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Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence

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Abstract

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism.

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Abbreviations

DMBQ:

2,4-Dimethoxybenzoquinone

IAA:

Indole acetic acid

6-BAP:

6-Benzylaminopurine

References

  • Albrecht H, Yoder JI, Phillips DA (1999) Flavonoids promote haustoria formation in the root parasite Triphysaria. Plant Physiol 119:585–591

    Article  PubMed  CAS  Google Scholar 

  • Atsatt P, Strong D (1970) The population biology of annual grassland hemiparasites: I. The host environment. Evolution 24:278–291

    Article  Google Scholar 

  • Baird WV, Riopel JL (1985) Surface characteristics of root haustorial hairs of parasitic Scrophulariaceae. Bot Gazet 146:63–69

    Article  Google Scholar 

  • Bendahmane A, Querci M, Kanyuka K, Baulcombe D (2000) Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant J 21:73–81

    Article  PubMed  CAS  Google Scholar 

  • Boisson-Dernier A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker D (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant Microbe Interact 14:695–700

    PubMed  CAS  Google Scholar 

  • Boone LS, Fate G, Chang M, Lynn DG (1995) Seed germination. In: Press MC, Graves JD (eds) Parasitic plants. Chapman & Hall, New York, pp 14–38

    Google Scholar 

  • CAMBIA (2006) Materials and methods available. Accessed 2006, http://www.cambia.org/daisy/cambia/materials.html

  • Chang J, Tai YS, Bernal A, Lavelle D, Staskawicz B, Michelmore R (2002) Functional analyses of the Pto resistance gene family in tomato and the identification of a minor resistance determinant in a susceptible haplotype. Mol Plant Microbe Interact 15:281–291

    PubMed  CAS  Google Scholar 

  • Chen I, Christie P, Dubnau D (2005) The ins and outs of DNA transfer in bacteria. Science 310:1456–1460

    Article  PubMed  CAS  Google Scholar 

  • Chilton M-D, Tepfer DA, Petit A, David C, Casse-Delbart F, Tempe J (1982) Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells. Nature 295:432–434

    Article  CAS  Google Scholar 

  • Cho H-J, Farrand S, Noel G, Widholm J (2000) High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode. Planta 210:195–204

    Article  PubMed  CAS  Google Scholar 

  • Christey M (2001) Use of Ri-mediated transformation for production of transgenic plants. In Vitro Cell Dev Biol Plant 37:687–670

    Article  CAS  Google Scholar 

  • Crane C, Wright E, Dixon R, Zeng-Yu W (2006) Transgenic Medicago truncatula plants obtained from Agrobacterium tumefaciens-transformed roots and Agrobacterium rhizogenes-transformed hairy roots. Planta 223:1344–1354

    Article  PubMed  CAS  Google Scholar 

  • Dörr I (1996) New results on interspecific bridges between parasites and their hosts. In: Moreno MT, Cubero J, Berber D, Joel D, Muselman L, Parker C (eds) Advances in parasitic plant research; sixth international parasitic weed symposium. Junta de Andalucía Consejería de Agricultura y Pesca, Córdoba, Spain, pp 196–201

    Google Scholar 

  • Dörr I (1997) How Striga parasitizes its host: a TEM and SEM study. Ann Bot 79:463–472

    Article  Google Scholar 

  • Dörr I, Kollman R (1995) Symplastic sieve elements continuity between Orobanche and its host. Botanica Acta 108:47–55

    Google Scholar 

  • Fraley RT, Rogers SC, Horsch BB (1986) Genetic transformation in higher plants. CRC Crit Rev Plant Sci 4:1–46

    Article  CAS  Google Scholar 

  • Gibson CC, Watkinson AR (1989) The host range and selectivity of a parasitic plant: Rhinanthus minor L. Oecologia 78:401–406

    Article  Google Scholar 

  • Goldwasser Y, Westwood JH, Yoder JI (2002) The use of Arabidopsis to study interactions between parasitic angiosperms and their plant hosts. In: Somerville C, Meyerowitz E (eds) The Arabidopsis book. American Society of Plant Biologists, Rockville

    Google Scholar 

  • Hansen J, Jørgensen J-E, Stougaard J, Marcker K (1989) Hairy roots—a short cut to transgenic root nodules. Plant Cell Rep 8:12–15

    Article  Google Scholar 

  • Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127

    Article  PubMed  CAS  Google Scholar 

  • Heide-Jorgensen H, Kuijt J (1993) Epidermal derivatives as xylem elements and transfer cells: a study of the host–parasite interface in two species of Triphysaria (Scrophulariaceae). Protoplasma 174:173–183

    Article  Google Scholar 

  • Heide-Jorgensen HS, Kuijt J (1995) The haustorium of the root parasite Triphysaria (Scrophulariaceae), with special reference to xylem bridge ultrastructure. Am J Bot 82:782–797

    Article  Google Scholar 

  • Hibberd JM, Dieter Jeschke W (2001) Solute flux into parasitic plants. J Exp Bot 52:2043–2049

    Article  PubMed  CAS  Google Scholar 

  • Hoagland DR, Arnon DI (1938) The water-culture method for growing plants without soil. Univ Calif Coll Agric Exp Sta Circ, Berkeley, pp 347–353

    Google Scholar 

  • Hoekema A, Hirsch P, Hooykaas P, Schilperoort R (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180

    Article  CAS  Google Scholar 

  • Holsters M, Silva B, Genetello C, Engler C, Vliet F, van de Block M, Villarroel R, van Montagu M, Schell J (1978a) Spontaneous formation of cointegrates of the oncogenic Ti plasmid and the wide host range P-plasmid RP4. Plasmid 1:456–446

    Article  CAS  Google Scholar 

  • Holsters M, Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978b) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187

    Article  CAS  Google Scholar 

  • Hwang C-F, Bhakta A, Truesdell G, Pudlo W, Williamson V (2000) Evidence for a role of the N terminus and leucine-rich repeat region of the Mi gene product in regulation of localized cell death. Plant Cell 12:1319–1329

    Article  PubMed  CAS  Google Scholar 

  • Jamison DS, Yoder JI (2001) Heritable variation in quinone-induced haustorium development in the parasitic plant Triphysaria. Plant Physiol 125:1870–1879

    Article  PubMed  CAS  Google Scholar 

  • Janssen B, Gardner R (1990) Localized transient expression of gus in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol Rep 14:61–72

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: B-glucuronidase as a sensitive and verstile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jensen J, Marcker K, Otten L, Schell J (1986) Nodule-specific expression of a chimaeric soybean leghaemoglobin gene in transgenic Lotus corniculatus. Science 321:669–674

    CAS  Google Scholar 

  • Johansen L, Carrington J (2001) Silencing on the spot induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126:930–938

    Article  PubMed  CAS  Google Scholar 

  • Kuijt J (1969) The biology of parasitic flowering plants. University of California Press, Berkeley

    Google Scholar 

  • Kuijt J, Toth R (1976) Ultrastructure of angiosperm haustoria—a review. Ann Bot 40:1121–1130

    Google Scholar 

  • Kumagai H, Kouchi H (2003) Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules. Mol Plant Microbe Interact 16:663–668

    PubMed  CAS  Google Scholar 

  • Lichtenstein C, Fuller S (1987) Vectors for the genetic engineering of plants. Genet Eng 6:103–183

    Google Scholar 

  • Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H, Bisseling T, Geurts R (2004) RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula. J Exp Bot 55:983–992

    Article  PubMed  CAS  Google Scholar 

  • Lynn DG, Chang M (1990) Phenolic signals in cohabitation: implications for plant development. Ann Rev Plant Phys Plant Mol Biol 41:497–526

    Article  CAS  Google Scholar 

  • Minlong C, Takayanagi K, Kamada H, Nishimura S, Handa T (2000) Transformation of Antirrhinum majus L by a rol-type multi-auto-transformation (MAT) vector system. Plant Sci 159:273–280

    Article  PubMed  CAS  Google Scholar 

  • Musselman LJ (1980) The biology of Striga, Orobanche, and other root parasitic weeds. Ann Rev Phytopath 18:463–489

    Article  Google Scholar 

  • Nickrent D (2005) Parasitic plant connection. Southern Illinois University, Carbondale, Accessed 2006, http://www.parasiticplants.siu.edu/usingPPC.html

  • Olmstead RG, DePamphilis CW, Wolfe AD, Young ND, Elisons WJ, Reeves PA (2001) Disintegration of the Scrophulariaceae. Am J Bot 88:348–361

    Article  PubMed  CAS  Google Scholar 

  • Parker C, Riches CR (1993) Parasitic weeds of the world: biology and control. CAB International, Wallingford

    Google Scholar 

  • Petit A, Stougaard J, Kühle A, Marcker K, Tempé J (1987) Transformation and regeneration of the legume Lotus corniculatus: a system for molecular studies of symbiotic nitrogen fixation. Mol Gen Genet 207:245–250

    Article  CAS  Google Scholar 

  • Riopel JL, Timko MP (1995) Haustorial initiation and differentiation. In: Press MC, Graves JD (eds) Parasitic plants. Chapman & Hall, London, pp 39–79

    Google Scholar 

  • Rogers SO, Bendish AJ (1988) Extraction of DNA from plant tissues. In: Gelvin SB, Schilperoort RA (eds) Plant Molecular Biology Manual. Kluwer Academic Publishers, Dordrecht, pp 1–10

    Google Scholar 

  • Ruzin S (1999) Plant microtechnique and microscopy. Oxford University Press, New York

    Google Scholar 

  • Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Schob H, Kunz C, Meins F (1997) Silencing of transgenes introduced into leaves by agroinfiltration: a simple, rapid method for investigating sequence requirements for gene silencing. Mol Gen Genet 256:581–585

    Article  PubMed  CAS  Google Scholar 

  • Sonti R, Chiurazzi M, Wong D, Davies C, Harlow G, Mount D, Signer E (1995) Arabidopsis mutant deficient in T-DNA integration. Proc Natl Acad Sci 92:11786–11790

    Article  PubMed  CAS  Google Scholar 

  • Subramanian C, Woo J-C, Cai X, Xu X, Servick S, Johnson C, Nebenführ A, von Arnim A (2006) A suite of tools and application notes for in vivo protein interaction assays using bioluminescence resonance energy transfer (BRET). Plant J (in press)

  • Tepfer D (1984) Transformation of several species of higher plants by Agrobacterium rhizogenes: sexual transmission of the transformed genotype and phenotype. Cell 37:959–967

    Article  PubMed  CAS  Google Scholar 

  • Tepfer D, Metzger L, Prost R (1989) Use of roots transformed by Agrobacterium rhizogenes in rhizosphere research: applications in studies of cadmium assimilation from sewage sludges. Plant Mol Biol 13:295–302

    Article  PubMed  CAS  Google Scholar 

  • Tomilov A, Tomilova N, Yoder JI (2004) In vitro haustorium development in roots and root cultures on the hemiparasitic plant Triphysaria versicolor. Plant Cell Tissue Organ Cult 77:257–265

    Article  Google Scholar 

  • Tomilov AA, Tomilova NB, Abdallah I, Yoder JI (2005) Localized hormone fluxes and early haustorium development in the hemiparasitic plant Triphysaria versicolor. Plant Physiol 138:1469–1480

    Article  PubMed  CAS  Google Scholar 

  • Torres MJ, Tomilov AA, Tomilova N, Reagan RL, Yoder JI (2005) Pscroph, a parasitic plant EST database enriched for parasite associated transcripts. BMC Plant Biol 24:24

    Article  CAS  Google Scholar 

  • Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium. J Gen Virol 26:33–48

    Article  PubMed  CAS  Google Scholar 

  • Vilaine F, Casse-Delbart F (1987) Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhizogenes. Mol Gen Genet 206:17–23

    Article  CAS  Google Scholar 

  • Visser R, Jacobsen E, Witholt B, Feenstra W (1989) Efficient transformation of potato Solanum-tuberosum using a binary vector in Agrobacterium rhizogenes. Theor Appl Genet 78:594–600

    Article  CAS  Google Scholar 

  • Wigchert SCM, Zwanenburg B (1999) A critical account on the inception of Striga seed germination. J Agric Food Chem V47:1320–1325

    Article  Google Scholar 

  • Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotech J 3:259–273

    Article  CAS  Google Scholar 

  • Yoder JI (2001) Host–plant recognition by parasitic Scrophulariaceae. Curr Opin Plant Biol 4:359–365

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant from the NSF No. 0236545. The authors wish to thank Tadeusz Wroblewski for advice and resources and Tatiana Filippova and Pradeepa Gunathilake for excellent technical assistance.

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Authors and Affiliations

  1. Department of Plant Sciences, University of California Davis, Davis, CA, 95616, USA

    Alexey Tomilov, Natalya Tomilova & John I. Yoder

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  1. Alexey Tomilov
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  2. Natalya Tomilova
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  3. John I. Yoder
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Correspondence to John I. Yoder.

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Alexey Tomilov and Natalya Tomilova made an equal contribution in the paper.

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Tomilov, A., Tomilova, N. & Yoder, J.I. Agrobacterium tumefaciens and Agrobacterium rhizogenes transformed roots of the parasitic plant Triphysaria versicolor retain parasitic competence. Planta 225, 1059–1071 (2007). https://doi.org/10.1007/s00425-006-0415-9

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