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Plant Virus Biodiversity and Ecology (PVBE)

Thorough understanding of virus biodiversity in an ecological context. Profound advances in understanding ecology of both viruses and higher organisms. Plant Virus Biodiversity and Ecology (PVBE). PVBE Team. Marilyn Roossinck Michael Palmer Rick Nelson Bruce Roe Kay Scheets Adam Zlotnick

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Plant Virus Biodiversity and Ecology (PVBE)

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  1. Thorough understanding of virus biodiversity in an ecological context Profound advances in understanding ecology of both viruses and higher organisms Plant Virus Biodiversity and Ecology (PVBE)

  2. PVBE Team Marilyn Roossinck Michael Palmer Rick Nelson Bruce Roe Kay Scheets Adam Zlotnick Jonathan Wren Jeanmarie Verchot-Lubicz Kelly Craven Andrew Doust Olga Blinkova Tracy Feldman Byoung Eun Min Nikki Charlton Vijay Muthukumar Veenita Grover Vaskar Thapa Graham Wiley Devinka Bamunusinghe Guoan Shen Judy Zhang Yan Song Shashank Bhide And many others: Marlee Pierce, Dan McGlinn, Fumiko Shirakura, Peter Earls, Hua Weng,Tao Ding,Drew Porter, Will Heise, Minh Ngo, Jennifer Hackett, Alexandra Kondos, Tsuyoshi Asako, Will Lowry, Zach Gales, Arron (Faculty, Postdoctorals, Graduate Students, Staff, Undergraduate Students)

  3. PVBE Overview • Plants harbor complex microbial worlds. • Metagenomics leads to identification of novel viruses. • Metagenomics provides insights into virus-plant interactions in ecosystems.

  4. Plant Microbial World: Overview • Plant-Lichen-Fungus-Virus • Plant-Plant-Fungus-Virus • Plant-Fungi & Plant-Bacteria

  5. Plant Microbial World: Lichens 5' 3' 5' Phenol:Chloroform 5' 3' 3' 5' Lichen ---> Total N.A. ---> dsRNA Olga Blinkova

  6. 5' 3' 3' 5' dsRNA Plant Microbial World: dsRNA Melt and anneal with RandomHexPrimer 5' 3' NNNNNN NNNNNN CCTCCTAGGCTTCC5' CCTCCTAGGCTTCC5' 5'CCTTCGGATCCTCC 5'CCTTCGGATCCTCC NNNNNN NNNNNN 3' 5' RT Reaction Add RT enzyme and dNTPs 5' 3' NNNNNN CCTCCTAGGCTTCC5' 5'CCTTCGGATCCTCC NNNNNN 3' 5' RNase treatment, remove primers First PCR cycle GGAGGATCCGAAGG3' CCTCCTAGGCTTCC5' 5'CCTTCGGATCCTCC 3'GGAAGCCTAGGAGG PCR amplification with tagged primer 5'TGGACCTTCGGATCCTCC M. Roossinck Guoan Shen

  7. Plant Microbial World: Lichen Viruses First report of evidence of lichen viruses! Olga Blinkova Yan Song http://bioinfosu.okstate.edu/pvbe/pvlist.php

  8. Plant Microbial World: Dodder • Parasitic plant • Fungi isolated from host and parasite: Alternaria, Stemphylium • dsRNA approach used on cultures of these fungi Tracy Feldman

  9. Plant Microbial World: Dodder • 14% of fungi isolated (from both plants) have viruses • Viruses tentatively detected (sequence fragments): • Endornaviridae (4), • Totiviridae (3), • Astroviridae (1), • Chrysoviridae (2), • Hypoviridae (1), • Narnaviridae (1), • Partitiviridae (1) Tracy Feldman

  10. Plant Microbial World: Plant VNA Degenerate Primers Viral Nucleic Acid (VNA) PCR products Vijay Muthukumar 1 kb+ Plant 295 Plant 248 Plant 162 Plant 289 Plant 296 Neg. control Tao (Colin) Ding Minh Ngo Mike Palmer, Vaskar Thapa Also: Dan McGlinn, Fumiko Shirakura and others Judy Zhang

  11. Plant Microbial World: Complexity Graham Wiley Bruce Roe

  12. Plant Microbial World: Bacteria & Fungi • Scattered species • Single specimens • 16S rDNA major; 23S rDNA also • Two major kinds: • Burkholderia cepacia complex • Escherichia • Frequently co-associated • Less frequent among reads • Largest in one Panicum • rDNA predominates also • small subunit: Pycnoporus sp. • large subunit: Donkioporia expansa Asclepias viridis Ruellia humilis Asplenium rhizophyllum Dichanthelium oligosanthes Sedum nutallianum Chara globularis Panicum virgatum Jennifer Hackett Alexandra Kondos

  13. Plant Microbial World: Conclusion Russian Doll Model of Life

  14. Metagenomics Overview • Approaches: • Lawn mower • Asclepias asymptomatic virus 1 • Other virus signatures identified • Challenges • Titer • Distant relationship • Unrelated to known groups

  15. Metagenomics: Lawn Mowing Degenerate Primers Viral Nucleic Acid (VNA) PCR products Baptisia australis Ceanothus americanus Croton monanthogynus Elymus virginicus Lespedeza procumbens Monarda fistulosa Physalis pumila Sporobolus compositus Clipped 30 cm x 30 cm Olga Blinkova Jennifer Hackett

  16. Metagenomics: Carmovirus Known Carmovirus TGP Carmovirus sequences • Which plant? Which plants? • Single plant species or multiple? • One segment / one subsample: statistically non-similar

  17. Metagenomics: Tymovirus? Known Tymovirus Asc. Asym Virus TGP1

  18. Metagenomics: Tymovirus! 248-1 248-2 337 1000 896 307 578 294 351 30 nm Particles Adam Zlotnick Neighbor Joining Tree • Infectivity: • Nicotiana benthamiana • Chenopodium amaranthicolor • Byoung Eun Min, Rick Nelson

  19. Metagenomics: Classification 05TGP00321 05TGP00321 05TGP00312 Taro bacilliform virus TPP Badnavirus

  20. Metagenomics: Pyrosequencing GGAGGATCCGAAGG3' CCTCCTAGGCTTCC5' 5'CCTTCGGATCCTCC 3'GGAAGCCTAGGAGG PCR amplification with tagged primer 5'TGGACCTTCGGATCCTCC Graham Wiley Bruce Roe

  21. Metagenomics: Pyrosequencing Viruses

  22. Metagenomics: Problems Reads Virus Total • Varying titers Vijay Muthukumar • Distantly related viruses • Unrelated viruses • High titer • Low titer Adam Zlotnick

  23. Ecosystems: Metagenomics Insights • Strategies • Frequent • Novel • Community • Benign • Specialist

  24. Ecosystems: Microarray Application? • Virus Signature Hybridization • Oligodeoxynucleotides • Genus specific • Short (20-25 nt) • 3’ T-Tailed • Degenerate and non-degenerate • Targets • Fluorescently tagged • Amplified from sample nucleic acid • Reverse approach? Veenita Grover, Soon also Teecie West

  25. Ecosystems: Viruses Frequent Yes No

  26. Ecosystems: Viruses Frequent

  27. Ecosystems: Viruses Novel • “Novel” viruses by VLP-VNA = 8 • “Novel” viruses by 454 and/or dsRNA = 11 • “Known” viruses by either method = 0 • Supports prediction Jonathan Wren

  28. Ecosystems: Viral Complexity

  29. Ecosystems: Virus “Specialists”? Generalist Specialist

  30. Ecosystems: Viruses Benign

  31. Ecosystems: Metagenomics Insights • Strategies • Frequent • Novel • Community • Specialist • Benign

  32. Summary • Plants host a complex microbial world. • Viruses are not inherently pathogens. • There are many fold more viruses than we know about now. • The future: Plant Virus Ecology. Join the Research Coordination Network!

  33. PVBE Team Marilyn Roossinck Michael Palmer Rick Nelson Bruce Roe Kay Scheets Adam Zlotnick Jonathan Wren Jeanmarie Verchot-Lubicz Kelly Craven Andrew Doust Olga Blinkova Tracy Feldman Byoung Eun Min Nikki Charlton Vijay Muthukumar Veenita Grover Vaskar Thapa Graham Wiley Devinka Bamunusinghe Guoan Shen Judy Zhang Yan Song Shashank Bhide And many others: Marlee Pierce, Dan McGlinn, Fumiko Shirakura, Peter Earls, Hua Weng,Tao Ding,Drew Porter, Will Heise, Minh Ngo, Jennifer Hackett, Alexandra Kondos, Tsuyoshi Asako, Will Lowry (Faculty, Postdoctorals, Graduate Students, Staff, Undergraduate Students)

  34. The Virus Players Asclepias tymovirus Asclepias comovirus Ambrosia potexvirus Ambrosia badnavirus Ambrosia endornavirus Panicum unclass. Closteroviridae Carmovirus (Origin unk.) Asplenium chrysovirus Cladonia mitovirus Cladonia chrysovirus Oxalis tymovirus Vitis oryzavirus Vitis cypovirus Viola cryptovirus Viola partitivirus Baptisia totivirus Alternaria endornavirus Alternaria hypovirus Stemphylium totivirus

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