Evolutionary Biology Evolutionary Biology of of Tospoviruses Tospoviruses in in Floral Crops Floral Crops J. W. Moyer J. W. Moyer North Carolina State North Carolina State University University Department of Plant Pathology Department of Plant Pathology NORTH CAROLINA STATE UNIVERSITY
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Evolutionary Biology of Tospoviruses in Floral Crops J. W. Moyer North Carolina State University Department of Plant Pathology NORTH CAROLINA STATE UNIVERSITY.
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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
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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
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Genomic OrganizationGenomic Organization
++
++
L
M
S
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Virus StructureVirus Structure
N 29K
L 330K
G2 58K
G1 78K
80 - 110 nm
L 330K
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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
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)
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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 ?
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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
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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
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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
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.
►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
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)
►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.
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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
► 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
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
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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
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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
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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).
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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.