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Activation of three pathogen-inducible promoters in transgenic citrus (Citrus sinensis Osbeck) after Xanthomonas axonopodis pv. citri infection and wounding

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Abstract

To improve the resistance of citrus to canker disease caused by Xanthomonas axonopodis pv. citri (Xac), it is important to identify gene promoters that are specifically induced by pathogen infection. Here, we evaluated the functionality of PPP1 and hsr203J (Nicotiana tabacum L.) and gst1 (potato) pathogen-inducible promoters to drive expression of the β-glucuronidase (GUS) reporter gene in transgenic citrus. The activities of these promoters in response to the Xac pathogen and wounding were determined quantitatively and qualitatively by fluorometric and histochemical GUS assays, and compared with that of the cauliflower mosaic virus (CaMV) 35S promoter. In citrus, the hsr203J promoter from tobacco was hardly activated by the Xac pathogen or wounding, whereas the PPP1 and gst1 promoters were rapidly and efficiently activated by both inducers. There was very little visible background expression from the PPP1 promoter, but a high level of background expression from the gst1 promoter. Because of its low background expression, the PPP1 promoter was more responsive to Xac and wounding than was the gst1 promoter. However, the gst1 promoter was more rapidly activated than the PPP1 promoter by Xac and wounding. The inducible activity of the two promoters was restricted to infection sites. Taken together, our results showed that the PPP1 promoter was the most efficient promoter among those evaluated in this study. Its strong responsiveness to Xac and wounding suggests that it would be a good candidate for expression of antibacterial transgenes specifically at infection sites to improve canker disease resistance in citrus.

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References

  • Ballester A, Cervera M, Pena L (2008) Evaluation of selection strategies alternative to nptII in genetic transformation of citrus. Plant Cell Rep 27:1005–1015

    Article  PubMed  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 Hortic 122:109–115

    Article  CAS  Google Scholar 

  • Bauer DW, Garr ER, Beer SV, Norelli JL, Aldwinckle HS (1999) New approaches to the development of transgenic plants resistant to fire blight. Plant Dis 87:756–765

    Google Scholar 

  • Belbahri L, Boucher C, Candresse T, Nicole M, Ricci P, Keller H (2001) A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant–pathogen interaction incompatible. Plant J 28:419–430

    Article  PubMed  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Butaye KM, Cammue BP, Delauré SL, De Bolle MF (2005) Approaches to minimize variation of transgene expression in plants. Mol Breed 16:79–91

    Article  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 

  • Cervera M, Pina J, Juârez J, Navarro L, Peña L (1998) Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. Plant Cell Rep 18:271–278

    Article  CAS  Google Scholar 

  • Chen C, Chen Z (2002) Potentiation of developmentally regulated plant defense response by AtWRKY18, a pathogen-induced Arabidopsis transcription factor. Plant Physiol 129:706–716

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • de Oliveira MLP, Lima Silva CC, Abe VY, Costa MGC, Cernadas RA, Benedetti CE (2013) Increased resistance against citrus canker mediated by a citrus MAP kinase. Mol Plant-Microbe Interact 26:1190–1199

    Article  PubMed  CAS  Google Scholar 

  • Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant–pathogen interactions. Nat Rev Genet 11:539–548

    Article  PubMed  CAS  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 

  • Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206

    Article  PubMed  CAS  Google Scholar 

  • Fitzgerald HA, Chern M-S, Navarre R, Ronald PC (2004) Overexpression of (At) NPR1 in rice leads to a BTH-and environment-induced lesion-mimic/cell death phenotype. Mol Plant-Microbe Interact 17:140–151

    Article  PubMed  CAS  Google Scholar 

  • Flachowsky H, Szankowski I, Fischer TC, Richter K, Peil A, Höfer M, Dörschel C, Schmoock S, Gau AE, Halbwirth H, Hanke M-V (2010) Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight. Planta 231:623–635

    Article  PubMed  CAS  Google Scholar 

  • Fu X, Chen C, Wang Y, Liu J, Moriguchi T (2011) Ectopic expression of MdSPDS1 in sweet orange (Citrus sinensis Osbeck) reduces canker susceptibility: involvement of H2O2 production and transcriptional alteration. BMC Plant Biol 11:55

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Gong X, Liu J (2013) Genetic transformation and genes for resistance to abiotic and biotic stresses in Citrus and its related genera. Plant Cell Tissue Organ Cult 113:137–147

    Article  CAS  Google Scholar 

  • Gough C, Hemon P, Tronchet M, Lacomme C, Marco Y, Roby D (1995) Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements. Mol Gen Genet 247:323–337

    Article  PubMed  CAS  Google Scholar 

  • Gurr SJ, Rushton PJ (2005) Engineering plants with increased disease resistance: how are we going to express it? Trends Biotechnol 23:283–290

    Article  PubMed  CAS  Google Scholar 

  • Gust AA, Brunner F, Nürnberger T (2010) Biotechnological concepts for improving plant innate immunity. Curr Opin Biotechnol 21:204–210

    Article  PubMed  CAS  Google Scholar 

  • Hammond-Kosack KE, Harrison K, Jones J (1994) Developmentally regulated cell death on expression of the fungal avirulence gene Avr9 in tomato seedlings carrying the disease-resistance gene Cf-9. Proc Natl Acad Sci USA 91:10445–10449

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • He Y, Chen S, Peng A, Zou X, Xu L, Lei T (2011) Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds. Sci Hortic 128:99–107

    Article  CAS  Google Scholar 

  • Helliwell EE, Wang Q, Yang Y (2013) Transgenic rice with inducible ethylene production exhibits broad-spectrum disease resistance to the fungal pathogens Magnaporthe oryzae and Rhizoctonia solani. Plant Biotechnol J 11:33–42

    Article  PubMed  CAS  Google Scholar 

  • Honée G, Melchers LS, Vleeshouwers VG, van Roekel JS, de Wit PJ (1995) Production of the AVR9 elicitor from the fungal pathogen Cladosporium fulvum in transgenic tobacco and tomato plants. Plant Mol Biol 29:909–920

    Article  PubMed  Google Scholar 

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

    PubMed Central  PubMed  CAS  Google Scholar 

  • Keller H, Pamboukdjian N, Ponchet M, Poupet A, Delon R, Verrier J-L, Roby D, Ricci P (1999) Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance. Plant Cell 11:223–235

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Kobayashi M, Yoshioka M, Asai S, Nomura H, Kuchimura K, Mori H, Doke N, Yoshioka H (2012) StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst. New Phytol 196:223–237

    Article  PubMed  CAS  Google Scholar 

  • Kumar D, Patro S, Ghosh J, Das A, Maiti IB, Dey N (2012) Development of a salicylic acid inducible minimal sub-genomic transcript promoter from Figwort mosaic virus with enhanced root-and leaf-activity using TGACG motif rearrangement. Gene 503:36–47

    Article  PubMed  CAS  Google Scholar 

  • Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16:319–331

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Malnoy M, Venisse J-S, Reynoird J, Chevreau E (2003) Activation of three pathogen-inducible promoters of tobacco in transgenic pear (Pyrus communis L.) after abiotic and biotic elicitation. Planta 216:802–814

    PubMed  CAS  Google Scholar 

  • Malnoy M, Reynoird JP, Borejsza-Wysocka EE, Aldwinckle HS (2006) Activation of the pathogen-inducible Gst1 promoter of potato after elicitation by Venturia inaequalis and Erwinia amylovora in transgenic apple (Malus × domestica). Transgenic Res 15:83–93

    Article  PubMed  CAS  Google Scholar 

  • Martini N, Egen M, Rüntz I, Strittmatter G (1993) Promoter sequences of a potato pathogenesis-related gene mediate transcriptional activation selectively upon fungal infection. Mol Gen Genet 236:179–186

    Article  PubMed  CAS  Google Scholar 

  • McDowell JM, Woffenden BJ (2003) Plant disease resistance genes: recent insights and potential applications. Trends Biotechnol 21:178–183

    Article  PubMed  CAS  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 

  • Miyata LY, Harakava R, Stipp LCL, Mendes BMJ, Appezzato-da-Glória B, Mourao Filho FDAA (2012) GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters. Plant Cell Rep 31:2005–2013

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nadal A, Montero M, Company N, Badosa E, Messeguer J, Montesinos L, Montesinos E, Pla M (2012) Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness. BMC Plant Biol 12:159–179

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Peng J, Bao Z, Li P, Chen G, Wang J, Dong H (2004) HarpinXoo and its functional domains activate pathogen-inducible plant promoters in Arabidopsis. Acta Botanica Sinica 46:1083–1090

    CAS  Google Scholar 

  • Pontier D, Godiard L, Marco Y, Roby D (1994) hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactions. Plant J 5:507–521

    Article  PubMed  CAS  Google Scholar 

  • Pontier D, Tronchet M, Rogowsky P, Lam E, Roby D (1998) Activation of hsr203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death. Mol Plant-Microbe Interact 11:544–554

    Article  PubMed  CAS  Google Scholar 

  • Reynoird JP, Abdul-Kader AM, Bauer DW, Borejsza-Wysocka E, Aldwinckle HS (2000) Activation of the potato GST1 and tobacco HSR203J promoters by Erwinia amylovora in transgenic Royal Gala apple and M. 26 apple rootstock. Plant Mol Biol Rep Suppl 18:22–81

    Google Scholar 

  • Rushton PJ, Reinstädler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen-and wound-induced signaling. Plant Cell 14:749–762

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  • Strittmatter G, Gheysen G, Gianinazzi-Pearson V, Hahn K, Niebel A, Rohde W, Tacke E (1996) Infections with various types of organisms stimulate transcription from a short promoter fragment of the potato gst1 gene. Mol Plant-Microbe Interact 9:68–73

    Article  PubMed  CAS  Google Scholar 

  • Venter M (2007) Synthetic promoters: genetic control through cis engineering. Trends Plant Sci 12:118–124

    Article  PubMed  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 strains. J Gen Virol 26:33–48

    Article  PubMed  CAS  Google Scholar 

  • Xu W, Yu Y, Ding J, Hua Z, Wang Y (2010) Characterization of a novel stilbene synthase promoter involved in pathogen-and stress-inducible expression from Chinese wild Vitis pseudoreticulata. Planta 231:475–487

    Article  PubMed  CAS  Google Scholar 

  • Yang Y, Shah J, Klessig DF (1997) Signal perception and transduction in plant defense responses. Genes Dev 11:1621–1639

    Article  PubMed  CAS  Google Scholar 

  • Yang L, Hu C, Li N, Zhang J, Yan J, Deng Z (2011) Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease. Plant Mol Biol 75:11–23

    Article  PubMed  CAS  Google Scholar 

  • Yevtushenko DP, Misra S (2007) Comparison of pathogen-induced expression and efficacy of two amphibian antimicrobial peptides, MsrA2 and temporin A, for engineering wide-spectrum disease resistance in tobacco. Plant Biotechnol J 5:720–734

    Article  PubMed  CAS  Google Scholar 

  • Yevtushenko DP, Romero R, Forward BS, Hancock RE, Kay WW, Misra S (2005) Pathogen-induced expression of a cecropin A-melittin antimicrobial peptide gene confers antifungal resistance in transgenic tobacco. J Exp Bot 56:1685–1695

    Article  PubMed  CAS  Google Scholar 

  • Zhang J, Peng Y, Guo Z (2008) Constitutive expression of pathogen-inducible OsWRKY31 enhances disease resistance and affects root growth and auxin response in transgenic rice plants. Cell Res 18:508–521

    Article  PubMed  CAS  Google Scholar 

  • Zhang X, Francis MI, Dawson WO, Graham JH, Orbović V, Triplett EW, Mou Z (2010) Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker. Eur J Plant Pathol 128:91–100

    Article  CAS  Google Scholar 

  • Zou X, Li D, Luo X, Luo K, Pei Y (2008) An improved procedure for Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata L. Raf.) via indirect organogenesis. In Vitro Cell Dev Biol Plant 44:169–177

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by grants from the Natural Science Foundation Project of CQ (cstc2011jjA80010 to X. Zou), the Earmarked Fund for China Agriculture Research System (CARS-27 to S. Chen), the National High Technology Research and Development Program of China (2011AA100205, to Y. He), and the National Key Technology Research and Development Program of the Ministry of Science and Technology (201003067-02-4 to S. Chen).

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The authors declare that they have no conflicts of interest.

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Authors and Affiliations

  1. Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing, 400712, People’s Republic of China

    Xiuping Zou, Erling Song, Aihong Peng, Yongrui He, Lanzhen Xu, Tiangang Lei, Lixiao Yao & Shanchun Chen

  2. National Center for Citrus Variety Improvement, Chongqing, 400712, People’s Republic of China

    Xiuping Zou, Aihong Peng, Yongrui He, Lanzhen Xu, Tiangang Lei, Lixiao Yao & Shanchun Chen

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  1. Xiuping Zou
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Correspondence to Xiuping Zou or Shanchun Chen.

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Zou, X., Song, E., Peng, A. et al. Activation of three pathogen-inducible promoters in transgenic citrus (Citrus sinensis Osbeck) after Xanthomonas axonopodis pv. citri infection and wounding. Plant Cell Tiss Organ Cult 117, 85–98 (2014). https://doi.org/10.1007/s11240-013-0423-y

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