ladder P. clandestina P. gonapodyides P. spp. P. humicola P. idaei P. inflatum P. iranica P. katsurae P. medii P. melonis P. quercina - 125 bp specific Amplicon for Species ID RFLP Analysis of Phytophthora Genus-

Molecular Techniques for Identification of Subpopulations • RAPDs • AFLPs – Phytophthora • Lamour and Hausbeck 2001, Ivors et al. 2004 – Pythium • Garzon et al. 2005a, b • Inter simple sequence repeats (ISRR) – Pythium • Vasseur et al. 2005 • Microsatellites – Phytophthora • Prospero et al. 2004, Ivors et al. 2006, Lees et al. 2006, Dobrowolski et al. 2002 – Pythium • Lee and Moorman 2007 • Micro/macro arrays to identify SNPs • Mitochondrial haplotypes – Phytophthora infestans

Species-Specific PCR for Pathogen Detection • Conventional vs real-time PCR – Due to less sensitivity and the time necessary for running the sample conventional PCR less common in diagnostic setting • Important to have multiplexed – Plant marker as internal control for DNA extraction – Genus-specific marker is desirable • Different chemistries for real-time PCR – TaqMan – perhaps most common – Scorpion – need less time to run cycle than TaqMan, so need less time to complete assay – Molecular beacons

Approaches to Enhance Specificity • Nested amplification – Advantage that in also increases sensitivity – Disadvantage that it adds a few steps and has more opportunities for errors • Locked nucleic acids – Allows higher annealing temperatures to be used • Padlocked probes – Szemes et al. 2005 • Analysis of hybridization melt kinetics – Anderson et al. 2006

Padlock Probes to Improve Specificity T1, T2 – species-specific sequences P1, P2 – forward and reverse primers Zip – sequences generated to be species-specific for TaqMan probe Szemes et al. 2005

Considerations when starting to use PCR markers reported in the literature • At least initially try using exact procedures reported • Validate technique in your lab – Amplification conditions – Block uniformity

Loop Mediated Isothermal Amplification • Reported as diagnostic for Phytophthora ramorum – Tomlinson et al. 2007 • Does not require a thermal cycler (just a temperature controlled block) • Can visualize results – On a gel by electrophoresis – Intercalation of a dye – Increased turbidity (production of Mg pyrophosphatase) – Real-time PCR • Some limitations – Less sensitive than TaqMan assay (10 pg vs 250 fg) – Commonly used dye has to be added at the end of the reaction as it inhibits the reaction

Using Mitochondrial Sequences for a Systematic Approach for Marker Development • See more sequence variation than in many nuclear regions • Target has high copy number • Want to identify region where variable sequences are flanked by conserved sequences to simplify marker development for additional species • Use in conjunction with plant and Phytophthora genus specific markers

Phytophthora ramorum Multiplex Amplification First Round Amplification cox II spacer cox I Phytophthora Plant Second Round Amplification P ramorum . Phytophthora Additional details: http://www.ars.usda.gov/Research/docs.htm?docid=8728

Genomic Sequencing of the MtDNA for Marker Development • Rather than looking at individual sequences one at a time, will approach this by looking at genomic sequences of the mitochondrial DNA – Identify conserved/variable regions to focus on – Look for gene order differences with related genera and plants to enhance specificity of the markers

Mitochondrial Genome Sequencing • Pythium spp. – 15 species – 18 genomes • 2 isolates for 3 species to evaluate intraspecific variation • Phytophthora spp. – 12 species – 13 genomes • 2 isolates of 1 species to evaluate intraspecific variation

Mitochondrial Genome Organization • Circular orientation – Some Pythium spp. have linear genomes • Inverted repeats? – Yes – Pythium, Saprolegnia, Achlya, Aplanopsis, Leptolegnia, Saparomyces – No – Phytophthora • Small inverted repeat (< 1.5 kb) present in P. ramorum and P. hibernalis

Pythium mtDNA Inverted Repeat Single Copy Region IR IR

Linear Mitochondrial Genomes of Pythium spp. • Occur as concatamers • Found in all species examined – For most species linear arrangements are present in very low amounts • Termini correspond to the small unique region • Termini have hairpin loop

Genome Sizes for Pythium spp. Small Inverted Large Genome Size Genome Size % Genome IR Species Uniquea Repeata Uniquea One arm IRa Totala P. catenulatum 2,704 24,964 10,253 37,921 62,885 79.4 P. graminicola 7,280 27,611 9,915 44,806 72,417 76.3 P. heterothallicum 3,368 21,269 13,066 37,703 58,972 72.1 P. myriotylum 3,900 28,342 12,148 44,390 72,732 77.9 P. nunn 3,304 22,346 13,103 38,754 61,100 73.1 P. oligandrum 1,372 30,911 10,291 42,574 73,485 84.1 P. sylvaticum 3,395 20,599 13,102 37,096 57,695 71.4 P. ultimum 2,711 21,954 13,068 37,733 59,687 73.6 a Sizes in bp

Ph. megasperma P. aphanidermatum P. deliense P. insidiosum Pythium spp. P. aristosporum P. arrhenomanes P. volutum P. vanterpoolii P. graminicola P. plurisporum P. catenulatum P. torulosum P. coloratum P. dissotocum P. dissimile P. myriotylum P. sulcatum P. pyrilobum Inflated P. grandisporangium Filamentous P. oligandrum P. australe P. opalinum Spherical P. iwayami P. paddicum Globose P. okanoganse P. irregulare P. lucens P. mamillatum P. spinosum P. irregulare P. sylvaticum P. erinacieus P. pulchrum P. rostratum P. minor P. nunn P. heterothallicum P. splendens P. ultimum HS P. ultimum P. ultimum v spor 10 changes

Phytophthora Mitochondrial Genome Organization • Lack an inverted repeat – Exceptions • P. megasperma, less than 0.9 kb based on Southern analysis (Schumard-Hudspeth and Hudspeth 1990) • P. ramorum, 1,150 bp (Martin et al. 2007) • P. hibernalis, ca. 1,500 bp • Has the same genes found in Pythium – Some differences in ORFs • Differences in gene order

Phytophthora ramorum rps 4 rps 8 rps 2 ymf 100 rps 14 rpl 6 rrnL KA N S Me P atp 8 Mf ymf 99 rpl 4 M rpl 5 rpl 16 G Y rps 3 0 rps 19 rrnS rpl 2 rps 13 35 K W rps 11 ymf96 5 K S C cox 2 orf 32 L Length: 39,314 bp cox 1 37 genes 30K i nvert ed r e peat nad 11 10 K 26 tRNAs for 19 AA orf 176 nad 1 7 ORFs, I unique R cob i nve nad 4L rt e d nad 9 r ep orf 176 atp 9 ea t R nad 3 25 K K 15 D nad 6 atp 6 cox 3 20 K rps 7 nad 5 rps 12 F RQ IV Inverted Repeat rps 10 atp 1 nad 2 -1,150 bp in length E nad 4 H ymf 98 nad 7 -Includes 528 bp ORF

100* 100* P cactorum . P. cactorum P hedraiandra . P idaei . P pseudotsugae . P. pseudotsugae 100* 100* P iranica . 100* P clandestina . P tentaculata . 100* . P infestans P. infestans Clade 1 P sp. “andina” . 92 P ipomoeae . 75 100* 1.0 90 P mirabilis . 60 P phaseoli . 1.0 P nicotianae . P. nicotianae 100* P arecae. 100* P palmivora . P. palmivora Clade 4 P sp. “quercetorum” . P megakarya . 94 53 68 . P quercina P. quercina 1.0 77 P citrophthora . P. citrophthora 1.0 P inflata . 100* P meadii . P botryosa . 100* P colocasiae . 100* P capsici . P. capsici 100* 100* 100* P mexicana . Clade 2 P sp. “glovera” . P tropicalis . P citricola . P. citricola 96 P multivesiculata . 86 P sp. “bisheria” . 1.0 100* P ilicis . P psychrophila . 92 38 100* P nemorosa . P. nemorosa Clade 3 1.0 P pseudosyringae . P. pseudosyringae 100* P heveae . P katsurae . P. heveae Clade 5 100* P humicola . 100* P inundata . 100* P sp. “personii” . 100* P gonapodyides . P. gonapodyides Clade 6 P megasperma . P. megasperma P sp. “asparagi” . 100* P cambivora . P. cambivora P alni . 100* P fragariae . P. fragariae P uliginosa . P europaea . 100* P cajani . 100* 100* P vignae . P sinensis . 100* P melonis . Clade 7 100* P pistaciae . P sojae . P. sojae 100* P sp. “niederhauserii” . 96 85 100* 1.0 P cinnamomi . P. cinnamomi P cinnamomi var. parvispora . 100* P richardiae . 100* P erythroseptica . P. erythroseptica P cryptogea . P sp. “kelmania” . P drechsleri . P. drechsleri 100* P sp. “sansomea” . 100* P trifolii . 82 P medicaginis . 100* 99 1.0 100* . P brassicae . P porri Clade 8 P primulae . P syringae . P. syringae 95 100* P ramorum . P. ramorum 53 1.0 P lateralis . P. lateralis P foliorum . P. foliorum 80 57 100* P insolita . . P hibernalis P. hibernalis 1.0 P polonica . 100* P sp. “basal species” . P sp. “cuyabensis” . 100* P fallax . P captiosa . 100* P quininea . Clades P macrochlamydospora . P. macrochlamydospora 9, 10 100* P boehmeriae . P kernoviae . Pythium vexans 0.01 Multilocus phylogeny of Blair et al. (2008)

Is gene order related to phylogenetic relationships in Phytophthora? • While some differences in gene order may be associated with phylogenetic relationships, many are not. • Interspecific comparisons of genomes reveals some regions are more variable than others – Gene order in some regions highly conserved in genus

Development of New Marker System for Phytophthora • Two conserved differences in gene order compared to Pythium have been identified • Both regions have been sequenced in 90+ isolates representing 60+ species to assess intra- and interspecific variation. • One region has been selected for further study based on the sequence data – Interspecific polymorphisms – Intraspecific sequence conservation – %GC of sequences

Phytophthora Multiplex Amplification Phytophthora amplicon ca. 190 bp gene gene Gene order differences between Phytophthora and Pythium - also with plant mtDNA from GenBank search

Phytophthora Multiplex Amplification Phytophthora amplicon ca. 190 bp gene gene Phytophthora TaqMan Probe gene gene Species-specific TaqMan Probe

Mitochondrial Haplotype Determination • Can intraspecific variation be used as haplotype markers to differentiate isolates? – P. infestans – Ia, Ib, IIa, IIb • Are there specific places in the genome that are more prone to variation to simplify looking for haplotype markers from a wider number of species? – Genomic rearrangements leading to intraspecific differences in gene order tend to occur at specific places. Is this also a region more prone to intraspecific variation as well?

Phytophthora ramorum Mitochondrial Haplotypes • Is there intraspecific variation in the sequences of the mitochondrial genome that can be used to assign haplotype? • Kroon et al. – SNP in cox1 gene • If so, can they be used as a marker to help monitor populations of the pathogen? Martin (2008) Current Genetics 54:23-34

Phytophthora ramorum Intraspecific Sequence Conservation • California vs European mtDNA genomic sequence – 13 single nucleotide polymorphisms – 1 insertion of 180 bp • Additional polymorphisms when looking at 40 other isolates – 15 new SNPs

Evaluation of Mitochondrial Haplotypes • Identification of SNPs – Designed primers to amplify and sequence regions that are variable in comparisons between the US and EU mt genomes. – Looked at other regions that were polymorphic in comparisons among other species. • Determination of haplotypes – Total of 7,496 bp (or 19% of the genome) examined – Looked at 40 isolates from geographically diverse areas

P. ramorum Mitochondrial Haplotypes Marker # Variable Bases mtDNA Haplotypes Prv-9 1 I – EU II - US , III – Washington Nursery ymf-16 2 I – EU , III – Washington Nursery II - US cox2 + spacer 3 III – Washington Nursery I = II Prv-1 2 III – Washington Nursery I = II Prv-8 2 I – EU II - US III – Washington Nursery Prv-11 2 I – EU II - US III – Washington Nursery Prv-13 8 I – EU II – US III – Washington Nursery cox1 4 I – EU II – US III – Washington Nursery Prv-14 4 I – EU IIa – US IIb – Oregon forest III – Washington Nursery

Non-Sequence Based Haplotype Determination • Melt curve analysis of amplicons – Using the Idaho Technology Light Scanner – Redesigned the amplification primers so a smaller amplicon was generated (for the most part less than 200 bp)

P. ramorum Mitochondrial Haplotype Melt Curve Analysis Haplotype I IIa , IIb III

Non-Sequence Based Haplotype Determination • Melt curve analysis of amplicons – Using the Idaho Technology Light Scanner – Redesigned the amplification primers so a smaller amplicon was generated (for the most part less than 200 bp) • Has worked well for most regions for differentiating haplotypes – Can differentiate IIa from IIb

Acknowledgements • MtDNA genomic sequencing – P. ramorum and P. sojae (Current Genetics 51:285-296) • J. Boore, D. Bensasson – JGI, Walnut Creek, CA • B. Tyler – VBI, VPI Blacksburg, VA – Pythium and other Phytophthora spp. • P. Richardson et al., JGI, Walnut Creek, CA • Thanks to the USDA-CSREES-NRI Plant Biosecurity Grant Program for supporting this work

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