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Transgenic ‘Hamlin’ sweet orange expressing csd1 or d4e1 genes exhibits decreased susceptibility to citrus canker disease

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Abstract

Currently, there are no resistant sweet orange cultivars to the citrus canker (cc) [Xanthomonas citri subsp. citri (Xcc)], and the production of transgenic plants resistant to cc can be an efficient solution in the search of improved cultivars. Several strategies can be applied in this scenario, including the expression of genes that code for antimicrobial peptides and resistance genes from Xcc-tolerant cultivars. We evaluated the reaction of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) transformed with a gene that codes for the D4E1 synthetic antimicrobial peptide, and ‘Hamlin’ sweet orange plants transformed with the gene that codes for a copper and zinc superoxide dismutase (csd1), isolated from Poncirus trifoliata, after inoculation with Xcc. Transgenic plants were propagated through grafting in ‘Rangpur’ lime (Citrus limonia Osbeck), and then inoculated with a Xcc suspension. Hydrogen peroxide production, activity of selected antioxidant enzymes and callose deposition were evaluated in the transgenic plants subjected to Xcc inoculation. The incidence and the severity of citrus canker were significantly reduced in several transgenic events, which also exhibited lower Xcc titers when compared with non-transgenic plants. Transgenic events which exhibited higher superoxide dismutase activities also showed increased accumulation of hydrogen peroxide in foliar tissues after Xcc inoculation. Epifluorescence microscopy analyses revealed a high callose deposition in the sieve tube elements of leaf tissues in some transgenic events. Callose accumulation is an important plant defense response to biotic stresses and, in csd1 transgenic lines, it was associated with lower Xcc populations.

Key message

Transgenic sweet orange expressing genes by two different disease resistance strategies exhibited less susceptibility to citrus canker with potential to be further studied and integrated into mitigation of this disease.

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Data availability

The datasets of the current study are presented in this article or are available upon a reasonable request.

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Acknowledgements

The authors thank Dr. Marcia Eugenia Amaral Carvalho, for her critical comments on the manuscript, and Dr. Beatriz Appezzato-da-Glória, for providing the structure for histological analysis. The authors also thank Dr. Beatriz Madalena Januzzi Mendes, for technical support during the production of the csd1 transgenic plants, and Dr. Caio Morais de Alcântara Barbosa, for support to produce and organize data into figures. Fundacao Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) INCTII-Citros and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support.

Funding

This work was supported by INCTII-Citros (CNPq 465440/2014-2; FAPESP 2014/50880-0), and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Author notes

  1. João Paulo Rodrigues Marques

    Present address: Departamento de Ciências Básicas, Faculdade de Zootecnica e Engenharia de Alimentos, Universidade de São Paulo. Av. Duque de Caxias, 225, Pirassununga, 13635-900, Brazil

Authors and Affiliations

  1. Departamento de Produção Vegetal, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias, 11, Piracicaba, 13418-900, Brazil

    Matheus Luís Docema, Lísia Borges Attílio, Liliane Cristina Liborio Stipp & Francisco de Assis Alves Mourão Filho

  2. Departamento de Ciências Biológicas, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias, 11, Piracicaba, 13418-900, Brazil

    Tatiana de Souza Moraes

  3. Laboratório de Instrumentação Nuclear, Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Av. Centenário, 303, Piracicaba, 13416-000, Brazil

    João Paulo Rodrigues Marques

  4. Laboratório de Bioquímica Fitopatológica, Instituto Biológico, Av. Conselheiro Rodrigues Alves, 1252, São Paulo, 04014-002, Brazil

    Ricardo Harakava

  5. Embrapa Mandioca e Fruticultura and Instituto Biológico, Av. Conselheiro Rodrigues Alves, 1252, São Paulo, 04014-002, Brazil

    Juliana Freitas-Astúa

  6. Departamento de Fitopatologia e Nematologia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias, 11, Piracicaba, 13418-900, Brazil

    José Belasque Junior

  7. Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Av. Pádua Dias, 11, Piracicaba, 13418-900, Brazil

    Ricardo Antunes Azevedo & Salete Aparecida Gaziola

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  1. Matheus Luís Docema
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Contributions

MLD: investigation, data curation, writing—original draft. TdSM: investigation, data curation. LBA: investigation, data curation, JPRM: investigation, data curation, writing—review & editing. LCLS: investigation, data curation, RH: conceptualization. JF-A: conceptualization. JBJ: conceptualization. RAA: writing—review & editing. SAG: investigation, writing—review & editing. FdAAMF: conceptualization, project administration, writing—review & editing.

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Correspondence to Francisco de Assis Alves Mourão Filho.

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Communicated by Wenwu Guo.

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Docema, M.L., Moraes, T.S., Attílio, L.B. et al. Transgenic ‘Hamlin’ sweet orange expressing csd1 or d4e1 genes exhibits decreased susceptibility to citrus canker disease. Plant Cell Tiss Organ Cult 152, 471–489 (2023). https://doi.org/10.1007/s11240-022-02420-3

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