1. 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

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

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

7. Plant Microbial World: Lichen Viruses Olga Blinkova First report of evidence of lichen viruses! 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 Mike Palmer, Vaskar Thapa Also: Dan McGlinn, Fumiko Shirakura and others Judy Zhang Viral Nucleic Acid (VNA) Degenerate Primers PCR products Vijay Muthukumar 1 kb+ Plant 295 Plant 248 Plant 162 Plant 289 Plant 296 Neg. control Minh NgoTao (Colin) Ding

11. Plant Microbial World: Complexity Graham WileyBruce Roe

12. Plant Microbial World: Bacteria & Fungi Asclepias viridis Ruellia humilis Asplenium rhizophyllum Dichanthelium oligosanthes Sedum nutallianum Chara globularis Panicum virgatum Scattered species Single specimens 16S rDNA major; 23S rDNA also Two major kinds: Burkholderia cepacia complex Escherichia Frequently co-associated Jennifer Hackett Less frequent among reads Largest in one Panicum rDNA predominates also small subunit: Pycnoporus sp. large subunit: Donkioporia expansa 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 Baptisia australis Ceanothus americanus Croton monanthogynus Elymus virginicus Lespedeza procumbens Monarda fistulosa Physalis pumila Sporobolus compositus Olga BlinkovaJennifer Hackett Viral Nucleic Acid (VNA) Degenerate Primers PCR products Clipped 30 cm x 30 cm

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

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

20. Metagenomics: Pyrosequencing Graham WileyBruce Roe GGAGGATCCGAAGG 3' CCTCCTAGGCTTCC5' 5'CCTTCGGATCCTCC 3'GGAAGCCTAGGAG G PCR amplification with tagged primer 5'TGGACCTTCGGATCCTCC

21. Metagenomics: Pyrosequencing Viruses Plant speciesViral Genus Asclepias viridisTymovirus Ophioglossum engelmanniiRetrovirus Oxalis violaceaTymovirus Vitis sp.Oryzavirus Vitis sp.Cypovirus Viola bicolorCryptovirus Viola bicolorPartitivirus Baptisia australisTotivirus

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

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

24. Ecosystems: Microarray Application? Veenita Grover, Soon also Teecie West 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?

25. Ecosystems: Viruses Frequent No.Species 42Asclepias viridis 12Ambrosia psilostachya 31Panicum virgatum 6Ruellia humilis 19Sorghastrum nutans 10Vernonia baldwinii 269All (144) Yes No

26. Ecosystems: Viruses Frequent VNA Sequences to date22 Plants with virus18 Plants with virus (non-retro)11 Plant viruses7 Fungal viruses1 Overall % Plants with non-retro viruses26%

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

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