Evolutionary biology of tospoviruses in floral crops
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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 Taxonomy TSWV is a member of the genus Tospovirus of the family Bunyaviridae . Other genera of Bunyaviridae : Bunyavirus

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Evolutionary biology of tospoviruses in floral crops l.jpg

Evolutionary Biology of Tospoviruses in Floral Crops

J. W. Moyer

North Carolina State University

Department of Plant Pathology

NORTH CAROLINA STATE UNIVERSITY


Taxonomy l.jpg
Taxonomy

  • TSWV is a member of the genus Tospovirus of the family Bunyaviridae. Other genera of Bunyaviridae:

    • Bunyavirus

    • Phlebovirus

    • Hantavirus

    • Nairovirus

NORTH CAROLINA STATE UNIVERSITY


Slide3 l.jpg

Members of the Genus Tospovirus

NORTH CAROLINA STATE UNIVERSITY


Genomic organization l.jpg

NSs

N

RdRp

L

+

NSm

G1/G2

M

+

S

Genomic Organization

NORTH CAROLINA STATE UNIVERSITY


Virus structure l.jpg

G2 58K

N 29K

G1 78K

L 330K

L 330K

80 - 110 nm

Virus Structure

NORTH CAROLINA STATE UNIVERSITY



Host range l.jpg
Host Range

  • Wide host range: exceeds 900 plant species, spanning both monocotyledonous and dicotyledonous plant species

  • Tremendous economic importance

NORTH CAROLINA STATE UNIVERSITY


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FloralCrop Movement

NORTH CAROLINA STATE UNIVERSITY


Genetics and evolution l.jpg
Genetics and evolution

  • Heterogeneity and rapid adaptability are two prominent phenotypic characteristics that distinguish TSWV from many other plant viruses

    • Existence of five strains of TSWV, separated from naturally occurring complexes (Norris, 1946)

    • Six strains named as A, B, C1, C2, D, and E separated from tomato plants (Best & Gallus, 1953)

    • First attempt to explain the “cross protection effect” with a new theory which implied transfer of character determinants from one virus particle to another (per se recombination)

NORTH CAROLINA STATE UNIVERSITY


Genetics and evolution10 l.jpg
Genetics and Evolution

  • Mechanisms of evolution:

    • Segment reassortment (Qiu, W.P., et al.)

    • Mutation

    • Defective interfering particles (DIs) (Resende, R.)

    • Recombination ?

  • Determinants of genetic structure:

    • Genetic drift ?

    • Selection ?

NORTH CAROLINA STATE UNIVERSITY


General objectives l.jpg
General Objectives

  • Identify collaborators in the floral crop industry from diverse geographic regions, from which representative samples of TSWV and INSV populations can be obtained for characterization

  • Characterize the diversity at the biological and molecular levels of representative populations

NORTH CAROLINA STATE UNIVERSITY


General objectives12 l.jpg
General Objectives

  • Determine the changes in the natural populations due to changes in host and vectors

  • Develop models of Tospovirus populations that can be used to identify the source of any given population: Attribution

NORTH CAROLINA STATE UNIVERSITY


Attribution l.jpg
Attribution

  • The ability to target the source as well as to identify the causal pathogen

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Objective 1 l.jpg
Objective 1

  • Developed a network of collaborators: Argentina, Brazil, Colombia, Germany, Italy, Japan, Uruguay, and the USA including the industry trough AGDIA

NORTH CAROLINA STATE UNIVERSITY


Objective 2 l.jpg
Objective 2

  • Technical steps toward analyses:

    • Improved RNA extraction methods for floral hosts.

    • PCR optimization for AU-rich regions of the genome (intergenic regions).

    • Evaluation of primers for amplification and sequencing of the whole genome; S RNA (7), M RNA (12) and L RNA (18). Number of primer pairs in parentheses.

    • Assembly and edition of nucleotide sequences with Vector NTi.

    • Alignments of nucleotide sequences with CLUSTAL X.

    • Edition of alignments with Genedoc 2.6.002

    • Construction of haplotype maps with Paup, v 4.0 beta 4.

    • Construction of Neighbor-joining phylograms with MEGA 2.1.

    • Test of genetic differentiation between subpopulations with Permtest.

    • Estimation of population genetics parameters, codon bias and examination of sequence divergence between species with DnaSP, v 3.51.

    • Test of recombination with LDhat.

    • Identification of selection pressures per codon with the CODEML program of the PAML package, v 3.0d.

NORTH CAROLINA STATE UNIVERSITY


Diversity between populations l.jpg
Diversity Between Populations

  • S RNA:

    • Direct sequencing of 13 isolates and subsequent analysis with data from the lab and GenBank.

      • NSs : 21 sequences

      • N : 41 sequences

  • M RNA

    • Direct sequencing of 13 isolates and subsequent analysis with data from the lab and GenBank.

      • NSm: 17 sequences

      • G1/G2: 19 sequences

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S rna l.jpg

GA

JAPAN

S RNA

NSs

N

CA

NC

SP

BU

NC

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M rna l.jpg
M RNA

NSm

G1/G2

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Region

Pi(Tot)

Pi(Sil)

(Tot)

(Sil)

ENC

NSs

0.0341

0.0906

0.0373

0.0907

46.666

N

0.0235

0.0789

0.0427

0.1319

46.654

NSm

0.0303

0.1127

0.0361

0.1309

43.102

G1/G2

0.035

0.1185

0.0463

0.1449

46.835


Geographic subdivision l.jpg
Geographic Subdivision

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Intraspecific polymorphism l.jpg
Intraspecific 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.

NORTH CAROLINA STATE UNIVERSITY

Region

Pi(Tot)

Pi(Sil)

(Tot)

(Sil)

ENC

Region

Pi(Tot)

Pi(Sil)

(Tot)

(Sil)

ENC

NSs

0.0341

0.0906

0.0373

0.0907

46.666

NSs

0.0341

0.0906

0.0373

0.0907

46.666

N

0.0235

0.0789

0.0427

0.1319

46.654

N

0.0235

0.0789

0.0427

0.1319

46.654

NSm

0.0303

0.1127

0.0361

0.1309

43.102

NSm

0.0303

0.1127

0.0361

0.1309

43.102

G1/G2

0.035

0.1185

0.0463

0.1449

46.835

G1/G2

0.035

0.1185

0.0463

0.1449

46.835


Population expansions geographic subdivision l.jpg
Population Expansions→Geographic subdivision

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Test for recombination l.jpg
Test for recombination

  • We used LDhat, which implements a coalescent-based method to detect and estimate recombination from gene sequences

  • The null hypothesis (no recombination) is rejected if the proportion (PLPT) is less than a significance level (<0.05)

  • NO EVIDENCE FOR RECOMBINATION FOR OUR DATA.

NORTH CAROLINA STATE UNIVERSITY


Interspecific divergence l.jpg
Interspecific Divergence

  • Neutral theory predicts that the ratio of silent to replacement substitutions should be the same for polymorphisms within species and fixed differences between species.

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Selection l.jpg
Selection

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Conclusions l.jpg
Conclusions

  • TSWV has a strong spatial structure, attributed to founder effects:

    • Significant genetic differentiation between subpopulations

    • Decrease of genetic variation within subpopulations and

    • Emergence of singletons in most of the analyzed loci

  • High intraspecific polymorphism

  • No evidence for recombination for the data analyzed

NORTH CAROLINA STATE UNIVERSITY


Objective 2 future l.jpg
Objective 2-Future

  • Define the structure of a single TSWV population (individual isolate): diversity within a population.

    • Virus sources: plants from field and greenhouse operations from:

      • Europe (Spain, Italy, Greece)

      • South Africa

      • Japan

      • U.S.A.

NORTH CAROLINA STATE UNIVERSITY


Slide27 l.jpg

Objective 2- Future

Sequencing

RFLP

PCR of individual

clones with inserts ( GC clamp in 5’ end of forward primer)

DGGE

Cloning

Heteroduplex analysis

Heteroduples analysis-SSCP

PCR or

/ nested PCR

Denaturation

cDNA

SSCP

Assymmetric PCR

Total RNA

Source

PCR with one primer

NORTH CAROLINA STATE UNIVERSITY


Objective 3 l.jpg
Objective 3

  • After defining the complexity of the structure certain selection pressures will be imposed:

    • Mechanical passages

    • Vector species

    • Host differentials

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Objective 329 l.jpg
Objective 3

  • Found middle (M) RNA of TSWV is responsible for thrips transmission by a novel viral genetic system

  • Analyzed M RNA sequences of non-transmissible and transmissible isolates

  • Found a nucleotide deletion in glycoprotein coding region of non-transmissible isolates and confirmed glycoprotein may play an important role in thrips transmission

  • Determined mutation frequencies of TSWV isolates maintained by repeated mechanical or thrips transmission

  • Found specificity of vectors may map with M RNA of TSWV

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Slide30 l.jpg

Progression of thrips transmission rate of TSWV per mechanical passage (Mc Nulty, 2001; unpublished data).

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Slide31 l.jpg

Isolation of non-transmissible isolates from population A mechanical passage (Mc Nulty, 2001; unpublished data).

T-

T-

T-

T-

T+

T-

T-

T-

T-

T-

T-

T-

T-

T-

T-

T+

Pop. A

(mechanically-maintained, low transmissibility)

Transmission Assay

Single Lesion Isolation

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Slide32 l.jpg

Results mechanical passage (Mc Nulty, 2001; unpublished data).

Isolation and identification of thrips non-transmissible and transmissible isolates from mechanically maintained RG2.

1. Transmission was confirmed by symptom development and RT-PCR-RFLP. Individual experiments are separated

by semicolon.

2. + means transmissible and – means non-transmissible.

NORTH CAROLINA STATE UNIVERSITY


Objective 4 l.jpg
Objective 4 mechanical passage (Mc Nulty, 2001; unpublished data).

  • Extend the Attribution studies to include INSV.

    • Other tospoviruses?

    • Other viruses?

  • Study the impact of host range and vector species in individual populations.

    • Molecular and biological dissections of individual populations.

    • Partnership with AGDIA for industrial application.

NORTH CAROLINA STATE UNIVERSITY


Slide34 l.jpg

Acknowledgements mechanical passage (Mc Nulty, 2001; unpublished data).

  • Purugganan

    Amy Lawton-Rauh

James Moyer

Jorge Abad

Jan Speck

S.-H. Sin

Stephen New

William Atchley

Michael J. Buck

Sebastian Guyader

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