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Evolutionary Biology of Tospoviruses in Floral Crops

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

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  1. Evolutionary Biology of Tospoviruses in Floral Crops J. W. Moyer North Carolina State University Department of Plant Pathology NORTH CAROLINA STATE UNIVERSITY

  2. 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

  3. Members of the Genus Tospovirus NORTH CAROLINA STATE UNIVERSITY

  4. NSs N RdRp L + NSm G1/G2 M + S Genomic Organization NORTH CAROLINA STATE UNIVERSITY

  5. G2 58K N 29K G1 78K L 330K L 330K 80 - 110 nm Virus Structure NORTH CAROLINA STATE UNIVERSITY

  6. NORTH CAROLINA STATE UNIVERSITY

  7. Host Range • Wide host range: exceeds 900 plant species, spanning both monocotyledonous and dicotyledonous plant species • Tremendous economic importance NORTH CAROLINA STATE UNIVERSITY

  8. FloralCrop Movement NORTH CAROLINA STATE UNIVERSITY

  9. 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

  10. 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

  11. 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

  12. 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

  13. Attribution • The ability to target the source as well as to identify the causal pathogen NORTH CAROLINA STATE UNIVERSITY

  14. 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

  15. 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

  16. 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 NORTH CAROLINA STATE UNIVERSITY

  17. GA JAPAN S RNA NSs N CA NC SP BU NC NORTH CAROLINA STATE UNIVERSITY

  18. M RNA NSm G1/G2 NORTH CAROLINA STATE UNIVERSITY 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

  19. Geographic Subdivision NORTH CAROLINA STATE UNIVERSITY

  20. 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

  21. Population Expansions→Geographic subdivision NORTH CAROLINA STATE UNIVERSITY

  22. 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

  23. 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. NORTH CAROLINA STATE UNIVERSITY

  24. Selection NORTH CAROLINA STATE UNIVERSITY

  25. 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

  26. 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

  27. 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

  28. Objective 3 • After defining the complexity of the structure certain selection pressures will be imposed: • Mechanical passages • Vector species • Host differentials NORTH CAROLINA STATE UNIVERSITY

  29. 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 NORTH CAROLINA STATE UNIVERSITY

  30. Progression of thrips transmission rate of TSWV per mechanical passage (Mc Nulty, 2001; unpublished data). NORTH CAROLINA STATE UNIVERSITY

  31. 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 NORTH CAROLINA STATE UNIVERSITY

  32. 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. NORTH CAROLINA STATE UNIVERSITY

  33. 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. NORTH CAROLINA STATE UNIVERSITY

  34. Acknowledgements • Purugganan Amy Lawton-Rauh James Moyer Jorge Abad Jan Speck S.-H. Sin Stephen New William Atchley Michael J. Buck Sebastian Guyader NORTH CAROLINA STATE UNIVERSITY

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