Sequencing and analysis of Phymatotrichopsis omnivora

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 • Originally classified a Basidiomycete

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. How does P.omnivora cause root rot? Fungus forms extensive growth around the hypocortex , destroys the periderm , moves through the medullary rays until it blocks the phloem and xylem disrupting the flow of nutrients and water. Horizontal section of Dicotyledonous root http://waynesword.palomar.edu/trjune99.htm

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

6. P.omnivora genome sequencing

7. 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

8. CHEF gel analysis of P.omnivora chromosomes in parallel with S.cerevisiae and S.pombe(Young Lab) 1 2 3 4 5 6 7 8 9 10 Lanes 7-10 P.ominvora Diffuse banding pattern characeristic of aneuploid heterokaryotic chromosomes 5.7Mb 4.6Mb 3.5Mb 2.2Mb 1.6Mb

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

10. 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’

11. Overview of 454 DNA preparation protocol • Anneal template to DNA Capture beads • 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

12. 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

13. P.omnivora assembly statistics (Newbler assembly)

14. 454 P.omnivora genome analysis schema ABI 3730 Assembly with Newbler and Phred/Phrap Predict genes using FgenesH, Predict tRNAs using tRNASCanSE Homology seartch against GenBank RNA genes Metabolic Reconstruction using KEGG, KOG and COGEME

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

16. Relative numbers of P.omnivora transporters belonging to various families

17. P.omnivora transport proteins • 28 drug efflux proteins • 19 calcium transporters • Transport proteins significant to its lifestyle 1 Bcmfs1, DHA14-like majorfacilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides 2 arsenite-translocating ATPases

18. EST (Expressed Sequence Tag) Sequencing 454 Library and Sequencing Isolate total RNA

19. Gene Expression of Known Function genes in vegetative mycelia in response to stress (Carbon and Nitrogen starvation)

20. Summary of Sequencing ESTs from mycelia in response to 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. • 5 proteins were found to be uniquely expressed that were not predicted in genomic contigs: • protein involved in membrane trafficking • Rab GDP dissociation factor • Mitochondrial NADP-dependant isocitrate dehydrogenase • ATP dependant chromatin remodelling protein • Methylated DNA protein cysteine methyl transferase

21. Gene Expression of Known Function genes in Sclerotia (resting structures)

22. Summary of Sequencing ESTs from Sclerotia • 60,493 reads were sequenced using the 454 GS 20 • High number of genes involved in fatty acid and carbohydrate metabolism • Detected 10 uniquely expressed proteins that were not predicted from genomic contigs: • Aconitase • Stomatin • NAD dependant formate dehydrogenase • Glycerol-3-phosphate dehydrogenase • Hydroxy proline rich surface glycoprotein • Mandelate racemase • Transaldolase • DNA primase small subunit • Thiamine biosynthesis protein NMT-1 • Histone H2A variant

23. Gene Expression in Spore mat (Consists of conidia /asexual spores) Genes involved in nucleosome formation, spore envelope

24. Summary of Sequencing ESTs from Spore mat • 61,702 reads were sequenced using the 454 GS 20 • High number of genes involved in chromatin remodelling and dynamics were expressed • Detected 5 uniquely expressed proteins that were not predicted from genomic contigs: • homoserine kinase • Carnitine O-acetyl transferase • Thiol specific antioxidant • Exocyst complex component Sec15 protein • Protein involved in vessicle trafficking

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

26. Disease and virulence genes found in genomic contigs and in response to host and non-host root exudate interaction • Delta latrotoxin gene • Snod Protein (detected in EST response to non-host root exudate) • PTH11 like protein, essential forpenetration structure formation and developmen Bg con • superoxide-generating NADPH oxidase • CAP20-like protein (Blumeriagraminis) • homologue of M.grisea pathogenicityprotein • NADPH oxidase • structural toxin protein homologue (detected in EST response to host root exudate) • superoxide generating NADPHoxidase cytosolic protein

27. Summary & Conclusions • The fungus P.omnivora is an obligate heterokaryote. • Preliminary assembly and analysis of the genome revealed high number of transporters. The high number of ABC type transporters is most likely to confer resistance to fungicides and other plant toxins. High number of calcium transporters may account for its ability to live in calcareous soils. • EST sequencing and analysis revealed processes most involved with each life stage of the fungus. Information obtained from EST sequencing added to the predicted gene pool of the fungus.

28. Future Work • Sequence each individual chromosome of P.omnivora using the shotgun/ paired-end approach on the 454 GS FLX • Obtain a more robust assembly using the latest version of Newbler. • Annotation, analysis and comparisons of the assembled P.omnivora genome using Genscan and FgenesH to predict protein coding genes followed by BLAST searches against GenBank and metabolic reconstruction using KEGG, KOG and COGEME.

29. 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. C.Rice, Dr. K. Duncan • Consortium for Legume Research

30. Devol, OK