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The use of stable and unstable green fluorescent proteins for studies in two bacterial models: Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris

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Abstract

Fluorescent proteins have been used to track plant pathogens to understand their host interactions. To be useful, the transgenic pathogens must present similar behaviour than the wild-type isolates. Herein, a GFP marker was used to transform two plant pathogenic bacteria, Agrobacterium and Xanthomonas, to localize and track the bacteria during infection. The transgenic bacteria were evaluated to determine whether they showed the same fitness than the wild-type strains or whether the expression of the GFP protein interfered in the bacterial activity. In Agrobacterium, the plasmid used for transformation was stable in the bacteria and the strain kept the virulence, while Xanthomonas was not able to conserve the plasmid and transformed strains showed virulence variations compared to wild-type strains. Although marking bacteria with GFP to track infection in plants is a common issue, works to validate the transgenic strains and corroborate their fitness are not usual. Results, presented here, confirm the importance of proper fitness tests on the marked strains before performing localization assays, to avoid underestimation of the microbe population or possible artificial effects in its interaction with the plant.

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Acknowledgements

We thank R. Vallejo, E. Ferragud and M. Sáez for their technical assistance and Instituto Valenciano de Investigaciones Agrarias (IVIA) for providing C58 and X1609 strains. The research was supported by the Florida Citrus Production Research Advisory Council, FDOC contract 00079694, and Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) project RTA2006-00149. We thank J.B. Jones for kindly providing plasmid pUFZ75.

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Authors and Affiliations

  1. Laboratorio de Bacteriología. Departamento de Protección Vegetal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Madrid, Spain

    Pilar Sabuquillo, Adela Gea & Jaime Cubero

  2. Área de Genética, Facultad de Ciencias, Instituto de Hortofruticultura Subtropical y Mediterránea ‘La Mayora’, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Málaga, Spain

    Isabel M. Matas & Cayo Ramos

  3. Instituto de Agrobiotecnología, CSIC-UPNA, Gobierno de Navarra, 31192, Mutilva, Navarra, Spain

    Isabel M. Matas

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  1. Pilar Sabuquillo
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Correspondence to Jaime Cubero.

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Communicated by Yusuf Akhter.

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Sabuquillo, P., Gea, A., Matas, I.M. et al. The use of stable and unstable green fluorescent proteins for studies in two bacterial models: Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris . Arch Microbiol 199, 581–590 (2017). https://doi.org/10.1007/s00203-016-1327-0

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