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Characterization of Insertion Sites in Rainbow Papaya, the First Commercialized Transgenic Fruit Crop

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Abstract

Inserts and insert sites in transgenic, papaya ringspot virus (PRSV)-resistant commercial papaya Rainbow and SunUp, were characterized as part of a petition to Japan to allow import of fresh fruit of these cultivars from the U.S. and to provide data for a larger study aimed at understanding the global impact of DNA transformation on whole genome structure. The number and types of inserts were determined by Southern analysis using probes spanning the entire transformation plasmid and their sequences determined from corresponding clones or sequence reads from the whole-genome shotgun (WGS) sequence of SunUp papaya. All the functional transgenes, coding for the PRSV coat protein (CP), neophosphotransferase (nptII) and β-glucuronidase (uidA) were found in a single 9,789 basepair (bp) insert. Only two other inserts, one consisting of a 290 bp nonfunctional fragment of the nptII gene and a 1,533 bp plasmid-derived fragment containing a nonfunctional 222 bp segment of the tetA gene were detected in Rainbow and SunUp. Detection of the same three inserts in samples representing transgenic generations five to eight (R5 to R8) suggests that the three inserts are stably inherited. Five out of the six genomic DNA segments flanking the three inserts were nuclear plastid sequences (nupts). From the biosafety standpoint, no changes to endogenous gene function based on sequence structure of the transformation plasmid DNA insertion sites could be determined and no allergenic or toxic proteins were predicted from analysis of the insertion site and flanking genomic DNA.

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Abbreviations

bp:

base pair

CP:

coat protein

DSB:

double-stranded break

ELISA:

enzyme-linked immunosorbent assay

FAO:

Food and Agriculture Organization (of the United Nations)

GE:

genetically engineered

GUS:

β-glucuronidase

IUIS:

International Union of Immunological Societies

kb:

kilobase pair

MAR:

matrix attachment regions

NHEJ:

nonhomologous end joining

nupt-DNA:

nuclear plastid DNA

nupts:

nuclear plastid sequence

numts:

nuclear mitochondrial sequence

ORF:

open reading frame

PCR:

polymerase chain reaction

PDR:

pathogen-derived resistance

PTGS:

post-transcriptional gene silencing

PRSV:

Papaya ringspot virus

SDAP:

Structural Database for Allergenic Proteins

T-DNA:

transferred DNA

Topo I:

Topoisomerase I

Topo II:

Topoisomerase II

WGS:

whole-genome shotgun

WHO:

World Health Organization

References

  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zheng Z et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. doi:10.1093/nar/25.17.3389

    Article  CAS  PubMed  Google Scholar 

  2. Brunaud V, Balzergue S, Dubreucq B, Aubourg S, Samson F et al (2002) T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Rep 3:1152–1157. doi:10.1093/embo-reports/kvf237

    Article  CAS  PubMed  Google Scholar 

  3. Dai S, Zheng P, Marmey P, Zhang S, Tian W et al (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33. doi:10.1023/A:1009687511633

    Article  CAS  Google Scholar 

  4. Drescher A, Ruf S, Calsa T Jr, Carrer H, Bock R (2000) The two largest chloroplast genome-encoded open reading frames of higher plants are essential genes. Plant J 22:97–104. doi:10.1046/j.1365-313x.2000.00722.x

    Article  CAS  PubMed  Google Scholar 

  5. FAO/WHO (2001) Evaluation of allergenicity of genetically modified foods. Report of a joint FAO/WHO expert consultation on allergenicity of foods derived from biotechnology. Available at: http://www.who.int/foodsafety/publications/biotech/en/ec_jan2001.pdf.

  6. Fermín GA (2002) Use, application, and technology transfer of native and synthetic genes to engineer single and multiple transgenic viral resistance. Ph.D. Thesis, Cornell University, Geneva, p 293

  7. Gonsalves D (1998) Control of papaya ringspot virus in papaya: A case study. Annu Rev Phytopathol 36:415–437. doi:10.1146/annurev.phyto.36.1.415

    Article  CAS  PubMed  Google Scholar 

  8. Gonsalves D, Ferreira S (2003) Transgenic papaya: A case for managing risks of Papaya ringspot virus in Hawaii. OnlinePlant Health Progress doi:10.1094/PHP-2003-1113-1003-RV

  9. Gonsalves D, Gonsalves C, Ferreira S, Pitz K, Fitch M, et al (2004) Transgenic virus resistant papaya: From hope to reality for controlling papaya ringspot virus in Hawaii. APSnet feature story for July, 2004 Online at: http://www.apsnet.org/online/feature/ringspot

  10. Gonsalves D (2006) Transgenic papaya: Development, release, impact, and challenges. Adv Virus Res 67:317–354. doi:10.1016/S0065-3527(06)67009-7

    Article  CAS  PubMed  Google Scholar 

  11. Gonsalves D, Vegas A, Prasartsee V, Drew R, Suzuki JY et al (2006) Developing papaya to control Papaya ringspot virus by transgenic resistance, intergeneric hybridization, and tolerance breeding. In: Janick J (ed) Plant breeding reviews. John Wiley and Sons, Inc., Hoboken, pp 35–73

    Google Scholar 

  12. Gonsalves D, Ferreira SA, Suzuki JY, Tripathi S (2008) Papaya. In: Kole C, Hall TC (eds) Tropical and subtropical fruits and nuts. Compendium of transgenic crop plants, vol. 5. Wiley-Blackwell, Oxford West Sussex Hoboken, pp 131-162

  13. Gonsalves D, Suzuki JY, Tripathi S, Ferreira SA (2008) Papaya ringspot virus. In: Mahy BWJ, van Regenmortel MHV (eds) Encyclopedia of virology. Elsevier Ltd, Oxford, pp 1–8

    Chapter  Google Scholar 

  14. Gorbunova V, Levy AA (1997) Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res 25:4650–4657. doi:10.1093/nar/25.22.4650

    Article  CAS  PubMed  Google Scholar 

  15. Gorbunova V, Levy AA (1999) How plants make ends meet: DNA double-strand break repair. Trends Plant Sci 4:263–269. doi:10.1016/S1360-1385(99)01430-2

    Article  PubMed  Google Scholar 

  16. Guo X, Ruan S, Hu W, Cai D, Fan L (2008) Chloroplast DNA insertions into the nuclear genome of rice: the genes, sites and ages of insertion involved. Funct Integr Genomics 8:101–108. doi:10.1007/s10142-007-0067-2

    Article  CAS  PubMed  Google Scholar 

  17. Heck GR, Armstrong CL, Astwood JD, Behr CF, Bookout JT et al (2005) Development and characterization of a CP4 EPSPS-based glyphosate-tolerant corn event. Crop Sci 45:329–339

    Article  CAS  Google Scholar 

  18. Huang CY, Ayliffe MA, Timmis JN (2004) Simple and complex nuclear loci created by newly transferred chloroplast DNA in tobacco. Proc Natl Acad Sci USA 101:9710–9715. doi:10.1073/pnas.0400853101

    Article  CAS  PubMed  Google Scholar 

  19. Huang CY, Grünheit N, Ahmadinejad N, Timmis JN, Martin W (2005) Mutational decay and age of chloroplast and mitochondrial genomes transferred recently to angiosperm nuclear chromosomes. Plant Physiol 138:1723–1733. doi:10.1104/pp.105.060327

    Article  CAS  PubMed  Google Scholar 

  20. Ivanciuc O, Schein CH, Braun W (2002) Data mining of sequences and 3D structures of allergenic proteins. Bioinformatics 18:1358–1364. doi:10.1093/bioinformatics/18.10.1358

    Article  CAS  PubMed  Google Scholar 

  21. Ivanciuc O, Schein CH, Braun W (2003) SDAP: Database and computational tools for allergenic proteins. Nucleic Acids Res 31:359–362. doi:10.1093/nar/gkg010

    Article  CAS  PubMed  Google Scholar 

  22. Kleter GA, Peijnenburg AACM (2002) Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE-binding linear epitopes of allergens. BMC Struct Biol 2:1–11. doi:10.1186/1472-6807-2-8

    Article  Google Scholar 

  23. Knoop V, Unseld M, Marienfeld J, Brandt P, Sunkel S et al (1996) copia-, gypsy- and LINE-Like retrotransposon fragments in the mitochondrial genome of Arabidopsis thaliana. Genetics 142:579–585

    CAS  PubMed  Google Scholar 

  24. Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95:7203–7208. doi:10.1073/pnas.95.12.7203

    Article  CAS  PubMed  Google Scholar 

  25. Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E et al (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52:247–258. doi:10.1023/A:1023941407376

    Article  CAS  PubMed  Google Scholar 

  26. Kohli A, Christou P (2008) Stable transgenes bear fruit. Nat Biotechnol 26:653–654. doi:10.1038/nbt0608-653

    Article  CAS  PubMed  Google Scholar 

  27. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M et al (2004) Versatile and open software for comparing large genomes. Genome Biol 5:R12. doi:10.1186/gb-2004-1185-1182-R1112, doi:10.1186/gb-2004-5-2-r12

    Article  PubMed  Google Scholar 

  28. Liebich I, Bode J, Frisch M, Wingender D (2002) S/MARt DB: a database on scaffold/matrix attached regions. Nucleic Acids Res 30:372–374. doi:10.1093/nar/30.1.372

    Article  CAS  PubMed  Google Scholar 

  29. Liere K, Maliga P (2001) Plastid RNA polymerases in higher plants. In: Anderson B, Aro EM (eds) Regulation of Photosynthesis. Kluwer Academic Publishers, Dordrecht, pp 29–49

    Google Scholar 

  30. Ling K, Namba S, Gonsalves C, Slightom JL, Gonsalves D (1991) Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene. Bio/Technol 9:752–758

    Article  CAS  Google Scholar 

  31. Liu X, Baird V (2001) Rapid amplification of genome DNA ends by NlaIII partial digestion and polynucleotide tailing. Plant Mol Biol Rep 19:261–267. doi:10.1007/BF02772898

    Article  Google Scholar 

  32. Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junction by thermal assymetric interlaced PCR. Plant J 8:457–463. doi:10.1046/j.1365-313X.1995.08030457.x

    Article  CAS  PubMed  Google Scholar 

  33. Lius S, Manshardt RM, Fitch MMM, Slightom JL, Sanford JC et al (1997) Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus. Mol Breed 3:161–168. doi:10.1023/A:1009614508659

    Article  Google Scholar 

  34. Makarevitch I, Somers DA (2006) Association of Arabidopsis topoisomerase IIA cleavage sites with functional genomic elements and T-DNA loci. Plant J 48:697–709. doi:10.1111/j.1365-313X.2006.02915.x

    Article  CAS  PubMed  Google Scholar 

  35. Manshardt RM (1998) ‘UH Rainbow’ papaya. University of Hawaii College of Tropical Agriculture and Human Resources New Plants for Hawaii-1, p2

  36. Martin W (2003) Gene transfer from organelles to the nucleus: Frequent and in big chunks. Proc Natl Acad Sci USA 100:8612–8614. doi:10.1073/pnas.1633606100

    Article  CAS  PubMed  Google Scholar 

  37. Matsuo M, Ito Y, Yamauchi R, Obokata J (2005) The rice nuclear genome continuously integrates, shuffles, and eliminates the chloroplast genome to cause chloroplast-nuclear DNA flux. Plant Cell 17:665–675. doi:10.1105/tpc.104.027706

    Article  CAS  PubMed  Google Scholar 

  38. Ming R, Moore PH, Zee F, Abbey CA, Ma H et al (2001) Construction and characterization of a papaya BAC library as a foundation for molecular dissection of a tree-fruit genome. Theor Appl Genet 102:892–899. doi:10.1007/s001220000448

    Article  CAS  Google Scholar 

  39. Ming R, Hou S, Feng Y, Yu Q, Dionne-Laporte A et al (2008) The draft genome of the transgenic tropical fruit tree papaya (Carica papaya Linnaeus). Nature 452:991–996. doi:10.1038/nature06856

    Article  CAS  PubMed  Google Scholar 

  40. NASS (2007) Papaya acreage survey 2007 results. In: National Agricultural Statistical Service, pp 1–8

  41. Pawlowski WP, Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. Proc Natl Acad Sci USA 95:12106–12110

    Article  CAS  PubMed  Google Scholar 

  42. Purcifull D, Edwardson J, Hiebert E, Gonsalves D (1984) Papaya ringspot virus. CMI/AAB Descriptions of plant viruses No 292 (No 84 Revised, July 1984) 8 pp CAB International, Wallingford, UK

  43. Richly E, Leister D (2004) NUPTs in sequenced eukaryotes and their genomic organization in relation to NUMTs. Mol Biol Evol 21:1972–1980. doi:10.1093/molbev/msh210

    Article  CAS  PubMed  Google Scholar 

  44. Ruf S, Kössel H, Bock R (1997) Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene. J Cell Biol 139:95–102. doi:10.1083/jcb.139.1.95

    Article  CAS  PubMed  Google Scholar 

  45. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8019. doi:10.1073/pnas.81.24.8014

    Article  CAS  PubMed  Google Scholar 

  46. Sawasaki T, Takahashi M, Goshima N, Morikawa H (1998) Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: Junction regions can bind to nuclear matrices. Gene 218:27–35. doi:10.1016/S0378-1119(98)00388-6

    Article  CAS  PubMed  Google Scholar 

  47. Shahmuradov IA, Akbarova YY, Solovyev VV, Aliyev JA (2003) Abundance of plastid DNA insertions in nuclear genomes of rice and Arabidopsis. Plant Mol Biol 52:923–934

    Article  CAS  PubMed  Google Scholar 

  48. Somers DA, Makarevitch I (2004) Transgene integration in plants: poking or patching holes in promiscuous genomes. Curr Opin Biotechnol 15:126–131. doi:10.1016/j.copbio.2004.02.007

    Article  CAS  PubMed  Google Scholar 

  49. Souza MT Jr, Tennant PF, Gonsalves D (2005) Influence of coat protein transgene copy number on resistance in transgenic line 63-1 against Papaya ringspot virus isolates. HortScience 40:2083–2087

    Google Scholar 

  50. Sugiura M (1992) The chloroplast genome. Plant Mol Biol 18:149–168. doi:10.1007/BF00015612

    Article  Google Scholar 

  51. Sugiura M, Hirose T, Sugita M (1998) Evolution and mechanism of translation in chloroplasts. Annu Rev Genet 32:437–459. doi:10.1146/annurev.genet.32.1.437

    Article  CAS  PubMed  Google Scholar 

  52. Suzuki JY, Tripathi S, Gonsalves D (2007) Virus-resistant transgenic papaya: Commercial development and regulatory and environmental issues. In: Punja SK, De Boer SH, Sanfaçon H (eds) Biotechnology and plant disease managment. CAB International, Wallingford, pp 436–461

    Chapter  Google Scholar 

  53. Szabados L, Kovács I, Oberschall A, Ábrahám E, Kerekes I et al (2002) Distribution of 1,000 sequenced T-DNA tags in the Arabidopsis genome. Plant J 32:233–242. doi:10.1046/j.1365-313X.2002.01417.x

    Article  CAS  PubMed  Google Scholar 

  54. Takano M, Egawa H, Ikeda J, Wakasa K (1997) The structure of integration sites in transgenic rice. Plant J 11:353–361. doi:10.1046/j.1365-313X.1997.11030353.x

    Article  CAS  PubMed  Google Scholar 

  55. Tatusova TA, Madden TL (1999) Blast 2 sequences—a new tool for comparing protein and nucleotide sequences. FEMS Microbiol Lett 174:247–250. doi:10.1111/j.1574-6968.1999.tb13575.x

    Article  CAS  PubMed  Google Scholar 

  56. Tennant P, Fermin G, Fitch MM, Manshardt RM, Slightom JL et al (2001) Papaya ringspot virus resistance of transgenic Rainbow and SunUp is affected by gene dosage, plant development, and coat protein homology. Eur J Plant Pathol 107:645–653. doi:10.1023/A:1017936226557

    Article  CAS  Google Scholar 

  57. Tennant P, Souza MT Jr, Gonsalves D, Fitch MM, Manshardt RM et al (2005) Line 63-1: a new virus-resistant transgenic papaya. HortScience 40:1196–1199

    Google Scholar 

  58. Tennant PF, Gonsalves C, Ling KS, Fitch M, Manshardt R et al (1994) Differential protection against papaya ringspot virus isolates in coat protein gene transgenic papaya and classically cross-protected papaya. Phytopathology 84:1359–1366. doi:10.1094/Phyto-84-1359

    Article  Google Scholar 

  59. Timmis JN, Ayliffe MA, Huang CY, Martin W (2004) Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5:123–135. doi:10.1038/nrg1271

    Article  CAS  PubMed  Google Scholar 

  60. Toyoshima Y, Onda Y, Shiina T, Nakahira Y (2005) Plastid transcription in higher plants. Crit Rev Plant Sci 24:59–81. doi:10.1080/07352680590910438

    Article  CAS  Google Scholar 

  61. Tripathi S, Suzuki J, Gonsalves D (2006) Development of genetically engineered resistant papaya for Papaya ringspot virus in a timely manner—A comprehensive and successful approach. In: Ronald P (ed) Plant-Pathogen interactions: Methods and protocols. The Humana, New Jersey, pp 197–240

    Chapter  Google Scholar 

  62. Van Droogenbroeck B, Maertens I, Haegeman A, Kyndt T, O’Brien C et al (2005) Maternal inheritance of cytoplasmic organelles in intergeneric hybrids of Carica papaya L. and Vasconcellea spp. (Caricaceae Dumort., Brassicales). Euphytica 143:161–168. doi:10.1007/s10681-005-3156-0

    CAS  Google Scholar 

  63. Vergunst AC, Hooykaas PJJ (1999) Recombination in the plant genome and its application in biotechnology. Crit Rev Plant Sci 18:1–31. doi:10.1016/S0735-2689(99)00385-8

    Article  CAS  Google Scholar 

  64. Wilson AK, Latham JR, Steinbrecher RA (2006) Transformation-induced mutations in transgenic plants: Analysis and biosafety implications. In: Biotechnology and genetic engineering review. Lavoisier/Intercept, Cachan, pp 209–234

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Authors and Affiliations

  1. USDA-ARS Pacific Basin Agricultural Research Center, P.O. Box 4459, 64 Nowelo St., Hilo, HI, 96720, USA

    Jon Y. Suzuki, Savarni Tripathi, Maureen M. M. Fitch, Paul H. Moore & Dennis Gonsalves

  2. Centro Jardín Botánico, Universidad de los Andes, Mérida, Venezuela

    Gustavo A. Fermín

  3. Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, Republic of China

    Fuh-Jyh Jan

  4. Advanced Studies in Genomics, Proteomics and Bioinformatics, University of Hawaii, Honolulu, HI, 96822, USA

    Shaobin Hou, Jimmy H. Saw & Maqsudul Alam

  5. Hawaii Agricultural Research Center, Aiea, Honolulu, HI, 96701, USA

    Christine M. Ackerman, Qingyi Yu & Ray Ming

  6. Center for Bioinformatics and Computational Biology, University of Maryland, College Park, MD, 20742, USA

    Michael C. Schatz & Steven L. Salzberg

  7. Plant and Environmental Protection Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822, USA

    Savarni Tripathi, Karen Y. Pitz & Stephen A. Ferreira

  8. Department of Plant Pathology, Cornell University, Geneva, New York, 14456, USA

    Marcela Yépes

  9. Department of Tropical Plant and Soil Sciences, College of Tropical Agriculture and Human Resources, University of Hawaii, Honolulu, HI, 96822, USA

    Richard M. Manshardt

  10. AureoGen Biosciences, Kalamazoo, MI, 49009, USA

    Jerry L. Slightom

  11. Department of Microbiology, University of Hawaii, Honolulu, HI, 96822, USA

    Jimmy H. Saw & Maqsudul Alam

  12. Department of Plant Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    Ray Ming

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  1. Jon Y. Suzuki
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Correspondence to Dennis Gonsalves.

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Suzuki, J.Y., Tripathi, S., Fermín, G.A. et al. Characterization of Insertion Sites in Rainbow Papaya, the First Commercialized Transgenic Fruit Crop. Tropical Plant Biol. 1, 293–309 (2008). https://doi.org/10.1007/s12042-008-9023-0

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