Transcript PVBE Tenet - Oklahoma State University–Stillwater

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 Roossinck
Michael Palmer
Rick Nelson
Bruce Roe
Kay Scheets
Adam Zlotnick
Jonathan Wren
Jeanmarie VerchotLubicz
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)
PVBE Overview
•Plants harbor complex microbial worlds.
•Metagenomics leads to
identification of novel viruses.
•Metagenomics provides insights into virusplant interactions in ecosystems.
Plant Microbial World: Overview
•Plant-Lichen-Fungus-Virus
•Plant-Plant-Fungus-Virus
•Plant-Fungi & Plant-Bacteria
Plant Microbial World: Lichens
5'
3'
5'
Phenol:Chloroform
5'
3'
Lichen ---> Total N.A. ---> dsRNA
Olga Blinkova
3'
5'
Plant Microbial World: dsRNA
5'
3'
3'
5'
dsRNA
Melt and anneal with RandomHexPrimer
5'
NNNNNN
NNNNNN
3'
NNNNNN
NNNNNN
3'
5'
RT Reaction
Add RT enzyme and
dNTPs
5'
3'
NNNNNN
NNNNNN
3'
5'
RNase treatment, remove primers
First PCR cycle
PCR amplification with tagged primer
5'TGGACCTTCGGATCCTCC
M. Roossinck
Guoan Shen
Plant Microbial World: Lichen Viruses
First report of evidence of lichen viruses!
Olga Blinkova
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
Viral
Nucleic
Acid (VNA)
Degenerate
Primers
PCR
products
1 kb+
Plant 295
Plant 248
Plant 162
Plant 289
Plant 296
Neg. control
Vijay Muthukumar
Mike Palmer, Vaskar Thapa
Also: Dan McGlinn, Fumiko
Shirakura and others
Judy Zhang
Tao (Colin) Ding Minh Ngo
Plant Microbial World: Complexity
Percent of Reads by "Organism"
100.0
Virus
Unclear
60.0
Plant
Fungus
Bacteria
40.0
Other
Animalia
20.0
vi
ja
y5
vi
ja
y5
7
vi
ja
y5
9
vi
ja
y2
vi
ja
y2
0
vi
ja
y2
1
vi
ja
y2
2
vi
ja
y2
3
vi
ja
y2
4
vi
ja
y2
7
vi
ja
y3
vi
ja
y3
5
vi
ja
y4
vi
ja
y4
5
vi
ja
y4
7
0.0
vi
ja
y1
vi
ja
y1
2
vi
ja
y1
3
vi
ja
y1
5
vi
ja
y1
7
vi
ja
y1
8
Percent of Total
80.0
Sample
Graham Wiley
Bruce Roe
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
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
Viral
Nucleic
Acid (VNA)
Clipped 30 cm x 30 cm
Olga Blinkova
Jennifer Hackett
Degenerate
Primers
Baptisia australis
Ceanothus americanus
Croton monanthogynus
Elymus virginicus
Lespedeza procumbens
Monarda fistulosa
Physalis pumila
Sporobolus compositus
PCR
products
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 Tymovirus
Asc. Asym Virus TGP1
Metagenomics: Tymovirus!
248-1
248-2
337
1000
896
578
294
30 nm Particles
307
351
Neighbor Joining Tree
Adam Zlotnick
Infectivity:
•Nicotiana benthamiana
•Chenopodium amaranthicolor
Byoung Eun Min, Rick Nelson
Metagenomics: Classification
05TGP00321
05TGP00321
9625564
9627246
905
18450268
946
27228718
Taro bacilliform virus
916
17. 89
TPP Badnavirus
15029533
589
18450258
986
882
27819377
62327614
0.1
05TGP00312
Metagenomics: Pyrosequencing
PCR amplification with tagged primer
5'TGGACCTTCGGATCCTCC
Graham Wiley
Bruce Roe
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: Problems
Reads
•Varying titers
Virus
Total
Vijay Muthukumar
•Distantly related viruses
•Unrelated viruses
•High titer
•Low titer
Adam Zlotnick
Ecosystems: Metagenomics Insights
•Strategies
•Frequent
•Novel
•Community
•Benign
•Specialist
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
Veenita Grover,
Soon also Teecie West
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
•“Novel” viruses by VLPVNA = 8
•“Novel” viruses by 454
and/or dsRNA = 11
•“Known” viruses by either
method = 0
•Supports prediction
Jonathan Wren
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 Roossinck
Michael Palmer
Rick Nelson
Bruce Roe
Kay Scheets
Adam Zlotnick
Jonathan Wren
Jeanmarie VerchotLubicz
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)
The Virus Players
Asclepias tymovirus
Oxalis tymovirus
Asclepias comovirus
Vitis oryzavirus
Ambrosia potexvirus
Vitis cypovirus
Ambrosia badnavirus
Viola cryptovirus
Ambrosia endornavirus
Viola partitivirus
Panicum unclass.
Closteroviridae
Baptisia totivirus
Carmovirus (Origin unk.)
Asplenium chrysovirus
Cladonia mitovirus
Cladonia chrysovirus
Alternaria endornavirus
Alternaria hypovirus
Stemphylium totivirus