Virology 376 (2008) 253–257

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Virology

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Rapid Communication A new and distinct species in the genus exists as an endogenous pararetroviral sequence in its host, Dahlia variabilis

Vihanga Pahalawatta, Keri Druffel, Hanu Pappu ⁎

Department of Plant Pathology, Washington State University, Pullman, WA, USA article info abstract

Article history: in certain genera in family were shown to integrate their genomic sequences into their Received 6 August 2007 host genomes and exist as endogenous pararetroviral sequences (EPRV). However, members of the genus Returned to author for revision Caulimovirus remained to be the exception and are known to exist only as episomal elements in the 13 September 2007 infected cell. We present evidence that the DNA genome of a new and distinct Caulimovirus species, associated Accepted 4 March 2008 with dahlia mosaic, is integrated into its host genome, dahlia (Dahlia variabilis). Using cloned viral genes as Available online 7 May 2008 probes, Southern blot hybridization of total plant DNA from dahlia seedlings showed the presence of viral DNA in the host DNA. Fluorescent in situ hybridization using labeled DNA probes from the D10 genome localized Keywords: the viral sequences in dahlia chromosomes. The natural integration of a Caulimovirus genome into its host and Pararetrovirus – Dahlia mosaic its existence as an EPRV suggests the co-evolution of this pathosystem. Caulimovirus © 2008 Elsevier Inc. All rights reserved.

Introduction genome, dahlia (Dahlia variabilis) and thus exists as an endogenous plant pararetrovirus (EPRV). Dahlia mosaic caulimovirus (DMV) is an important viral pathogen of dahlia in the US and several parts of the world. The physical map of the Results and discussion double-stranded DNA genome of DMV (DMV-Portland) associated with dahlia mosaic was reported (Richins and Shepherd, 1983), and its The most characteristic symptoms of the dahlia mosaic disease genomic sequence representing six open reading frames is available in include mosaic, vein-banding systemic chlorosis, stunting, and leaf GenBank. DMV-Portland is a typical Caulimovirus species based on its distortion. Symptoms vary depending on the cultivar, and cultivars genome organization. Studies on the incidence of DMV-Portland in with few or no symptoms are common (Fig. 1). The dsDNA genome of dahlia samples from the US and Europe revealed 21% and 53% of the DMV-D10 was cloned and sequenced. It consists of 7046 bp and its respective samples tested to be infected with DMV-Portland (Paha- genome organization is typical of the members of the genus Cauli- lawatta et al., 2007b). However, our surveys of dahlias to determine movirus with a few exceptions (Fig. 2a). The (ORF) II the incidence of DMV revealed the overwhelming presence of another in members of the genus Caulimovirus codes for an aphid transmission caulimovirus, designated DMV-D10, that was found to be distinct from factor which is not present in the genome of DMV-D10 (Pahalawatta et DMV-Portland (Pahalawatta et al., 2008), and 98% of the samples al., 2008). Another notable divergence from the typical caulimovirus tested were positive for DMV-D10 irrespective of the symptom genome organization is that DMV-D10 has a truncated coat protein expression (Pahalawatta et al., 2007b). Further tests revealed the (Fig. 2a). Phylogenetic analyses of the DMV-D10 ORFs showed that it is presence of DMV-D10 sequences in all plant parts including leaves, a distinct species within the Caulimovirus genus (Fig. 2b). roots, seeds, flower petals and pollen, and DMV-D10 could be detected Total genomic plant DNA extracted from dahlia and other known in tissue culture-derived dahlia . Moreover, our studies have hosts of DMV was used for Southern blot hybridization using cloned also shown that DMV-D10 is transmitted through seed to 100% of the DNA of DMV-D10 as probes. Hybridization was observed with both progeny plants resulting in establishment of infection and symptom undigested and restriction endonuclease-digested dahlia genomic expression (Pahalawatta et al., 2007a). These results indicated the DNA extracted from seedlings, seed coat as well as roots using DMV- possibility of integration of DMV-D10 viral sequences into the host D10 specific probes (Figs. 3a and b). The hybridization signal for DNA plant genome. We present evidence for the first ever finding of a DNA extracts from dahlia seed coat was very weak, possibly due to the genome of a Caulimovirus species that is integrated into its host difficulty of obtaining quality genomic DNA. None of the hybridization signals were seen to occur at ~7 kb, which was expected for genomic DNA of DMV-D10. In contrast, all of the hybridization signals from the ⁎ Corresponding author. Department of Plant Pathology, PO Box 646430, Washington State University, Pullman, WA 99164-6430, USA. Fax: +1 509 335 9581. undigested DNA occurred at high molecular weights which are ex- E-mail address: [email protected] (H. Pappu). pected for dahlia genomic DNA (Figs. 3a and b). A single hybridization

0042-6822/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2008.03.003 254 Rapid Communication

Fig. 1. Progression of symptoms caused by Dahlia mosaic caulimovirus (DMV)-D10 in Dahlia variabilis. Clockwise from top left: 4-week old seedling without any symptoms, veinal chlorosis and vein clearing, mosaic and mottling, and flower breaking symptoms.

signal at 2.5 kb was expected from the EcoRI digestion if the viral Petunia vein clearing virus (PVCV) in Petunia spp (Richert-Pöggeler et dsDNA genome was present as an episomal element. However, the al., 1997, 2003; Harper et al., 2003), Tobacco vein clearing virus (TVCV) probe representing ORF I of DMV-D10 had hybridized to multiple in spp (Jakowitsch et al., 1999; Lockhart et al., 2000; Gregor fragments of digested dahlia DNA (Fig. 3a). No hybridization was et al., 2004), and Rice tungro bacilliform virus (RTBV) in Oryza spp detected with genomic DNA samples from hosts susceptible to DMV or (Kunii et al., 2004) genomes (reviewed by Staginnus and Richert- from unrelated plant taxa (Fig. 3c). Poggeler, 2006). Although integration events were observed for these In addition to the unequivocal molecular evidence of the existence viral sequences, many of these viruses do not encode an integrase of DMV D10 as an EPRV presented above, the localization of DMV-D10 function. The integration of viral sequences is hypothesized to have sequences in dahlia chromosomes was demonstrated using fluores- occurred by illegitimate recombination (Jakowitsch et al., 1999; Mette cent in situ hybridization (FISH). A mixture of digoxigenin-labeled et al., 2002). DNA probes representing the ORFs I and VI of DMV-D10 were used to While members of certain genera of the family Caulimoviridae hybridize to metaphase chromosomes isolated from dahlia root tips. were shown to integrate their DNA into their hosts and exist as EPRVs, The probes hybridized to several chromosomes. Similarly labeled genus Caulimovirus remained an exception, though the type species probes from ORFs I and VI of DMV-Portland did not show any Cauliflower (CaMV) was the first plant virus that was hybridization. This was expected since DMV-Portland is not known to shown to contain a DNA genome (Hull, 2001). Our results provided exist as an EPRV (data not shown). unequivocal evidence that a Caulimovirus species exists as an EPRV. Integration of viral DNA into bacterial and animal host chromo- Prior to the experimental evidence presented here, several lines of somes is a common occurrence. In contrast, there are limited reports evidence, including the presence of D10 sequences in over 98% of the of integration of viral sequences in a plant genome. The integrated samples of numerous dahlia varieties tested from several parts of the viral sequences or EPRVs that have been described so far were all world (Pappu et al., 2005; Pahalawatta et al., 2007b), 100% seed and derived from viruses with DNA genomes or from viruses that have a pollen transmission (Pahalawatta et al., 2007b) suggested this DNA phase in their replication cycle. These include the single- possibility. The Southern and FISH data confirm that this new and stranded DNA geminiviruses (Bejarano et al., 1996; Ashby et al., distinct species in the genus Caulimovirus exists as an EPRV. Though 1997) and the double-stranded DNA viruses (reviewed by Harper CaMV, the type species in the genus Caulimovirus, is not known to et al., 2002; Staginnus and Richert-Poggeler, 2006). The family Cauli- exist as an integrated sequence in its hosts in nature, it was shown that moviridae, consisting of six genera, contains plant viruses that it could integrate, under experimental conditions, into the genome of replicate by reverse transcription (Hull, 2001). Four viruses represent- through a mechanism involving homologous ing three of the genera in this family were reported to have integrants recombination (Schoelz et al., 2005). in the respective host plant genomes. These include Banana streak It remains to be seen if D10 exists as an episomal element in the virus (BSV) in Musa spp.(Harper et al., 1999; Ndowora et al., 1999), infected plant, in addition to its existence as an EPRV. From the Rapid Communication 255

Fig. 2. Characteristics and interviral relationships of Dahlia mosaic caulimovirus (DMV)-D10 genome. a, Comparison of the genome structure and organization of Dahlia mosaic caulimovirus (DMV)-D10 with that of Cauliflower mosaic caulimovirus (CaMV), the type member of the genus Caulimovirus. Coding regions with the same color are considered homologs. Roman numerals signify open reading frames (ORFs). ORF VII Protein of unknown function; ORF I Movement protein; ORF II Aphid transmission factor; ORF III Nucleic acid binding protein; ORF IV Coat protein; ORF V polyprotein; ORF VI Inclusion body protein. The region of the genome covered by the ORF I probe is indicated, along with the sizes (kb) of the undigested and EcoRI restriction enzyme digested fragments. b, Phylograms drawn from ClustalW alignments of the open reading frame VI (inclusion body protein) of selected members of the family Caulimoviridae. Species within the genus Caulimovirus are indicated by a parenthesis to the right of the species acronyms. BRRV—Blueberry red ring spot virus, CaMV—Cauliflower mosaic virus, CERV—Carnation etched ring virus, CYLV—Cestrum yellow leaf virus, ComYMV—Commelina yellow mottle virus, DMV—Dahlia mosaic virus, FMV— Figwort mosaic virus,HLV—Horseradish latent virus, MMV—Mirabilis mosaic virus, PCSV—Peanut chlorotic streak virus, SbCMV—Soybean chlorotic streak virus, SVBV—Strawberry vein banding virus. Bootstrap values are indicated at branching points in the phylogram as a percentage of 1000 iterations.

evolutionary point of view, it is not known when this integration into infected plants using electron microscope were also unsuccessful. the plant genome might have taken place. Testing wild dahlia species Therefore, it is possible that DMV-D10 exists primarily as an EPRV and collected from the center of origin may provide some clues toward is vertically transmitted. Tobacco vein clearing virus was reported to be answering this question. Our preliminary data suggests that certain transmitted exclusively through vertical transmission (Lockhart et al., wild dahlia species collected from the highlands of Mexico, considered 2000). Interestingly, dahlia plants positive only for DMV-D10 to be the center of diversity for Dahlia, were infected by DMV (Eid et sequences exhibited a range of disease symptoms ranging from mild al., in press). In our studies on the genome structure and organization to severe mosaic (Fig. 1). Therefore, it is possible that the EPRV is of DMV-D10, we were unable to detect the presence of a gene that is capable of initiating infection and produce disease symptoms. Studies homologous to the aphid transmission factor that is coded by all on other EPRVs have shown that the expression of episomal infections known species in the genus Caulimovirus (Pahalawatta et al., 2008). and the degree of severity of symptoms are correlated with en- Furthermore, the D10 genome coded for a truncated coat protein and vironmental conditions such as day length and temperature (Harper we were not successful in transmitting this virus to other hosts et al., 2002). It remains to be seen if environmental factors affect the through mechanical inoculation. Efforts to localize virions in D10- expression of infectious episomal DMV-D10 genome. 256 Rapid Communication

integration event would provide important insights into the co- evolution of caulimoviruses as EPRVs and their plant hosts.

Materials and methods

DNA extraction

Total genomic DNA was extracted from dahlia seedlings, germinated in covered Petri dishes to avoid exposure to the aphid vector which has been previously reported to transmit the virus in a non-persistent manner. To determine the presence of the virus in dahlia seeds, seeds were soaked in water and the seed coat was separated from the embryos. The separated seed coats and embryos were used for DNA extraction. Genomic DNA was also extracted from dahlia roots. In addition, total genomic DNA was extracted from leaves of Zinnia elegans, Verbesina encelioides and potato plants. Total genomic DNA extractions were made using the DNeasy plant mini kit (Qiagen Inc, Valencia, CA) according to manufacturer's instructions. Dahlia seedling DNA extracts were digested overnight at 37 °C with EcoRI.

Southern blot hybridization

DMV-D10 specific primer pairs V416 (5′- TTA CCA CTT CTA ACA AAA GG -3′)/ C1070 (5′- TCC ATA CAT GTC TAC TTT TTC GG -3′) and ORF4 CP1-1431F (5′- TGC ATA AAA TGA GTT CTA TC -3′)/ORF4 CP1- 1926C (5′- TGA ACT TGT TCA TCA TTA TC -3′) were used in PCR amplification using DNA extracts from DMV-D10 infected dahlia plants as template. The PCR reaction included a final volume of 2 µl 10× PCR buffer and a final concentration of 0.15 mM dNTPs, 2 mM

MgCl2 and 0.6 µM of each primer in a total reaction volume of 20 µl. PCR amplification was performed in a DNA thermocycler (Bio-Rad, Hercules, CA) programmed for 3 min at 94 °C for initial denaturation

and 50 cycles each consisting of 30 s at 94 °C, 20 s at Tm and 1 min extension per 1000 bp product at 72 °C, followed by a final extension for 7 min at 72 °C. The PCR products were analyzed by 1.2% agarose gel electrophoresis in 1× TAE buffer. The PCR products were puri- fied using the Qiaquick PCR purification kit (Qiagen Inc). Purified PCR products (25 ng) were 32 P labeled using the Prime-a-gene® Fig. 3. Southern blot hybridization analysis of total plant genomic DNA using a, probe labeling kit (Promega, Madison, WI). DNA extracts (10–20 µg) representing the open reading frame (ORF) I of Dahlia mosaic caulimovirus (DMV)-D10. were loaded on 0.8% agarose gels. Electrophoresis was performed Total genomic DNA extracts from dahlia seedlings UD — undigested; RD — digested with using 1× TAE buffer running at 20 V overnight. DNA was transferred — EcoRI. b, probe representing ORF IV of DMV-D10. Total genomic DNA extracts from DS overnight onto Hybond N+ membrane (GE Healthcare Bio-Sciences dahlia seedlings; SC — seed coat; R — root; P — potato; C — control (PCR product used to produce the labeled probe). c, probe representing open reading frame I (ORF I) of Dahlia Corp, Piscataway, NJ). Prehybridization, hybridization and membrane mosaic virus (DMV)-D10. Total genomic DNA extracts from DS — dahlia seedlings; V — washes were carried out at 65 °C. Membranes were exposed to film Verbesina; Z — Zinnia. The relative positions of the ORFs in the DMV genome are shown (Kodak™ X-Omat AR, 20×25 cm) for 6 days and the film developed. in Fig. 2a. Fluorescent in situ hybridization

Chromosomes were prepared from actively growing root tips of Studies on Musa and Oryza genomes revealed the identification of dahlia seedlings as described (Gatt et al., 1998). In situ hybridization the junctions of virus sequence integration (Ndowora et al., 1999; using Digoxigenin-labeled DNA probes spanning ORF I and ORF VI Kunii et al., 2004) due to the availability of genomic libraries for these from DMV-D10 was done following the protocols by Schwarzacher plant hosts. However similar resources are not yet available for the and Heslop-Harrison, 2000. dahlia genome. Clonal propagation of vegetative tissues, stem cuttings and bulbs have been considered to be the primary reason for the spread Acknowledgments of viruses in dahlias. The most effective means for managing viruses in vegetatively propagated plants is through testing and elimination of Research was supported in part by the Samuel Smith and Patricia virus-infected material and use of virus-free stock for propagation. Smith Endowment for Dahlia Virus Research, created by the American Meristem tip culture was used to eliminate viruses from infected stock. Dahlia Society. Funding from the USDA Northwest Nursery Crop However, in instances where the viral genome is integrated into the host Research Center is gratefully acknowledged. We thank Robert Brugge- genome, a similar approach may not be effective. Under these man, Arron Carter and Robert Zemetra for assistance. PPNS No. 0483, circumstances, knowledge of the environmental conditions that trigger Department of Plant Pathology, College of Agricultural, Human and symptom development will play an essential role in developing virus Natural Resource Sciences, Agricultural Research Center, Project # management strategies. This is the first report of the existence of a WNPO 0545, Washington State University, Pullman, WA 99164-6430, Caulimovirus species as an EPRV. Further studies on the nature of this USA. Rapid Communication 257

References vertically and related to sequences integrated in the host genome. J. Gen. Virol. 81, 1579–1585. Mette, M.F., Kanno, T., Aufsatz, W., Jakowitsch, J., van der Winden, J., Matzke, M.A., Ashby, M.K., Warry, A., Bejarano, E.R., Khashoggi, A., Burrell, M., Lichtenstein, C., 1997. Matzke, A.J.M., 2002. Endogenous viral sequences and their potential contribution Analysis of multiple copies of geminiviral DNA in the genome of four closely related to heritable virus resistance in plants. EMBO J. 21, 461–469. Nicotiana species suggests a unique integration event. Plant Mol. Biol. 35, 313–321. Ndowora, T., Dahal, G., LaFleur, D., Harper, G., Hull, R., Olszewski, N.E., Lockhart, B., 1999. Bejarano, E.R., Khashoggi, A., Witty, M., Lichtenstein, C., 1996. Integration of multiple Evidence that badnavirus infection in Musa can originate from integrated repeats of geminiviral DNA into the nuclear genome of tobacco during evolution. pararetroviral sequences. Virology 255, 214–220. Proc. Natl. Acad. Sci. U. S. A. 93, 759–764. Pahalawatta, V., Druffel, K., Pappu, H.R., 2007a. Seed transmission of Dahlia mosaic virus Eid, S.G., Druffel, K.L., Saar, D.E., Pappu, H.R., in press. Incidence and diversity of in Dahlia pinnata. Plant Dis. 91, 88–91. caulimoviruses in wild dahlia species from the highlands of Mexico. Phytopathol- Pahalawatta, V., Miglino, R., Druffel, K.L., Jodlowska, A., van Schadewijk, A.R., Pappu, H.R., ogy 98 (supplement): (abstr.). 2007b. Incidence and relative distribution of distinct caulimoviruses associated Gatt, M., Ding, H., Hammett, K., Murray, B., 1998. Polyploidy and evolution in wild and with dahlia mosaic in Dahlia variabilis. Plant Dis. 91, 1194–1197. cultivated Dahlia species. Annal. Appl. Bot. 81, 647–656. Pahalawatta, V., Druffel, K.L., Wyatt, S.D., Eastwell, K.C., Pappu, H.R., 2008. Genome Gregor, W., Mette, M.F., Staginnus, C., Matzke, M.A., Matzke, A.J.M., 2004. A distinct structure and organization of a novel and distinct species of genus Caulimovirus endogenous pararetrovirus family in Nicotiana tomentosiformis, a diploid progeni- (Family Caulimoviridae) associated with dahlia mosaic. Arch. Virol. 153, 733–738. tor of polyploid tobacco. Plant Physiol. 134, 1191–1199. Pappu, H.R., Wyatt, S.D., Druffel, K.L., 2005. Dahlia mosaic virus: Molecular detection and Harper, G., Osuji, J.O., Heslop-Harrison, J.S., Hull, R., 1999. Integration of banana streak distribution in dahlia in the US. HortScience 40, 697–699. badnavirus into the Musa genome: molecular and cytogenetic evidence. Virology Richert-Pöggeler, K.R., Shepherd, R.J., 1997. Petunia vein clearing virus:aplant 255, 207–213. pararetrovirus with core sequences for an integrase function. Virology 236, Harper, G., Hull, R., Lockhart, B., Olszewski, N., 2002. Viral sequences integrated into 137–146. plant genomes. Annu. Rev. Phytopathol. 40, 119–136. Richert-Pöggeler, K.R., Noreen, F., Schwarzacher, T., Harper, G., Hohn, T., 2003. Induction Harper, G., Richert-Pöggeler, K.R., Hohn, T., Hull, R., 2003. Detection of petunia vein- of infectious petunia vein clearing (pararetro) virus from endogenous provirus in clearing virus: model for the detection of DNA viruses in plants with homologous petunia. EMBO J. 22, 4836–4845. endogenous pararetrovirus sequences. J. Virol. Methods 107, 177–184. Richins, R.D., Shepherd, R.J., 1983. Physical maps of the genome of Dahlia mosaic virus and Hull, R., 2001. Matthews Plant Virology, 4th Edition. Academic Press. 1056 pp. Mirabilis mosaic virus — two members of the Caulimovirus group. Virology 124, Jakowitsch, J., Mette, M.F., van der Winden, J., Matzke, M.A., Matzke, A.J.M., 1999. 208–214. Integrated pararetroviral sequences define a unique class of dispersed repetitive Schwarzacher, T., Heslop-Harrison, P., 2000. Practical in-situ hybridization. BIOS DNA in plants. Proc. Natl. Acad. Sci. U. S. A. 96, 13241–13246. Scientific Publishers, Oxford, UK. 203 pp. Kunii, M., Kanda, M., Nagano, H., Uyeda, I., Kishima, Y., Sano, Y., 2004. Reconstruction Schoelz, J.E., Wiggins, B.E., Balaji, B., Palukaitis, P.F., Squires, J., 2005. Integration of of putative DNA virus from endogenous rice tungro bacilliform virus-like Cauliflower mosaic virus into the genome of Arabidopsis thaliana through a sequences in the rice genome: implications for integration and evolution. BMC mechanism involving homologous recombination. XIIIth Intl. Cong. of Virology. Genomics 5, 80. July 23–28. San Francisco, CA. Lockhart, B.E., Menke, J., Dahal, G., Olszewski, N.E., 2000. Characterization and genomic Staginnus, C., Richert-Poggeler, K.R., 2006. Endogenous pararetroviruses: two faceted analysis of tobacco vein clearing virus, a plant pararetrovirus that is transmitted travelers in the plant genome. Trends Plant Sci. 11, 485–491.