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GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

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Abstract

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the β-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of ‘Hamlin’, ‘Pera’, and ‘Valencia’ sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

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References

  • Albrecht U, Bowman KD (2008) Gene expression in Citrus sinensis (L.) Osbeck following infection with the bacterial pathogen Candidatus Liberibacter asiaticus causing Huanglongbing in Florida. Plant Sci 175:291–306

    Article  CAS  Google Scholar 

  • Azevedo FA, Mourão Filho FAA, Mendes BMJ, Almeida WAB, Schinor EH, Pio R, Barbosa JM, Guidetti-Gonzalez S, Carrer H, Lam E (2006) Genetic transformation of Rangpur lime (Citrus limonia Osbeck) with the bO (bacterio-opsin) gene and its initial evaluation for Phytophthora nicotianae resistance. Plant Mol Biol Report 24:185–196

    Article  CAS  Google Scholar 

  • Barbosa-Mendes JM, Mourão Filho FAA, Bergamin Filho A, Harakava R, Beer SV, Mendes BMJ (2009) Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker. Sci Hort 122:109–115

    Article  CAS  Google Scholar 

  • Bolar JP, Norelli JL, Wong KW, Hayes CK, Harman GE, Aldwinckle HS (2000) Expression of endochitinase from Trichoderma harziannum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathol 90:72–77

    Article  CAS  Google Scholar 

  • Boscariol RL, Monteiro M, Takahashi EK, Chabregas SM, Vieira MLC, Vieira LGE, Pereira LFP, Mourão Filho FAA, Cardoso SC, Christiano RSC, Bergamin Filho A, Barbosa JM, Azevedo FA, Mendes BMJ (2006) Attacin A gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis ‘Hamlin’. J Am Soc Hort Sci 131:530–536

    CAS  Google Scholar 

  • Bostwick DE, Dannenhoffer JM, Skaggs MI, Lister RM, Larkins BA, Thompson GA (1992) Pumpkin phloem lectin genes are specifically expressed in companion cells. Plant Cell 4:1539–1548

    PubMed  CAS  Google Scholar 

  • Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 88:7–37

    Google Scholar 

  • Cardoso SC, Barbosa-Mendes JM, Boscariol-Camargo RL, Christiano RSC, Bergamin Filho A, Vieira MLC, Mendes BMJ, Mourão Filho FAA (2010) Transgenic sweet orange (Citrus sinensis L. Osbeck) expressing the attacin A gene for resistance to Xanthomonas citri subsp. citri. Plant Mol Biol Rep 28:185–192

    Article  CAS  Google Scholar 

  • Collinge DB, Lund OS, Thordal-Christensen H (2008) What are the prospects for genetically engineered, disease resistant plants? Eur J Plant Pathol 121:217–231

    Article  CAS  Google Scholar 

  • Distefano G, La Malfa S, Vitale A, Lorito M, Deng Z, Gentile A (2008) Defence-related gene expression in transgenic lemon plants producing an antimicrobial Trichoderma harzianum endochitinase during fungal infection. Transgenic Res 17:873–879

    Article  PubMed  CAS  Google Scholar 

  • Domínguez A, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2000) Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep 19:427–433

    Article  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Dutt M, Ananthakrishnan G, Jaromin MK, Brlansky RH, Grosser JW (2012) Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiol 32:83–93

    Article  PubMed  CAS  Google Scholar 

  • Fagoaga C, Rodrigo I, Conejero V, Hinarejos C, Tuset JJ, Arnau J, Pina JA, Navarro L, Peña L (2001) Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5. Mol Breed 7:175–185

    Article  CAS  Google Scholar 

  • Fagoaga C, López C, Mendoza AH, Moreno P, Navarro L, Flores R, Peña L (2006) Post-transcriptional gene silencing of the p23 silencing supressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Mol Biol 60:153–165

    Article  PubMed  CAS  Google Scholar 

  • Faize M, Sourice S, Dupuis F, Parisi L, Gautier MF, Chevreau E (2004) Expression of wheat puroindoline-b reduces scab susceptibility in transgenic apple (Malus × domestica Borkh.). Plant Sci 167:347–354

    Article  CAS  Google Scholar 

  • Febres VJ, Niblett CL, Lee RF, Moore GA (2003) Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Rep 21:421–428

    PubMed  CAS  Google Scholar 

  • Febres VJ, Lee RF, Moore GA (2008) Transgenic resistance to Citrus tristeza virus in grapefruit. Plant Cell Rep 27:93–104

    Article  PubMed  CAS  Google Scholar 

  • Gentile A, Deng Z, La Malfa S, Distefano G, Domina F, Vitale A, Polizzi G, Lorito M, Tribulato E (2007) Enhanced resistance to Phoma tracheiphila and Botrytis cinerea in transgenic lemon plants expressing a Trichoderma harzianum chitinase gene. Plant Breed 126:146–151

    Article  CAS  Google Scholar 

  • Gómez G, Pallás V (2001) Identification of an in vitro ribonucleoprotein complex between a viroid RNA and a phloem protein from cucumber plants. Mol Plant Microbe Interact 14:910–913

    Article  PubMed  Google Scholar 

  • Gottwald JR, Krysan PJ, Young JC, Evert RF, Sussman MR (2000) Genetic evidence for the in plant role of phloem-specific plasma membrane sucrose transporters. Proc Natl Acad Sci USA 97:13979–13984

    Article  PubMed  CAS  Google Scholar 

  • Guo H, Chen X, Zhang H, Fang R, Yuan Z, Zhang Z-S, Tian Y (2004) Characterization and activity enhancement of the phloem-specific pumpkin PP2 gene promoter. Transgenic Res 13:559–566

    Article  PubMed  CAS  Google Scholar 

  • Gutiérrez-E MA, Luth D, Moore GA (1997) Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of Citrus tristeza virus. Plant Cell Rep 16:745–753

    Article  Google Scholar 

  • Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol 5:387–405

    Article  CAS  Google Scholar 

  • Kayim M, Ceccardi TL, Beretta MJG, Barthe GA, Derrick KS (2004) Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. × Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation. Plant Cell Rep 23:377–385

    Article  PubMed  CAS  Google Scholar 

  • Kim J, Sagaram US, Burns JK, Wang N (2009) Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 99:50–57

    Article  PubMed  Google Scholar 

  • Martin T, Frommer WB, Salanoubat M, Willmitzer L (1993) Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs. Plant J 4:367–377

    Article  PubMed  CAS  Google Scholar 

  • Mendes BMJ, Boscariol RL, Mourão Filho FAA, Almeida WAB (2002) Agrobacterium-mediated transformation of ‘Hamlin’ sweet orange. Pesqui Agropecu Bras 37:955–961

    Article  Google Scholar 

  • Mendes BMJ, Cardoso SC, Boscariol-Camargo RL, Cruz RB, Mourão Filho FAA, Bergamin Filho A (2010) Reduction in susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis expressing the rice Xa21 gene. Plant Pathol 59:68–75

    Article  CAS  Google Scholar 

  • Mitani N, Kobayashi S, Nishizawa Y, Kuniga T, Matsumoto R (2006) Transformation of trifoliate orange with rice chitinase gene and the use of the transformed plant as a rootstock. Sci Hort 108:439–441

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15:473–497

    Google Scholar 

  • Murashige T, Tucker DPH (1969) Growth factor requirements of citrus tissue culture. Proc 1st Int Citrus Symp, pp 1155–1161

  • Owens RA, Blackburn M, Ding B (2001) Possible involvement of the phloem lectin in long-distance viroid movement. Mol Plant Microbe Interact 14:905–909

    Article  PubMed  CAS  Google Scholar 

  • Paoli LG, Boscariol-Camargo RL, Harakava R, Mendes BMJ, Mourão Filho FAA (2007) Transformação genética de laranja ‘Valência’ com o gene cecropin MB39. Pesqui Agropecu Bras 42:1663–1666

    Article  Google Scholar 

  • Peña L, Cervera M, Juárez J, Navarro A, Pina JA, Navarro L (1997) Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep 16:731–737

    Article  Google Scholar 

  • Peña L, Cervera M, Fagoaga C, Romero J, Ballester A, Soler N, Pons E, Rodríguez A, Peris J, Juárez J, Navarro L (2008) Citrus. In: Chittaranjan K, Hall TC (eds) Compendium of transgenic crops plants: transgenic tropical and subtropical fruits and nuts. Blackwell Publishing Ltd, Oxford, pp 1–61

    Google Scholar 

  • Ravelonandro M, Scorza R, Callahan A, Levy L, Jacquet C, Monsion M, Damstreegt V (2000) The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model. Virus Res 71:63–69

    Article  PubMed  CAS  Google Scholar 

  • Read SM, Northcote DH (1983) Chemical and immunological similarities between the phloem proteins of three genera of the Cucurbitaceae. Planta 158:119–127

    Article  CAS  Google Scholar 

  • Sauer N (2007) Molecular physiology of higher plant sucrose transporters. FEBS Lett 581:2309–2317

    Article  PubMed  CAS  Google Scholar 

  • Sauer N, Stolz J (1994) SUC1 and SUC2: two sucrose transporters from Arabidopsis thaliana, expression and characterization in baker’s yeast and identification of the histidine-tagged protein. Plant J 6:67–77

    Article  PubMed  CAS  Google Scholar 

  • Scorza R, Callahan A, Levy L, Damsteegt V, Weeb K, Ravelonandro M (2001) Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum pox potyvirus coat protein gene. Transgenic Res 10:201–209

    Article  PubMed  CAS  Google Scholar 

  • Singer SD, Hily J-M, Cox KD (2011) The sucrose synthase-1 promoter from Citrus sinensis directs expression of the β-glucuronidase reporter gene in phloem tissue and in response to wounding in transgenic plants. Planta 234:623–637

    Article  PubMed  CAS  Google Scholar 

  • Stadler R, Sauer N (1996) The Arabidopsis thaliana AtSUC2 gene is specifically expressed in companion cells. Bot Acta 109:299–306

    CAS  Google Scholar 

  • Tennant PF, Gonsalves C, Ling KS, Fitch M, Manshardt R, Slightom JL, Gonsalves D (1994) Differential protection against Papaya ringspot virus isolates in coat protein gene transgenic papaya and classically cross-protected papaya. Phytopathol 84:1359–1366

    Article  Google Scholar 

  • Truernit E, Sauer N (1995) The promoter of the Arabidopsis thaliana SUC2 sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem: evidence for phloem loading and unloading by SUC2. Planta 196:564–570

    Article  PubMed  CAS  Google Scholar 

  • Wang M, Boulter D, Gatehouse JA (1992) A complete sequence of the rice sucrose synthase-1 (RSs1) gene. Plant Mol Biol 19:881–885

    Article  PubMed  CAS  Google Scholar 

  • Yang NS, Russel D (1990) Maize sucrose synthase-1 promoter directs phloem cell-specific expression of Gus gene in transgenic tobacco plants. Proc Natl Acad Sci USA 87:4144–4148

    Article  PubMed  CAS  Google Scholar 

  • Yang ZN, Ingelbrecht IL, Louzada E, Skaria M, Mirkov TE (2000) Agrobacterium-mediated transformation of the commercially importance grapefruit cultivar Rio Red (Citrus paradisi Macf.). Plant Cell Rep 19:1203–1211

    Article  CAS  Google Scholar 

  • Zanek MC, Reyes CA, Cervera M, Peña EJ, Velázquez K, Costa N, Plata MI, Gray O, Peña L, García ML (2008) Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus. Plant Cell Rep 27:57–66

    Article  PubMed  CAS  Google Scholar 

  • Zhao Y, Liu Q, Davis RE (2004) Transgene expression in strawberries driven by a heterologous phloem-specific promoter. Plant Cell Rep 23:224–230

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the M.Sc. student fellowship to the first author. The second, fourth, fifth and sixth authors acknowledge CNPq for research fellowships. The authors thank Fundo de Defesa da Citricultura (Fundecitrus) and CNPq for financial support (Proc. 474184/2008-0). The authors thank Centro APTA Citros Sylvio Moreira for kindly providing part of the germplasm material for the experiment. The authors thank Dr. Marcio Gilberto Cardoso Costa for providing the gene construct utilized as the positive control in the histochemical GUS assay. The authors also acknowledge Ms. Flavia Zambon and Meire Menezes Bassan for technical support, and Dr. Jay Lee Schell for critical comments.

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Authors and Affiliations

  1. Departamento de Produção Vegetal, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, SP, 13418-900, Brazil

    Luzia Yuriko Miyata, Liliane Cristina Libório Stipp & Francisco de Assis Alves Mourão Filho

  2. Instituto Biológico, Seção de Bioquímica Fitopatológica, São Paulo, SP, 04014-002, Brazil

    Ricardo Harakava

  3. Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, SP, 13416-000, Brazil

    Beatriz Madalena Januzzi Mendes

  4. Departamento de Ciências Biológicas, Universidade de São Paulo, Escola Superior de Agricultura “Luiz de Queiroz”, Piracicaba, SP, 13418-900, Brazil

    Beatriz Appezzato-da-Glória

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  1. Luzia Yuriko Miyata
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  2. Ricardo Harakava
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Correspondence to Francisco de Assis Alves Mourão Filho.

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Communicated by L. Peña.

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Miyata, L.Y., Harakava, R., Stipp, L.C.L. et al. GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters. Plant Cell Rep 31, 2005–2013 (2012). https://doi.org/10.1007/s00299-012-1312-2

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