Download The Incidence and Diversity of Plant Viruses

The Incidence and Diversity of
Plant Viruses in
the Tallgrass Prairie Preserve
-Vaskar Thapa, Ulrich Melcher, Daniel McGlinn, Marilyn Roossink,
Drew Porter, Rita Marvelli, Tracy Feldman, and Michael Palmer
Outline
1) What is PVBE?
2) Methods
• Field methods
• Laboratory methods
3) Results
• Incidence of plant viruses
• Diversity of plant viruses
4) Summary
Background: What is PVBE?
• Plant Virus Biodiversity and Ecology is
one of two scientific theme areas,
awarded for research by the National
Science Foundation (NSF)-Oklahoma
Experimental Program to stimulate
Competitive Research (EPSCoR).
• Funding provided 2005 to 2008.
Objective for PVBE
 To discover diversity and ecological functions of
plant viruses in natural systems.
Underlying hypothesis
The distribution and evolution of viruses are
determined by complex environmental interactions
among many factors including distributions of
hosts, vectors, other viruses and climate.
Working team
University of Tulsa joining soon
Specializing in
Ecology
Virology
Molecular biology
Genomics
Structural biology and
Bioinformatics.
Research site
The Nature Conservancy’s Tallgrass Prairie Preserve in
Osage County, Oklahoma
• Representation of intact native Tallgrass Prairie landscape
• 15,000 hectares
• more than 700 plant species
• 12 vegetation types
Palmer, M. W., P. Earls, and J. R. Arévalo. 2000. The vegetation of the Tallgrass Prairie Preserve (unpublished report).
Focus of this presentation
 Preliminary
results from plants collected since
May 2005
 Analysis of double-stranded (ds) RNA from the
plant collected
Methods
Field methods
Plants for voucher herbarium
Sample collection for dsRNA isolation
Plant samples for intensive study
Laboratory methods
ds RNA isolation
Field methods
Plant sample for voucher herbarium
• Sample from each species
• Collection irrespective of
symptoms
• Collection from sites with
abundance of target species
• Record of GPS location
• Habitat and individual plant photos
• Two repositories for herbarium –OSU and TGPP
Field methods ..
Sample collection for ds RNA isolation
• 10 grams of young leaves
• Transported to the laboratory in a container
with ice packs
• Stored in cold room at 4 ْ C before processing
for dsRNA isolation
Field methods ..
Plant samples for intensive study
• Six of the most frequent
plants in tallgrass prairie
vegetation
• Represent major taxonomic
groups
• Multiple samples from 20
random plots
Ambrosia psilostachya, Cuman ragweed
Asclepias viridis, green antelopehorn
Panicum virgatum, switchgrass
Sorghastrum nutans, Indiangrass
Ruellia humilis, fringeleaf wild petunia
Vernonia baldwini, baldwin's ironweed
Laboratory method for double-stranded RNA isolation
Mix vigorously
to form
emulsion
Young leaves (5 g)
Centrifuge
Grind in
liquid
Transfer into 50 ml tube
nitrogen
containing 10 ml
extraction buffer and 10
ml Ph:Ch
Transfer top phase into
new tube
Final aqueous
phase
Repeat Ph:Ch extraction
Decant and
resuspend in 0,1
mM EDTA / 0.3
M NaOAC
Centrifuge
to pellet
dsRNA
Wash in 6 time
with application
buffer
Precipitate with
NaOAc and
EtOH overnight
at -20ْ C
Transfer eluate to
a 15 ml tube
1 2 3 4 Lad 1 kb
Transfer to a
microcentrifuge tube and
fill with cold ethanol to
reprecipitate
Add absolute
proof ethanol
(16,5% of
aqueous
volume)
Add elution
buffer
Resuspend in 50 mkl 0.1 mM
EDTA
bp
12,2
2
1,6
Check the dsRNA by
electrophoresis on a
1.5% agarose gel in
0.5X TBE II
Pass through
enocolumn
containing
CF11powder
cellulose binding
dsRNA (if ethanol
concentration is
16,5%)
Vernonia baldwinii (line 1) and
Flavoparmelia sp. (line 4) have no dsRNA.
Total NA (for bar
coding and making
hybridization target)
Buffers
Extraction buffer: Application buffer:
0.1 M NaCl
0.1 M NaCl
50 mV Tris, pH 8
50 mM Tris, pH8
1 mV EDNA, pH 8
0.5 mV EDTA, pH8
1% SDS
16.5% Ethanol
Elution buffer:
0.1 M NaCl
50 mV EDTA, pH 8
1
506,5
396
344
298
Ambrosia psilostachya (line 2) and Parmelia
sp1. (line 3) show bands for dsRNA
Protocol for ds RNA isolation adopted from M. Roossinck, 2005
Results
• 635 specimen from 485
species, 307 genera and 91
families collected.
• 592 specimens analyzed for
48%
52%
ds RNA
• gels of 592 these specimens
• 308 of the are putatively
Positive for dsRNA
positive for dsRNA (i.e.
probable viruses)
Negative for dsRNA
sc
le
pi
ad
ac
ea
e
Cy
pe
ra
ce
ae
A
ca
nt
he
ce
ae
Fa
ba
ce
ae
pi
ac
ea
e
300
250
Po
ac
ea
e
A
ste
ra
ce
84
ae
ot
he
rf
am
ili
es
A
A
Number of specimen
Distribution within the top plant families
2 = 37.39, p = 0.00
Negative
Positive
200
150
100
50
0
Double-stranded RNA in native and exotic species
2 = 0.06, p = 0.8
120%
Percentage of specimen
100%
80%
Negative
60%
Positive
40%
20%
0%
Native
Exotic
Nativity
Distribution of dsRNA in different life forms
2 = 1.23, p = 0.87
120%
Percentage of specimen
100%
80%
Negative
60%
Positive
40%
20%
0%
Perennial
Annual
Tree
Life forms
Shurb
Vine
Distribution of dsRNA in different taxonomic groups
2 = 13.81,
p = 0.00
120%
Percentage of specimen
100%
80%
Negative
60%
Positive
40%
20%
0%
Dicot
Monocot
Fern
Distribution of dsRNA in six selected species
2 = 9.76,
p = 0.08
Ruellia humilis
Vernonia baldwinii
Asclepias viridis
Positive
Negative
Sorghastrum nutans
Panicum virgatum
Ambrosia psilostachya
0%
20%
40%
60%
80%
100%
120%
Viral Diversity
• Too early to comment on plant
virus diversity in TGPP
• Gel analysis shows wide
variation in banding patterns
• Different banding patterns within
and across species.
Caveats
• The results are preliminary, based on a limited
sample
• dsRNA is not unique for plant virus, it may be from
fungal or arthropod viruses
• Viruses of low titer may have been missed
• DNA viruses are not assessed.
• The reading of the gels has some subjectivity; this
will be resolved in the sequencing phase of PVBE
Conclusions
• 50% of plant samples contain dsRNA, indicating
viruses are widespread in nature.
• Viruses are frequent in all growth forms, life
histories, and taxonomic groups.
Acknowledgments
Following persons who help us in plant collection
Pete Earls
Fumiko Shirakura
M.Hara
Will Lowry
Shyam Thomas
Ray Moranz
Josh Lofton
Mari Carmen Cobo
Laxman Karki
Katie Lewis
Rest of all team members of PVBE