Plant Virus Biodiversity and Ecology (PVBE)

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Plant Virus Biodiversity and Ecology (PVBE)
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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)
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PVBE Overview

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

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Plant Microbial World Overview

• Plant-Lichen-Fungus-Virus
• Plant-Plant-Fungus-Virus
• Plant-Fungi Plant-Bacteria

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Plant Microbial World Lichens
5'
3'
5'
PhenolChloroform
5' 3'
3' 5'
Lichen ---gt Total N.A. ---gt dsRNA
Olga Blinkova
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Plant Microbial World dsRNA
5' 3'
3' 5'
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'TGGACCTTCGG
ATCCTCC
M. Roossinck
Guoan Shen
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Plant Microbial World Lichen Viruses
First report of evidence of lichen viruses!
Olga Blinkova
Yan Song
http//bioinfosu.okstate.edu/pvbe/pvlist.php
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Plant Microbial World Dodder

• Parasitic plant
• Fungi isolated from host and parasite
  Alternaria, Stemphylium
• dsRNA approach used on cultures of these fungi

Tracy Feldman
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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
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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
Minh Ngo
Tao (Colin) Ding
Mike Palmer, Vaskar Thapa Also Dan McGlinn,
Fumiko Shirakura and others
Judy Zhang
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Plant Microbial World Complexity
Graham Wiley
Bruce Roe
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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
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Plant Microbial World Conclusion
Russian Doll Model of Life
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Metagenomics Overview

• Approaches
• Lawn mower
• Asclepias asymptomatic virus 1
• Other virus signatures identified
• Challenges
• Titer
• Distant relationship
• Unrelated to known groups

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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
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Metagenomics Carmovirus
Known Carmovirus TGP Carmovirus sequences

• Which plant? Which plants?
• Single plant species or multiple?
• One segment / one subsample statistically
  non-similar

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Metagenomics Tymovirus?
Known Tymovirus Asc. Asym Virus TGP1
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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

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Metagenomics Classification
05TGP00321
05TGP00321
05TGP00312
Taro bacilliform virus
TPP Badnavirus
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Metagenomics Pyrosequencing
GGAGGATCCGAAGG3' CCTCCTAGGCTTCC5'
5'CCTTCGGATCCTCC 3'GGAAGCCTAGGAGG
PCR amplification with tagged primer 5'TGGACCTTCGG
ATCCTCC
Graham Wiley
Bruce Roe
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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
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Metagenomics Problems
Reads Virus Total

• Varying titers

Vijay Muthukumar

• Distantly related viruses
• Unrelated viruses
• High titer
• Low titer

Adam Zlotnick
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Ecosystems Metagenomics Insights

• Strategies
• Frequent
• Novel
• Community
• Benign
• Specialist

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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
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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
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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
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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
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Ecosystems Viral Complexity
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Ecosystems Virus Specialists?
Generalist
Specialist
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Ecosystems Viruses Benign
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Ecosystems Metagenomics Insights

• Strategies
• Frequent
• Novel
• Community
• Specialist
• Benign

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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!

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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)
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The Virus Players
Asclepias tymovirus Asclepias comovirus Ambrosia
potexvirus Ambrosia badnavirus Ambrosia
endornavirus Panicum unclass. Closteroviridae Car
movirus (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