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. Genomic Organization + + L M S NORTH CAROLINA STATE UNIVERSITY

5. Virus Structure N 29K L 330K G 2 58K G1 78K 80 - 110 nm L 330K 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. Floral Crop Movement

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. S RNA CA NC SP BU NC NSsN GA JAPAN NORTH CAROLINA STATE UNIVERSITY

18. M RNA 46.8350.14490.04630.11850.035G1/G2 43.1020.13090.03610.11270.0303NSm 46.6540.13190.04270.07890.0235N 46.6660.09070.03730.09060.0341NSs ENC (Sil) (Tot) Pi(Sil)Pi(Tot)Region NSmG1/G2 NORTH CAROLINA STATE UNIVERSITY

19. Geographic Subdivision RegionSubpop.KstKsP-value NSs CA vs Bul. 0.688890.0103750.0* N Bul. vs GA 0.5076460.0116680.0* Bul. vs CA 0.5652850.007990.0* CA vs GA 0.4567760.0067810.0* NSm CA vs NC 0.1907890.0178910.006* G1/G2 0.2653680.0185820.001* 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. 46.8350.14490.04630.11850.035G1/G2 43.1020.13090.03610.11270.0303NSm 46.6540.13190.04270.07890.0235N 46.6660.09070.03730.09060.0341NSs ENC (Sil) (Tot) Pi(Sil)Pi(Tot)Region 46.8350.14490.04630.11850.035G1/G2 43.1020.13090.03610.11270.0303NSm 46.6540.13190.04270.07890.0235N 46.6660.09070.03730.09060.0341NSs ENC (Sil) (Tot) Pi(Sil)Pi(Tot)Region Region Pi (Tot) Pi (Sil) (Tot) (Sil) NSs0.034050.09060.03730.09069 N0.023520.078850.042710.13188 NSm0.030320.112720.036120.13085 G1/G20.034990.118530.046260.14486 NORTH CAROLINA STATE UNIVERSITY

21. Population Expansions → Geographic subdivision Population Expansions → Geographic subdivision NSsPi(Tot)Pi(Sil) ∂ (Tot) ∂ (Sil) Tajimas D Fu & Li D Fu & Li F California 0.0060.0160.00640.0193-0.4955-0.7047-0.725 Bulgaria 0.01660.0340.01850.0404-0.532-0.734-0.808 NPi(Tot)Pi(Sil) ∂ (Tot) ∂ (Sil) Tajimas D Fu & Li D Fu & Li F California 0.00220.00850.00320.0121-1.524*-1.61*-1.73* Bulgaria 0.01310.0290.01890.0411-1.645*-1.779*-1.95* Georgia 0.01020.039390.01280.0478-1.0002-1.116-1.22 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 (P LPT ) 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. RegionFSFRPSPR G value NSs221266101630.00032* N12616594400.0* NSm12313288230.0* G1/G24895003771300.0* NORTH CAROLINA STATE UNIVERSITY

24. Selection Region M2 vs M3 M7 vs M8 Pos. sites dN/dS NSs14.6632* 15.228 *** 458, 460 0.3671 N8.092*11.586**10,174,2550.2207 NSm0.0020.598-------------0.0811 G1/G21.156.364*5300.1326 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. Source Total RNA cDNA PCR or / nested PCR Cloning RFLP Sequencing PCR of individual clones 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 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 Pop. A (mechanically- maintained, low transmissibility) T+ T- T+ Single Lesion Isolation Transmission Assay NORTH CAROLINA STATE UNIVERSITY

32. Results Isolation and identification of thrips non-transmissible and transmissible isolates from mechanically maintained RG2. Single lesion isolates Transmission (infected/inoculated) 1 Transmissibility2 0/30 ; 0/30 -2-2-2-2 11 - 28 - 37 9/30 ; 8/30 + 41 7/30 ; 6/30 + 43 6/30 ; 7/30 + 81 0/30 ; 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.

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