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

PVBE Team

Marilyn RoossinckMichael PalmerRick NelsonBruce RoeKay ScheetsAdam ZlotnickJonathan WrenJeanmarie Verchot-Lubicz

Kelly CravenAndrew DoustOlga BlinkovaTracy FeldmanByoung Eun MinNikki Charlton

Vijay MuthukumarVeenita GroverVaskar ThapaGraham WileyDevinka Bamunusinghe

Guoan ShenJudy ZhangYan SongShashank 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)

PVBE Overview

•Plants harbor complex microbial worlds.

•Metagenomics leads to

identification of novel viruses.

•Metagenomics provides insights into virus-

plant interactions in ecosystems.

Plant Microbial World: Overview

•Plant-Lichen-Fungus-Virus

•Plant-Plant-Fungus-Virus

•Plant-Fungi & Plant-Bacteria

Plant Microbial World: Lichens

Olga Blinkova

Lichen ---> Total N.A. ---> dsRNA

5'

5'

3'

5'3'

3'5'

Phenol:Chloroform

Plant Microbial World: dsRNA5'3'

3'5'

dsRNA

5'

3'

3'

5'

5'CCTTCGGATCCTCC

5'CCTTCGGATCCTCCNNNNNN NNNNNN

NNNNNNNNNNNNCCTCCTAGGCTTCC5'

CCTCCTAGGCTTCC5'

5' 3'CCTCCTAGGCTTCC5'

NNNNNN

3' 5'

5'CCTTCGGATCCTCCNNNNNN

Melt and anneal with RandomHexPrimer

RT ReactionAdd RT enzyme anddNTPs

RNase treatment, remove primersFirst PCR cycle

GGAGGATCCGAAGG3'CCTCCTAGGCTTCC5'

5'CCTTCGGATCCTCC3'GGAAGCCTAGGAGG

PCR amplification with tagged primer5'TGGACCTTCGGATCCTCC

M. Roossinck Guoan Shen

Plant Microbial World: Lichen Viruses

Olga Blinkova

First report of evidence of lichen viruses!

Yan Song

http://bioinfosu.okstate.edu/pvbe/pvlist.php

Plant Microbial World: Dodder

•Parasitic plant

•Fungi isolated from host and parasite: Alternaria, Stemphylium

•dsRNA approach used on cultures of these fungi

Tracy Feldman

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

Plant Microbial World: Plant VNA

Mike Palmer, Vaskar Thapa

Also: Dan McGlinn, Fumiko Shirakura and others

Judy Zhang

ViralNucleic Acid (VNA)

Degenerate

Primers

PCR products

Vijay Muthukumar

1 kb

+

Pla

nt

295

Pla

nt

248

Pla

nt

162

Pla

nt

289

Pla

nt

296

Neg

. co

ntr

ol

Minh NgoTao (Colin) Ding

Plant Microbial World: ComplexityPercent of Reads by "Organism"

0.0

20.0

40.0

60.0

80.0

100.0

vijay1

vijay12

vijay13

vijay15

vijay17

vijay18

vijay2

vijay20

vijay21

vijay22

vijay23

vijay24

vijay27

vijay3

vijay35

vijay4

vijay45

vijay47

vijay5

vijay57

vijay59

Sample

Perc

en

t o

f To

tal Virus

Unclear

Plant

Fungus

Bacteria

Other

Animalia

Graham Wiley Bruce Roe

Plant Microbial World: Bacteria & Fungi

Asclepias viridisRuellia humilisAsplenium rhizophyllumDichanthelium oligosanthesSedum nutallianumChara globularisPanicum 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

Plant Microbial World: Conclusion

Russian Doll Model of Life

Metagenomics Overview

Approaches:

•Lawn mower

•Asclepias asymptomatic virus 1

•Other virus signatures identified

•Challenges

•Titer

•Distant relationship

•Unrelated to known groups

Metagenomics: Lawn Mowing

Baptisia australisCeanothus americanusCroton monanthogynusElymus virginicusLespedeza procumbensMonarda fistulosaPhysalis pumilaSporobolus compositus

Olga Blinkova Jennifer Hackett

ViralNucleic Acid (VNA)

Degenerate

Primers

PCR products

Clipped 30 cm x 30 cm

Metagenomics: Carmovirus

Known Carmovirus

TGP Carmovirus sequences

•Which plant? Which plants?

•Single plant species or multiple?

•One segment / one subsample: statistically non-similar

Metagenomics: Tymovirus?

Known TymovirusAsc. Asym Virus TGP1

Metagenomics: Tymovirus!

30 nm Particles Adam Zlotnick

Neighbor Joining Tree

351

307

337248-2

248-1

896578

294

1000

Infectivity:•Nicotiana benthamiana•Chenopodium amaranthicolor

Byoung Eun Min, Rick Nelson

Metagenomics: Classification

0.1

9625564

9627246

18450268

905

27228718

17.89

916

946

15029533

18450258

27819377

986

589

62327614

882

Taro bacilliform virus

TPP Badnavirus

05TGP00321 05TGP00321 05TGP00312

Metagenomics: Pyrosequencing

Graham Wiley Bruce Roe

GGAGGATCCGAAGG3'CCTCCTAGGCTTCC5'

5'CCTTCGGATCCTCC3'GGAAGCCTAGGAGG

PCR amplification with tagged primer5'TGGACCTTCGGATCCTCC

Metagenomics: Pyrosequencing Viruses

Plant species Viral Genus

Asclepias viridis Tymovirus

Ophioglossum engelmannii Retrovirus

Oxalis violacea Tymovirus

Vitis sp. Oryzavirus

Vitis sp. Cypovirus

Viola bicolor Cryptovirus

Viola bicolor Partitivirus

Baptisia australis Totivirus

Metagenomics: ProblemsReads

Virus Total•Varying titers

•Distantly related viruses

•Unrelated viruses•High titer•Low titer

Adam Zlotnick

Vijay Muthukumar

Ecosystems: Metagenomics Insights

•Strategies

•Frequent

•Novel

•Community

•Benign

•Specialist

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?

Ecosystems: Viruses Frequent

No. Species

42 Asclepias viridis

12 Ambrosia psilostachya

31 Panicum virgatum

6 Ruellia humilis

19 Sorghastrum nutans

10 Vernonia baldwinii

269 All (144)

Yes No

Ecosystems: Viruses Frequent

VNA Sequences to date 22

Plants with virus 18

Plants with virus (non-retro) 11

Plant viruses 7

Fungal viruses 1

Overall % Plants with non-retro viruses 26%

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

Ecosystems: Viral Complexity

Ecosystems: Virus “Specialists”?

Generalist Specialist

Ecosystems: Viruses Benign

Ecosystems: Metagenomics Insights

•Strategies

•Frequent

•Novel

•Community

•Specialist

•Benign

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!

PVBE Team

Marilyn RoossinckMichael PalmerRick NelsonBruce RoeKay ScheetsAdam ZlotnickJonathan WrenJeanmarie Verchot-Lubicz

Kelly CravenAndrew DoustOlga BlinkovaTracy FeldmanByoung Eun MinNikki Charlton

Vijay MuthukumarVeenita GroverVaskar ThapaGraham WileyDevinka Bamunusinghe

Guoan ShenJudy ZhangYan SongShashank 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)

The Virus PlayersAsclepias 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