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Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease

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Abstract

Transgenic expression of the pepper Bs2 gene confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv) pathogenic strains which contain the avrBs2 avirulence gene in susceptible pepper and tomato varieties. The avrBs2 gene is highly conserved among members of the Xanthomonas genus, and the avrBs2 of Xcv shares 96% homology with the avrBs2 of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. A previous study showed that the transient expression of pepper Bs2 in lemon leaves reduced canker formation and induced plant defence mechanisms. In this work, the effect of the stable expression of Bs2 gene on citrus canker resistance was evaluated in transgenic plants of Citrus sinensis cv. Pineapple. Interestingly, Agrobacterium-mediated transformation of epicotyls was unsuccessful when a constitutive promoter (2× CaMV 35S) was used in the plasmid construction, but seven transgenic lines were obtained with a genetic construction harbouring Bs2 under the control of a pathogen-inducible promoter, from glutathione S-transferase gene from potato. A reduction of disease symptoms of up to 70% was observed in transgenic lines expressing Bs2 with respect to non-transformed control plants. This reduction was directly dependent on the Xcc avrBs2 gene since no effect was observed when a mutant strain of Xcc with a disruption in avrBs2 gene was used for inoculations. Additionally, a canker symptom reduction was correlated with levels of the Bs2 expression in transgenic plants, as assessed by real-time qPCR, and accompanied by the production of reactive oxygen species. These results indicate that the pepper Bs2 resistance gene is also functional in a family other than the Solanaceae, and could be considered for canker control.

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Acknowledgements

This project was supported by Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) and the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, PICT-2006-02082; PICT-2011-1833). We especially thank Dr. Beer for providing the pCPP1300 transformation plasmid, Dr. Pardo (ITANOA, Argentina) for the assistance in figures edition and Dr. Welin (ITANOA, Argentina), Dr. Felker (UCLA, EEUU) and Ms. Montivero for the critical reading of this manuscript and for reviewing the English version.

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    1. Estación Experimental Agroindustrial Obispo Colombres (EEAOC) – Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA), Av. William Cross 3150, T4101XAC, Las Talitas, Tucumán, Argentina

      Lorena Noelia Sendín, Ingrid Georgina Orce, Rocío Liliana Gómez, Ramón Enrique, Carlos Froilán Grellet Bournonville, Aldo Sergio Noguera, Atilio Pedro Castagnaro & María Paula Filippone

    2. Instituto de Ciencia y Tecnología Dr. Cesar Milstein, Fundación Pablo Cassará, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Saladillo 2468, C1440FFX, Buenos Aires, Argentina

      Adrián Alberto Vojnov

    3. Facultad de Bioquímica y Farmacia-Instituto de Biología Celular y Molecular de Rosario, Universidad Nacional de Rosario, Suipacha 590, S2002LRK, Rosario, Argentina

      María Rosa Marano

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    1. Lorena Noelia Sendín
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    Correspondence to María Paula Filippone.

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    Lorena Noelia Sendín and Ingrid Georgina Orce have contributed equally to this work.

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    11103_2017_586_MOESM1_ESM.jpg

    Supplementary material Fig. S1 Molecular characterization of Bs2-transgenic lines. Analysis of copy number of L28 and L46 plants by means of Southern blot using HindIII enzyme with Bs2-probe; NTC: Non-transgenic control plant; C+: pGST1-BS2 (JPG 1109 KB)

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    Sendín, L.N., Orce, I.G., Gómez, R.L. et al. Inducible expression of Bs2 R gene from Capsicum chacoense in sweet orange (Citrus sinensis L. Osbeck) confers enhanced resistance to citrus canker disease. Plant Mol Biol 93, 607–621 (2017). https://doi.org/10.1007/s11103-017-0586-8

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