A generalist tymovirus of the tallgrass prairie

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A generalist tymovirus of the tallgrass prairie

• Michael Palmer1
• Andrew Doust1
• Rick Nelson2
• Min Byoung2
• Vaskar Thapa1
• Marilyn Roossinck2
• Ulrich Melcher1
• 1Oklahoma State University
• 2Samuel Roberts Noble Foundation

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Plant Virus Biodiversity and Ecology

• PVBE
• Methods
• Preliminary Results
• Confusions

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Disclaimer I am not a virologist
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Plant Virus Biodiversity and Ecology (PVBE)

• An attempt to document the presence of viruses in
  plants of the Nature Conservancys Tallgrass
  Prairie Preserve, and to generate testable
  hypotheses

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PVBE People
Virologists, ecologists, geneticists,
bioinformaticians, computer scientists
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Known viruses species are mostly from cultivated
plants
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Plants of the Tallgrass Prairie Preserve
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Sandstone outcrops
crosstimbers
Sandstone
Tallgrass Prairie
savannas
Acidity, sand
Limestone or bottomland
mesic forest
Shortgrass prairie / limestone outcrops
Forest cover
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PVBE schema
Extract nucleic acids and sequence
Mine data and interpret
Propose and test ecological hypotheses
Sample plants
Current status
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• Plants are sampled without regard for symptoms

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PVBE schema
Extract nucleic acids and sequence
Mine data and interpret
Propose and test ecological hypotheses
Broad survey
target plant species Ambrosia
psilostachya Asclepias viridis Panicum
virgatum Sorghastrum nutans Vernonia
baldwinii Ruellia humilis
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Selected target species
Ambrosia psilostachya, western ragweed
Panicum virgatum, switchgrass
Asclepias viridis, antelope-horn milkweed
Sorghastrum nutans, Indiangrass
Ruellia humilis, wild/prairie petunia
Vernonia baldwini, western ironweed
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Vegetation monitoring plots
10m
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PVBE schema
Extract nucleic acids and sequence
Mine data and interpret
Propose and test ecological hypotheses
Virus-like particle isolation
Broad survey
target plant species Ambrosia
psilostachya Asclepias viridis Panicum
virgatum Sorghastrum nutans Vernonia
baldwinii Ruellia humilis
dsRNA isolation with 454 pyrosequencing
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PVBE schema
Extract nucleic acids and sequence
Mine data and interpret
Propose and test ecological hypotheses
Virus-like particle isolation
Broad survey
target plant species Ambrosia
psilostachya Asclepias viridis Panicum
virgatum Sorghastrum nutans Vernonia
baldwinii Ruellia humilis
dsRNA isolation with 454 pyrosequencing
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Laboratory method for double-stranded RNA
purification
Mix vigorously to form emulsion
Young leaves (5 g)
Centrifuge
Grind in liquid nitrogen
Transfer top phase into new tube
Transfer into 50 ml tube containing 10 ml
extraction buffer and 10 ml PhCh
Final aqueous phase
Repeat PhCh extraction
Centrifuge to pellet dsRNA
Add absolute proof ethanol (16,5 of aqueous
volume)
Add elution buffer
Decant and resuspend in 0,1 mM EDTA / 0.3 M NaOAC
Wash in 6 time with application buffer
Pass through enocolumn containing CF11powder
cellulose binding dsRNA (if ethanol concentration
is 16,5)
Total NA (for bar coding and making hybridization
target)
Precipitate with NaOAc and EtOH overnight at -20?
C
Transfer eluate to a 15 ml tube
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2
3
4
Lad 1 kb
Buffers
Transfer to a microcentrifuge tube and fill with
cold ethanol to reprecipitate
Application buffer 0.1 M NaCl 50 mM Tris,
pH8 0.5 mV EDTA, pH8 16.5 Ethanol
Extraction buffer 0.1 M NaCl 50 mV Tris, pH 8 1
mV EDNA, pH 8 1 SDS
Resuspend in 50 mkl 0.1 mM EDTA
bp
Elution buffer 0.1 M NaCl 50 mV EDTA, pH 8
Vernonia baldwinii (line 1) and Flavoparmelia
sp. (line 4) have no dsRNA. Ambrosia
psilostachya (line 2) and Parmelia sp1. (line 3)
show bands for dsRNA
12,2
2
1,6
Check the dsRNA by electrophoresis on a 1.5
agarose gel in 0.5X TBE II
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506,5
396
344
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Protocol for ds RNA isolation adopted from M.
Roossinck, 2005
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454 sequencing
Sophisticated, high-throughput system but
easier said than done
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Preliminary (2006) Results

• 592 specimens analyzed
• 296 plant species in 307 genera and 91 families
• 308 specimens are positive for dsRNA (i.e.
  probable viruses)

2007-2008 Results

• not yet tabulated on project database
• high rate of novelties detected by main methods
• many fungal viruses

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• BLAST results
• One virus is predominantly found in Asclepias
  viridis that appears that is distinct from, but
  has high similarity to,
• Kennedya yellow mosaic tymovirus

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From ICTVdB
Tymovirus

• Icosahedral virion
• ssRNA
• Genome 6000-7000nt
• Infects dicots
• Symptoms none to severe appear in leaves
• Transmitted by beetles

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Asclepias viridis
Other species
Virus-like particle
dsRNA (454)
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Tymovirus in Asclepias viridis
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Asclepias viridis intensely sampled from 3 plots,
(and sequenced from selections of two)
Plot 208 (Swale)
2005, 2006, 2007
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(-, -,)
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Distance (m)
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0
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-15
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70m
-5
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Probability of equal infection in each transect
lt0.01
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Plot 307 (Sand creek)
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-15
(,,)
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Distance (m)
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Distance (m)
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25m
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60m
-25
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50m
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40m
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-5
Plot 343 (Stadia)
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(,-,)
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-10
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-15
Distance (m)
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30m
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Distance (m)
2
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Red- Tymovirus infection Black no Tymovirus
0
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-35
-25
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-5
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buffer
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-5
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buffer
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10m
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-15
Distance (m)
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Presence of tymovirus in Asclepias viridis over
time and space
Plot 2005 2006 2007
208 Negative Negative Positive (36,18/50)
307 Positive Positive Positive (66, 33/50)
343 Positive Negative Positive (82, 41/51)
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Tymovirus presence in A. viridis tubers in plot
208 (RT-PCR)
Plot 208 (Swale)
(Plant/ tuber)
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48
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10
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Distance (m)
15
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0
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buffer
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-15
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-5
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-5
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buffer
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-10
Distance (m)
-15
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Most viruses have relatively low proportion of
reads in VLPs
Exception a new tymovirus, but only in Asclepias
viridis.
Virus Genus Plant species plant specimens Reads/ specimen Read
Ascovirus Artemisia ludoviciana 1 3 2.0
Badnavirus Ambrosia psilostachya 2 10 1.3
  Sorghastrum nutans 1 3 1.0
Chrysovirus Asplenium rhizophyllum 1 27 11.9
Comovirus Asclepias viridis 1 4 0.7
Flexivirus Ambrosia psilostachya 2 97 25.4
Metapneumovirus Desmanthus illinoensis 1 3 1.5
Coccolithovirus Festuca subverticillata 1 2 0.8
  Sorghastrum nutans 1 2 0.8
Ranavirus Panicum virgatum 1 2 0.7
lambda-like phage Ambrosia psilostachya 1 2 0.4
Tymovirus Amorpha fruticosa 1 2 0.7
  Asclepias viridis 10 315 85.7
  Asplenium rhizophyllum 1 33 14.6
  Cephalanthus occidentalis 1 4 1.1
  Desmanthus illinoensis 1 7 3.5
Interpretation high titers
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Tymovirus reads from Virus-like Particles
(positives only)
Few reads mean that the genome is poorly sampled
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RT-PCR from capsid
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Asclepias only
Interspecific comparison
Capsid comparison
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Analysis

• Principal Components Analysis (PCA), with each
  position represented by four dummy variables (the
  four nucleotides).
• AACAAGGACTACGGAGAACCCGTGGAA
• The realized dimensionality is much less than
  sequence length x 4, because most positions are
  not variable.

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Common to all PCA results

• 50 of variation can be explained by the first
  four axes
• Axes higher than 2 do not add anything to the
  interpretation

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Tymovirus from Asclepias viridis only
PCA Axis 2
PCA Axis 1
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Capsid comparison
Location effects still evident
Plot 307 group
Plot 343 group
PCA Axis 2
PCA Axis 1
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Interspecific comparisons
Some Non-Asclepias might be outliers, but this
might be a methodological artifact, and the
variation within Asclepias is huge relative to
interspecific variation.
PCA Axis 2
PCA Axis 1
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• There are relatively few well-supported clades
• Non-Asclepias samples are all nested within
  well-supported clades of Asclepias samples
• Does this imply other non-Asclepias hosts are
  sinks?

Outgroup
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• Confusions
• Why does tymovirus reach high level only in
  Asclepias?
• Are non-Asclepias sink populations? (note
  there are several orders of magnitude more
  non-Asclepias than Asclepias in the TGPP.)
• Are there two different strains?
• How does transmission within and between species
  occur? (milkweed beetles are specialists)
• Are the sequence differences phylogenetic?
• Are there relevant structural differences in the
  RNA?
• Is the relationship parasitic, commensal, or
  mutualistic?

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