evolutionary biology of tospoviruses in floral crops j. w. moyer north carolina state university...

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


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


Genomic OrganizationGenomic Organization

++

++

L

M

S


Virus StructureVirus Structure

N 29K

L 330K

G2 58K

G1 78K

80 - 110 nm

L 330K


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



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)


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 ?


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


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


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


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


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


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


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


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


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.


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


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.


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



►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


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


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).


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


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.


► 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.


James Moyer Jorge Abad

Jan SpeckS.-H. SinStephen New

M.Purugganan

Amy Lawton-Rauh

Sebastian GuyaderSebastian Guyader

William AtchleyMichael J. Buck

AcknowledgementsAcknowledgements


evolutionary biology of tospoviruses in floral crops j. w. moyer north carolina state university...

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