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

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Taxonomy

• TSWV is a member of the genus Tospovirus of the
  family Bunyaviridae. Other genera of
  Bunyaviridae
• Bunyavirus
• Phlebovirus
• Hantavirus
• Nairovirus

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Members of the Genus Tospovirus

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Genomic Organization
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Virus Structure
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Host Range

• Wide host range exceeds 900 plant species,
  spanning both monocotyledonous and dicotyledonous
  plant species
• Tremendous economic importance

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Floral Crop Movement
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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)

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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 ?

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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

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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

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Attribution

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

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Objective 1

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

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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.

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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
NSs
N
CA
NC
SP
BU
NC
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M RNA
NSm
G1/G2
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?(Sil)
?(Tot)
Pi(Sil)
Pi(Tot)
Region
46.666
0.0907
0.0373
0.0906
0.0341
NSs
46.654
0.1319
0.0427
0.0789
0.0235
N
43.102
0.1309
0.0361
0.1127
0.0303
NSm
46.835
0.1449
0.0463
0.1185
0.035
G1/G2
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Geographic Subdivision
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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.

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ENC
?(Sil)
?(Tot)
Pi(Sil)
Pi(Tot)
Region
ENC
?(Sil)
?(Tot)
Pi(Sil)
Pi(Tot)
Region
46.666
0.0907
0.0373
0.0906
0.0341
NSs
46.666
0.0907
0.0373
0.0906
0.0341
NSs
46.654
0.1319
0.0427
0.0789
0.0235
N
46.654
0.1319
0.0427
0.0789
0.0235
N
43.102
0.1309
0.0361
0.1127
0.0303
NSm
43.102
0.1309
0.0361
0.1127
0.0303
NSm
46.835
0.1449
0.0463
0.1185
0.035
G1/G2
46.835
0.1449
0.0463
0.1185
0.035
G1/G2
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Population Expansions?Geographic subdivision
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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 (lt0.05)
• NO EVIDENCE FOR RECOMBINATION FOR OUR DATA.

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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
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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

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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.

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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
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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 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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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
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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Results
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.
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Objective 4

• 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.

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Acknowledgements

• 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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