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Establishment of protoplast preparation protocol and genetic transformation system for Sclerotiophoma versabilis

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Abstract

Going into deep study about the molecular mechanism of pathogenesis in Sclerotiophoma versabilis required high-efficiency transformation system. S. versabilis is a pathogenic fungus that causes severe leaf spot disease in Pseudostellaria heterophylla, a famous traditional Chinese medicinal plant. Here, we report an appropriate system for the production and regeneration of protoplasts with high viability, and then developed a stable and efficient genetic transformation system based on PEG-mediated transformation of protoplasts for S. versabilis. The optimum condition for protoplast preparation is 3 g of the prepared mycelia resuspended in 30 mL 1 M KCl (pH 5.8) containing 25 mg/mL lysing enzyme, incubated at 30 °C and 85 rpm for 3 h. The regeneration rate of the protoplasts was more than 90%. S. versabilis was highly sensitive to hygromycin B and geneticin, but showed high degree of resistance to zeocin. The sGFP gene was successfully transformed into the S. versabilis protoplasts using a modified PEG-mediated transformation method. PCR amplification and southern blot hybridization. GFP signal was detected in both mycelia and conidia of the transformants by confocal microscopy assay. The transformants showed no phenotypic difference from the wild type strain, which furthers the observation of infection process under the fluorescence microscope. The procedures for protoplast isolation, regeneration and transformation provide a foundation for understanding the mechanism of pathogenesis in S. versabilis.

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Acknowledgements

We thank Wilfred Mabeche Anjago, Jules Biregeya, Penguo Lin and Jiyu Su for their technical support, and Justice Norvienyeku and Huawei Zheng for helpful discussions.

Funding

The research is supported by grant from Scientific Key Project of Ningde Normal University (2019ZDK18).

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Author notes

  1. Yun-bo Kuang and Felix Abah contribute equally to this work.

Authors and Affiliations

  1. The Engineering Technology Research Centre of Characteristic Medicinal Plants of Fujian, College of Life Sciences, Ningde Normal University, Ningde, 352100, China

    Yun-bo Kuang & Zu-yun Ye

  2. Fujian University Key Laboratory for Plant-Microbe Interaction, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, 350002, China

    Yun-bo Kuang, Felix Abah, Yakubu Saddeeq Abubakar & Zong-hua Wang

  3. Fujian University Engineering Research Centre of Marine Biology and Drugs, Fuzhou Institute of Oceanography, Minjiang University, Fuzhou, 350108, China

    Zong-hua Wang

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  1. Yun-bo Kuang
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Contributions

WZ and KY conceived and designed research. FA and KY conducted experiments, FA performed the analysis and drafted the manuscript. KY and AYS reviewed the manuscript. ZY provided samples and new equipment. All authors read and approved the manuscript.

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Correspondence to Yun-bo Kuang.

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The authors consciously ensure that for the manuscript, "Establishment of Protoplast Preparation Protocol and Genetic Transformation System for Sclerotiophoma versabilis", the following are fulfilled: (1) This manuscript is the authors' original work and has not been previously published elsewhere. (2) The paper is not currently being considered for publication elsewhere. (3) The paper properly credits the meaningful contributions of co-authors and co-researchers. (4) The results are appropriately placed in prior and existing research contexts. (5) All resources used in this research were adequately disclosed. (6) All authors were personally and actively involved in substantial work leading to the paper and will take public responsibility for its content.

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Key message

The conditions for protoplast production and regeneration of Sclerotiophoma versabilis were investigated, then an efficient genetic transformation system based on polyethylene glycol -mediated transformation of protoplasts was developed.

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Kuang, Yb., Abah, F., Abubakar, Y.S. et al. Establishment of protoplast preparation protocol and genetic transformation system for Sclerotiophoma versabilis. J Plant Pathol 106, 165–173 (2024). https://doi.org/10.1007/s42161-023-01534-7

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