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Evolutionary Biology of Evolutionary Biology of TospovirusesTospoviruses in Floral in Floral
CropsCrops
J. W. MoyerJ. W. MoyerNorth Carolina State UniversityNorth Carolina State University
Department of Plant PathologyDepartment of Plant Pathology
NORTH CAROLINA STATE UNIVERSITY
TaxonomyTaxonomy
►TSWV is a member of the genus TSWV is a member of the genus TospovirusTospovirus of the family of the family BunyaviridaeBunyaviridae. Other genera of . Other genera of BunyaviridaeBunyaviridae:: BunyavirusBunyavirus PhlebovirusPhlebovirus HantavirusHantavirus NairovirusNairovirus
NORTH CAROLINA STATE UNIVERSITY
Members of the Genus Tospovirus
Serogroup Species Abbreviation Geographical Distribution Vectors
Tomato Spotted Wilt Virus TSWV (I) World-Wide, except for extreme
regions
Frankliniella bispinosa F. intonsa (FI)
F. fusca F. occidentalis (FO)
F. shultzei (FS) Thrips setosus T. tabaci (?)
Groundnut Ringspot Virus GRSV (II) South Africa, Brazil, Argentine FO, FS
Tomato spotted wilt
Tomato Chlorotic Spot Virus TCSV (II) Argentine, Brazil FI, FO, FS Zucchini Lethal Chlorotic
Virus ZLCV (IX) Brazil ?
Chrysanthemum Stem
Necrosis Virus CSNV (VIII) Brazil, the Netherlands FO, FS
Watermelon Silver Mottle Virus
WSMV (IV) Japan, Taiwan Thrips palmi (TP), FS
Watermelon Bud Necrosis Virus
WBNV (IV) India TP Watermelon silver
mottle Groundnut (Peanut) Bud
Necrosis Virus GBNV,
PBNV (IV) India FO
Impatiens Necrotic Spot Virus
INSV (III) USA, the Netherlands, Italy,
France, Portugal FO
Iris Yellow Spot Virus IYSV (VI) Brazil, Israel, the Netherlands,
USA T. tabaci
Peanut Chlorotic Fan-spot Virus
PCFV (X) Taiwan TP, Scirtothrips dorsalis (SD)
Peanut Yellow Spot Virus PYSV (V) India, Thailand TP, SD
Physalis Severe Mottle virus PSMV (VII) Thailand T. tabaci (?)
Potato Stem Necrosis Virus PSNV (IV) India TP (?), T. flavus
Ungrouped
Melon Yellow Spot virus MYSV Japan TP
NORTH CAROLINA STATE UNIVERSITY
Genomic OrganizationGenomic Organization
++
++
L
M
S
NORTH CAROLINA STATE UNIVERSITY
Virus StructureVirus Structure
N 29K
L 330K
G2 58K
G1 78K
80 - 110 nm
L 330K
NORTH CAROLINA STATE UNIVERSITY
NORTH CAROLINA STATE UNIVERSITY
Host Range Host Range
►Wide host range: exceeds 900 plant Wide host range: exceeds 900 plant species, spanning both species, spanning both monocotyledonous and dicotyledonous monocotyledonous and dicotyledonous plant species plant species
►Tremendous economic importanceTremendous economic importance
NORTH CAROLINA STATE UNIVERSITY
NORTH CAROLINA STATE UNIVERSITY
FloralFloral Crop MovementCrop Movement
Genetics and evolutionGenetics and evolution► Heterogeneity and rapid adaptability are Heterogeneity and rapid adaptability are
two prominent phenotypic characteristics two prominent phenotypic characteristics that distinguish TSWV from many other that distinguish TSWV from many other plant virusesplant viruses
Existence of five strains of TSWV, separated from naturally Existence of five strains of TSWV, separated from naturally occurring complexes (Norris, 1946)occurring complexes (Norris, 1946)
Six strains named as A, B, C1, C2, D, and E separated from Six strains named as A, B, C1, C2, D, and E separated from tomato plants (Best & Gallus, 1953)tomato plants (Best & Gallus, 1953)
First attempt to explain the “cross protection effect” with a First attempt to explain the “cross protection effect” with a
new theory which implied transfer of character new theory which implied transfer of character determinants from one virus particle to another (per se determinants from one virus particle to another (per se recombination)recombination)
NORTH CAROLINA STATE UNIVERSITY
Genetics and EvolutionGenetics and Evolution► Mechanisms of evolution:Mechanisms of evolution:
Segment reassortment (Qiu, W.P., et al.)Segment reassortment (Qiu, W.P., et al.) MutationMutation Defective interfering particles (DIs) Defective interfering particles (DIs)
(Resende, R.)(Resende, R.) Recombination ?Recombination ?
► Determinants of genetic structure:Determinants of genetic structure: Genetic drift ?Genetic drift ? Selection ?Selection ?
NORTH CAROLINA STATE UNIVERSITY
General ObjectivesGeneral Objectives
► Identify collaborators in the floral Identify collaborators in the floral crop industry from diverse crop industry from diverse geographic regions, from which geographic regions, from which representative samples of TSWV and representative samples of TSWV and INSV populations can be obtained for INSV populations can be obtained for characterization characterization
► Characterize the diversity at the Characterize the diversity at the biological and molecular levels of biological and molecular levels of representative populationsrepresentative populations
NORTH CAROLINA STATE UNIVERSITY
General ObjectivesGeneral Objectives
►Determine the changes in the natural Determine the changes in the natural populations due to changes in host populations due to changes in host and vectorsand vectors
►Develop models of Develop models of TospovirusTospovirus populations that can be used to populations that can be used to identify the source of any given identify the source of any given population: Attributionpopulation: Attribution
NORTH CAROLINA STATE UNIVERSITY
AttributionAttribution
►The ability to target the source as The ability to target the source as well as to identify the causal well as to identify the causal pathogenpathogen
NORTH CAROLINA STATE UNIVERSITY
Objective 1Objective 1
►Developed a network of collaborators: Developed a network of collaborators: Argentina, Brazil, Colombia, Germany, Argentina, Brazil, Colombia, Germany, Italy, Japan, Uruguay, and the USA Italy, Japan, Uruguay, and the USA including the industry trough AGDIAincluding the industry trough AGDIA
NORTH CAROLINA STATE UNIVERSITY
Objective 2Objective 2► Technical steps toward analyses:Technical steps toward analyses:
Improved RNA extraction methods for floral hosts.Improved RNA extraction methods for floral hosts. PCR optimization for AU-rich regions of the genome (intergenic PCR optimization for AU-rich regions of the genome (intergenic
regions).regions). Evaluation of primers for amplification and sequencing of the Evaluation of primers for amplification and sequencing of the
whole genome; S RNA (7), M RNA (12) and L RNA (18). Number of whole genome; S RNA (7), M RNA (12) and L RNA (18). Number of primer pairs in parentheses.primer pairs in parentheses.
Assembly and edition of nucleotide sequences with Vector NTi.Assembly and edition of nucleotide sequences with Vector NTi. Alignments of nucleotide sequences with CLUSTAL X.Alignments of nucleotide sequences with CLUSTAL X. Edition of alignments with Genedoc 2.6.002Edition of alignments with Genedoc 2.6.002 Construction of haplotype maps with Paup, v 4.0 beta 4.Construction of haplotype maps with Paup, v 4.0 beta 4. Construction of Neighbor-joining phylograms with MEGA 2.1.Construction of Neighbor-joining phylograms with MEGA 2.1. Test of genetic differentiation between subpopulations with Test of genetic differentiation between subpopulations with
Permtest.Permtest. Estimation of population genetics parameters, codon bias and Estimation of population genetics parameters, codon bias and
examination of sequence divergence between species with examination of sequence divergence between species with DnaSP, v 3.51.DnaSP, v 3.51.
Test of recombination with LDhat.Test of recombination with LDhat. Identification of selection pressures per codon with the CODEML Identification of selection pressures per codon with the CODEML
program of the PAML package, v 3.0d.program of the PAML package, v 3.0d.NORTH CAROLINA STATE UNIVERSITY
Diversity Between Diversity Between PopulationsPopulations
►S RNA:S RNA: Direct sequencing of 13 isolates and Direct sequencing of 13 isolates and
subsequent analysis with data from the lab and subsequent analysis with data from the lab and GenBank. GenBank.
►NSs : 21 sequencesNSs : 21 sequences►N : 41 sequencesN : 41 sequences
►M RNA M RNA Direct sequencing of 13 isolates and subsequent Direct sequencing of 13 isolates and subsequent
analysis with data from the lab and GenBankanalysis with data from the lab and GenBank..►NSm: 17 sequencesNSm: 17 sequences►G1/G2: 19 sequencesG1/G2: 19 sequences
NORTH CAROLINA STATE UNIVERSITY
S RNAS RNA RANUNCULUS
ASTER
T0208101
CHRYS1
T0200801
T0200802
T0200803
AF020659
PEANUT
T11B
SP2
SP3
D13926
AJ418778
AJ418777
AJ418779
AJ418780
AJ418781
AF020660
T0208601
DAHLIA
53
66
65
88
96
55
95
100
99
100
51
63
96
59
78
100
51
CACA
NCNC
SPSP
BUBU
NCNC
NSsNSs NN
GAGA
JAPANJAPAN
AF020659
AFO64470
AF064471
AFO64472
AF048716
AFO64474
AF048715
AFO64473
AFO48714
AFO64469
PEANUT
T0200801
ASTER
RANUNCULUS
T0208101
T0200803
T0200802
CHRYS1
X61799
T11B
SP2
X94550
SP3
Z36882
D00645
ABO38342
ABO38341
AB010997
AF020660
DAHLIA
T0208601
AJ297611
AJ418779
AJ296602
AJ296601
D13926
AJ418778
AJ297609
AJ297610
AJ296598
AJ296600
98
98
83
74
68
57
76
85
67
63
95
78
57
54
88
81
65
95
67
54 87
58
NORTH CAROLINA STATE UNIVERSITY
M RNAM RNANSmNSm G1/G2G1/G2
AF208498
T0200801
T0200803
T0200802
CHRYS1
0208101
AB010996
15001
15103
15702
14802
14903
SP3
SP2
X93603
AF208497
T020860198
100
97
97
96
64
73
61
97
88
97
15804
15702
14802
15107
14903
15001
15103
AF208498
AB010996
CHRYS1
t0208101
t0200801
t0200802
t0200803
SP3
SP2
002050
AF208497
t0208601100
100
100
84
100
98
100
100
100
9565
55
72
NORTH CAROLINA STATE UNIVERSITY
Geographic SubdivisionGeographic Subdivision
RegionRegion Subpop.Subpop. KstKst KsKs P-valueP-value
NSsNSs CA vs Bul.CA vs Bul. 0.688890.68889 0.0103750.010375 0.0*0.0*
NN Bul. vs GABul. vs GA 0.5076460.507646 0.0116680.011668 0.0*0.0*
Bul. vs CABul. vs CA 0.5652850.565285 0.007990.00799 0.0*0.0*
CA vs GACA vs GA 0.4567760.456776 0.0067810.006781 0.0*0.0*
NSmNSm CA vs NCCA vs NC 0.1907890.190789 0.0178910.017891 0.006*0.006*
G1/G2G1/G2 CA vs NCCA vs NC 0.2653680.265368 0.0185820.018582 0.001*0.001*NORTH CAROLINA STATE UNIVERSITY
Intraspecific polymorphismIntraspecific polymorphism►High intraspecific polymorphism
Estimation of the population parameters Pi(Sil), and (Sil), revealed a high polymorphism for each coding region, in agreement with the high mutation rates of RNA viruses.
RegionRegion Pi (Tot)Pi (Tot) Pi (Sil)Pi (Sil) (Tot) (Sil)
NSsNSs 0.034050.03405 0.09060.0906 0.03730.0373 0.090690.09069
NN 0.023520.02352 0.078850.07885 0.042710.04271 0.131880.13188
NSmNSm 0.030320.03032 0.112720.11272 0.036120.03612 0.130850.13085
G1/G2G1/G2 0.034990.03499 0.118530.11853 0.046260.04626 0.144860.14486NORTH CAROLINA STATE UNIVERSITY
Population ExpansionsPopulation Expansions→→Geographic subdivisionGeographic subdivision
NSs Pi(Tot) Pi(Sil) ∂(Tot) ∂(Sil) TajimasD
Fu & LiD
Fu & LiF
California 0.006 0.016 0.0064 0.0193 -0.4955 -0.7047 -0.725
Bulgaria 0.0166 0.034 0.0185 0.0404 -0.532 -0.734 -0.808
N Pi(Tot) Pi(Sil) ∂(Tot) ∂(Sil) TajimasD
Fu & LiD
Fu & LiF
California 0.0022 0.0085 0.0032 0.0121 -1.524* -1.61* -1.73*
Bulgaria 0.0131 0.029 0.0189 0.0411 -1.645* -1.779* -1.95*
Georgia 0.0102 0.03939 0.0128 0.0478 -1.0002 -1.116 -1.22
NORTH CAROLINA STATE UNIVERSITY
Test for recombinationTest for recombination
►We used LDhat, which implements a We used LDhat, which implements a coalescent-based method to detect coalescent-based method to detect and estimate recombination from gene and estimate recombination from gene sequences sequences
►The null hypothesis (no recombination) The null hypothesis (no recombination) is rejected if the proportion (Pis rejected if the proportion (PLPTLPT) is ) is less than a significance level (<0.05)less than a significance level (<0.05)
►NO EVIDENCE FOR RECOMBINATION NO EVIDENCE FOR RECOMBINATION FOR OUR DATA. FOR OUR DATA.
NORTH CAROLINA STATE UNIVERSITY
Interspecific DivergenceInterspecific Divergence
► Neutral theory predicts that the ratio of Neutral theory predicts that the ratio of silent to replacement substitutions should be silent to replacement substitutions should be the same for polymorphisms within species the same for polymorphisms within species and fixed differences between species.and fixed differences between species.
RegionRegion FSFS FRFR PSPS PR PR G valueG value
NSsNSs 221221 266266 101101 6363 0.000320.00032**
NN 126126 165165 9494 4040 0.0*0.0*
NSmNSm 123123 132132 8888 2323 0.0*0.0*
G1/G2G1/G2 489489 500500 377377 130130 0.0*0.0*NORTH CAROLINA STATE UNIVERSITY
SelectionSelection
RegionRegion M2 vs M2 vs M3M3
M7 vs M7 vs M8M8
Pos. sitesPos. sites dN/dSdN/dS
NSsNSs 14.663214.6632**
15.22815.228****** 458, 460458, 460 0.3670.36711
NN 8.092*8.092* 11.586**11.586** 10,174,210,174,25555
0.2200.22077
NSmNSm 0.0020.002 0.5980.598 -------------------------- 0.0810.08111
G1/G2G1/G2 1.151.15 6.364*6.364* 530530 0.1320.13266
NORTH CAROLINA STATE UNIVERSITY
ConclusionsConclusions
► TSWV has a strong spatial structure, TSWV has a strong spatial structure, attributed to founder effects:attributed to founder effects: Significant genetic differentiation between Significant genetic differentiation between
subpopulationssubpopulations Decrease of genetic variation within Decrease of genetic variation within
subpopulations and subpopulations and Emergence of singletons in most of the analyzed Emergence of singletons in most of the analyzed
loci loci
► High intraspecific polymorphismHigh intraspecific polymorphism► No evidence for recombination for the data No evidence for recombination for the data
analyzedanalyzedNORTH CAROLINA STATE UNIVERSITY
Objective 2-FutureObjective 2-Future►Define the structure of a single TSWV Define the structure of a single TSWV
population (individual isolate): diversity population (individual isolate): diversity within a population.within a population. Virus sources: plants from field and Virus sources: plants from field and
greenhouse operations from:greenhouse operations from:►Europe (Spain, Italy, Greece)Europe (Spain, Italy, Greece)►South AfricaSouth Africa►JapanJapan►U.S.A.U.S.A.
NORTH CAROLINA STATE UNIVERSITY
Source
Total RNA
cDNA
PCR or / nested PCR
Cloning
RFLP
Sequencing
PCR of individualclones with inserts ( GC clamp in 5’ end of forward primer)
DGGE
Heteroduplex analysis
Heteroduples analysis-SSCP
Denaturation
Assymmetric PCR
PCR with one primer
SSCP
Objective 2- Future
NORTH CAROLINA STATE UNIVERSITY
Objective 3Objective 3
►After defining the complexity of the After defining the complexity of the structure certain selection pressures structure certain selection pressures will be imposed:will be imposed: Mechanical passages Mechanical passages Vector speciesVector species Host differentialsHost differentials
NORTH CAROLINA STATE UNIVERSITY
Objective 3Objective 3► Found middle (M) RNA of TSWV is responsible for thrips Found middle (M) RNA of TSWV is responsible for thrips
transmission by a novel viral genetic systemtransmission by a novel viral genetic system
► Analyzed M RNA sequences of non-transmissible and Analyzed M RNA sequences of non-transmissible and transmissible isolatestransmissible isolates
► Found a nucleotide deletion in glycoprotein coding region Found a nucleotide deletion in glycoprotein coding region of non-transmissible isolates and confirmed glycoprotein of non-transmissible isolates and confirmed glycoprotein may play an important role in thrips transmissionmay play an important role in thrips transmission
► Determined mutation frequencies of TSWV isolates Determined mutation frequencies of TSWV isolates
maintained by repeated mechanical or thrips transmissionmaintained by repeated mechanical or thrips transmission
► Found specificity of vectors may map with M RNA of TSWVFound specificity of vectors may map with M RNA of TSWV
NORTH CAROLINA STATE UNIVERSITY
Transmission Efficiency
010203040
50607080
0 5 10 15 20
Mechanical Passage
Tra
nsm
issi
on
rat
e (%
)
Mechanical
Progression of thrips transmission rate of TSWV per mechanical passage (Mc Nulty, 2001; unpublished data).
NORTH CAROLINA STATE UNIVERSITY
Isolation of non-transmissible isolates from population A
Pop. A
(mechanically-maintained, low transmissibility)
T+
T-
T-
T-
T-T-
T-T-
T-
T-
T-
T-
T-
T-
T-
T+
Single Lesion Isolation Transmission AssayNORTH CAROLINA STATE UNIVERSITY
NORTH CAROLINA STATE UNIVERSITY
Results
Isolation and identification of thrips non-transmissible and transmissible isolates from mechanically maintained RG2.
Single lesion Single lesion isolatesisolates Transmission (infected/inoculated)Transmission (infected/inoculated)11 TransmissibilityTransmissibility
22 0/30 ; 0/300/30 ; 0/30 --22
1111 0/30 ; 0/300/30 ; 0/30 --
2828 0/30 ; 0/300/30 ; 0/30 --
3737 9/30 ; 8/309/30 ; 8/30 ++
4141 7/30 ; 6/307/30 ; 6/30 ++
4343 6/30 ; 7/306/30 ; 7/30 ++
8181 0/30 ; 0/300/30 ; 0/30 --
1. Transmission was confirmed by symptom development and RT-PCR-RFLP. Individual experiments are separated by semicolon.2. + means transmissible and – means non-transmissible.
Objective 4Objective 4
► Extend the Attribution studies to include Extend the Attribution studies to include INSV.INSV. Other tospoviruses?Other tospoviruses? Other viruses?Other viruses?
► Study the impact of host range and vector Study the impact of host range and vector species in individual populations.species in individual populations. Molecular and biological dissections of Molecular and biological dissections of
individual populations. individual populations. Partnership with AGDIA for industrial application.Partnership with AGDIA for industrial application.
NORTH CAROLINA STATE UNIVERSITY
James Moyer Jorge Abad
Jan SpeckS.-H. SinStephen New
M.Purugganan
Amy Lawton-Rauh
Sebastian GuyaderSebastian Guyader
William AtchleyMichael J. Buck
AcknowledgementsAcknowledgements
NORTH CAROLINA STATE UNIVERSITY