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Sequencing and analysis of Phymatotrichopsis omnivora

Sequencing and analysis of Phymatotrichopsis omnivora the fungal causative agent of root rot in plants Simone Macmil Dr. Roe’s Laboratory. Background. Phymatotrichopsis omnivora = Phymatotrichum omnivorum Recalcitrant soilborne fungus Deep, long-lived inoculum

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Sequencing and analysis of Phymatotrichopsis omnivora

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  1. Sequencing and analysis of Phymatotrichopsis omnivora the fungal causative agent of root rot in plants Simone Macmil Dr. Roe’s Laboratory

  2. Background • Phymatotrichopsis omnivora =Phymatotrichum omnivorum • Recalcitrant soilborne fungus • Deep, long-lived inoculum • Limited to alkaline, calcareous soils of SW USA and N Mexico • Root rot of >2000 dicots • Monocots immune • Belongs to the family of Ascomycetes.

  3. 20 19 19 16 19 20 7 9 18 19 9 12 29 17 4 20 19 16 6 8 11 7 1 19 28 2 20 3 26 10 27 25 26 5 20 9 22 23 20 13 19 19 20 14 15 Phylogeography of P. omnivora ITS Haplotypes

  4. Life cycle of P.omnivora Fungal hyphae Plant infection Blocked Vegetative Stage: Fungal growth in soil occurs as fungal hyphae. Moisture Sclerotia Spore Mat Plant death Moisture Sclerotial Stage: resting structures developed deep underground.

  5. How big is this genome? “An active parasexual cycle combined with the presence of many possible nuclei within individual hyphal cells and within a mycelium might help to explain the survival and consequent genetic flexibility of P.omnivorum, despite its lack of a known sexual cycle or functional asexual spore stage”. Hosford R & Gries. (1966) The nuclei and parasexuality in Phymatotrichumomnivorum. Amer.J. Bot53,570-579

  6. Phymatotrichopsis omnivora CHEF analysis (Young Lab) 5.7 Mb 4.6 Mb 3.5 Mb P.omnivora shows diffuse banding pattern characeristic of aneuploid heterokaryotic chromosomes

  7. P. omnivora telomeres Po Po Po Po OKAlf8 OKAlf8 E2368 E2368 NFOK NFOK OKAlf8 OKAlf8 E2368 E2368 NFOK NFOK Alf8 Alf8 Alf8 Alf8 23.0 23.0 6.5 2.3 EcoRI BglII Probed with Tubulin2 0.5 BglII EcoRI Probed with (TTAGGG)6

  8. Multinucleate, heterokaryotic hyphae of P. omnivora • 3-20 nuclei per young hyphal cells • Frequent hyphal fusions

  9. Biotin Overview of 454 DNA preparation protocol Nebulization Quantitate on Caliper AMS-90 DNA End Repair 5’ 3’ 3’ 5’ 5’ 3’ Adaptor Ligation (A&B) 3’ 5’ 5’ 3’ DNA End Repair 3’ 5’

  10. Overview of 454 DNA preparation protocol • Emulsify beads and PCR reagents in water-in-oil microreactors • - “B” primer is in solution and attached to capture bead • - “A” primer is biotinylated • emPCR Amplification • Break Microreactors • Enrich for DNA positive beads • Load onto 454 Plates Before PCR After PCR

  11. T C G A T dNTP Base Addition PP i Pyrosequencing Bead dTTP Polymerase A A T C G G C A T G C T A A A A G T C A T APS Annealed Primer Sulfurylase Luciferase ATP luciferin Light + oxy luciferin

  12. P.omnivora assembly statistics (Newbler assembly ver 2.0)

  13. 454 P.omnivora genome analysis schema ABI 3730 Assembly with Newbler Predict genes using FgenesH, Predict tRNAs using tRNAScanSE Homology search against GenBank Metabolic Reconstruction using KEGG, KOG and COGEME

  14. Repeats found in P.omnivora searchedagainst Repbase Interspersed Repeats

  15. Distribution of Metabolic processes (from proteins predicted in genomic contigs)

  16. Relative numbers of P.omnivora, S.cerevisae, and N.crassa transporters in various families

  17. P.omnivora Transport Proteins • 28 drug efflux proteins • 19 calcium transporters • Transport proteins significant to its lifestyle • Bcmfs1, DHA14-like major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides • Arsenite-translocating ATPases

  18. ^^^^^^ ^^^^^^ Paired-end sequencing on the 454 of CHEF gel extracted and amplified chromosomes EcoRI EcoRI EcoRI ^^^^^^ Excise gel bands containing chromosomal DNA Methylation Ligation of sticky ends and Circularization Chromosomal DNA extraction and isothermal amplification using the Multiple Displacement Amplification Reaction Hairpin adaptor ligation of ends Nebulization ^^^^^^ Generation of 454 shotgun library and sequencing Ecor I digestion Hydroshear amplified DNA to 2-4 Kb pieces

  19. Distribution of cellular processes in chromosomes 2 and 3

  20. Distribution of cellular processes in chromosomes 4 and 5

  21. EST (Expressed Sequence Tag) Sequencing Construct the 454 Library and Sequence Isolate total RNA

  22. Overview of EST analysis - Draft Contigs + singlets BlastX against KEGG and COG databases extract_kegg_cogs_1_pl Metabolic reconstruction ESTanalysis_script2_FZN2_pl Overall Expression Profile Using COG’s schema and KEGG’s metabolism Contigs and Singlets with No Hit tBlastX against EST Append the Overall Expression File

  23. ESTs studies from different life stages of P.omnivora

  24. Gene Expression of Known Function genes in vegetative mycelia 84,708 reads were sequenced using the GS FLX. High number of house keeping genes were found to be expressed

  25. Gene Expression of Known Function genes in vegetative mycelia in response to stress (Carbon and Nitrogen starvation) 10,720 reads were sequenced using the 454 GS 20 High number of mitochondrial genes expressed along with genes involved in gluconeogenesis and proteolysis.

  26. Gene Expression of Known Function genes in Sclerotia (resting structures) 60,493 reads were sequenced using the 454 GS 20 High number of genes involved in fatty acid and carbohydrate metabolism

  27. Gene Expression in Spore Mat(that consists of conidia /asexual spores) 61,702 reads were sequenced using the 454 GS 20 High number of genes involved in chromatin remodelling and conidial development were expressed

  28. Comparison of Gene Expression of P.omnivora mycelia during Interaction with the Host vs non-Host Root Exudate

  29. Comparison of gene expression of P.omnivora mycelia during interaction with host vs non-host root exudate

  30. cDNA Analysis and Conclusions • Energy metabolism was observed for each stage in the life cycle. • In response to stress, fungal mycelia resort to gluconeogenesis and protein degradation. • Sclerotial resting structures were found to be mainly involved in carbohydrate metabolism and fatty acid biosynthesis. • Spore mats were found to be metabolically active, histones H3& H4 involved in the condensation of chromosomes were observed. • Genes involved in lipid biosynthesis, polyketide biosynthesis and signal transduction were found to be expressed only during host-root exudate interaction hence account for putative pathogenicity factors of P.omnivora. • Genes involved in butanoate metabolism, porphyrin & ubiquinone biosynthesis were found during interaction with non-host root exudate only and may contribute to the saprophytic life style of the fungus.

  31. Disease and Virulence genes found in Genomic contigs and ESTs in response to Host and non-host Root Exudate Interaction • Similar to delta latrotoxin gene from Bortryotina • Pathogensis related Snod Protein1 (detected in EST response to non-host root exudate) • Superoxide-generating NADPH oxidase • CAP20-like protein (involved in virulence, penetration, and appressorium formation in Blumeria graminis) • Homologue of M. grisea pathogenicity protein • Structural toxin protein homologue (detected in EST response to host root exudate) • Superoxide generating NADPH oxidase cytosolic protein

  32. Summary & Conclusions • The fungus P.omnivora has numerous nuclei in it’s mycelia, high number of repeat elements with a genome size of ~74 Mb characteristic of an obligate heterokaryote. • Preliminary assembly and analysis of the genome revealed high number of transporter genes. • The high number of ABC type transporters most likely confers resistance to fungicides, heavy metals and other plant toxins. • The high number of calcium transporters may account for P. omnivora’s ability to live in calcareous soils. • The expression of specific cDNAs (ESTs) under different conditions reveals specific mRNAs that are involved with each unique stage of fungal growth and development cycle.

  33. Acknowledgements • Dr. Bruce A. Roe • Dr. F. Najar, Steve Kenton • Graham Wiley, ChumMei Qu, Ping Wang, Yanbo Xing, Doug White • All other members of Dr. Roe’s lab • Dr. C. Young and Dr. S.Marek at the Noble Foundation and OSU repectively • Graduate Committee members: Dr. P.Cook, Dr.A. West, Dr. P. Klebba, Dr. K. Duncan, Dr. C. Rice, • Oklahoma Dept. of Agriculture for funding the Oklahoma Consortium for Legume Research

  34. Devol, OK

  35. Library ssDNA 3’ 5’ B A Emulsify with A&B primers, A’ is Biotinylated on 5’ 16B’=A’ 5’ 3’ 5’ 3’ 3’ 5’ B’ A’ 3’ 5’ 94oC Hot Start 5’ 3’ 5’ 3’ 3’ 5’ B’ A’

  36. 3’ 5’ Free B’ Extends Along Free Template 5’ Free Biotinylated A’ Extends 5’ [ ] X10^6 emPCR Final Result 5’ 3’

  37. [ ] emPCR Final Result X10^6 5’ 3’ Bind to Streptavidin Coated Enrichment Bead Use Magnet to Sequester Beads Then Melt the DNA Anneal Sequencing Primer 3’ 5’ Load on 454

  38. 700 bp IGS ITS2 ITS1 1700 bp 1400 bp 5.8S SSU (18S) RNA LSU (28S) RNA 2900 bp • Molecular Systematics of P. omnivoraMarek, Hansen, Romanish, and Thorn (2008) • 129 isolates of Phymatotrichopsis omnivora • Genes sequenced • Nuclear Ribosomal Genes (rDNA) • RNA pol II subunit-2 (rpb2) 2.9Kb

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