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Protoplast-mediated transformation in Sporisorium scitamineum facilitates visualization of in planta developmental stages in sugarcane

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Abstract

Background

Sporisorium scitamineum is the causative agent of smut disease in sugarcane. The tricky life cycle of S. scitamineum consists of three distinct growth stages: diploid teliospores, haploid sporidia and dikaryotic mycelia. Compatible haploid sporidia representing opposite mating types (MAT-1 and MAT-2) of the fungus fuse to form infective dikaryotic mycelia in the host tissues, leading to the development of a characteristic whip shaped sorus. In this study, the transition of distinct stages of in vitro life cycle and in planta developmental stages of S. scitamineum are presented by generating stable GFP transformants of S. scitamineum.

Methods and results

Haploid sporidia were isolated from the teliospores of Ss97009, and the opposite mating types (MAT-1 and MAT-2) were identified by random mating assay and mating type-specific PCR. Both haploid sporidia were individually transformed with pNIIST plasmid, harboring an enhanced green fluorescent protein (eGFP) gene and hygromycin gene by a modified protoplast-based PEG-mediated transformation method. Thereafter, the distinct in vitro developmental stages including fusion of haploid sporidia and formation of dikaryotic mycelia expressing GFP were demonstrated. To visualize in planta colonization, transformed haploids (MAT-1gfp and MAT-2gfp) were fused and inoculated onto the smut susceptible sugarcane cultivar, Co 97009 and examined microscopically at different stages of colonization. GFP fluorescence-based analysis presented an extensive fungal colonization of the bud surface as well as inter- and intracellular colonization of the transformed S. scitamineum in sugarcane tissues during initial stages of disease development. Noticeably, the GFP-tagged S. scitamineum led to the emergence of smut whips, which established their pathogenicity, and demonstrated initial colonization, active sporogenesis and teliospore maturation stages.

Conclusion

Overall, for the first time, an efficient protoplast-based transformation method was employed to depict clear-cut developmental stages in vitro and in planta using GFP-tagged strains for better understanding of S. scitamineum life cycle development.

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Acknowledgements

The authors are grateful to The Director, ICAR-Sugarcane Breeding Institute for providing facilities and continuous encouragement. The financial support received from Department of Biotechnology (DBT), Department of Science and Technology - Science and Engineering Research Board (DST-SERB) and Indian Council of Agricultural Research (ICAR), New Delhi are greatly acknowledged. We greatly acknowledge Dr. Rajeev Sukumaran, CSIR- National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala for sharing the pNIIST plasmid.

Funding

This work was financially supported by the Department of Biotechnology (DBT) (Sanction No. BT/PR12883/BPA/118/142/2015) and Department of Science and Technology - Science and Engineering Research Board (DST-SERB) (Sanction Order No. EMR/2016/006055) in the form of research grants to the corresponding author.

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Authors and Affiliations

  1. Plant Pathology Section, Division of Crop Protection, ICAR-Sugarcane Breeding Institute, Coimbatore, Tamil Nadu, 641007, India

    V. N. Agisha, N. M. R. Ashwin, R. T. Vinodhini, Kumaravel Nalayeni, Amalraj Ramesh Sundar, Palaniyandi Malathi & Rasappa Viswanathan

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  1. V. N. Agisha
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  2. N. M. R. Ashwin
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  3. R. T. Vinodhini
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  4. Kumaravel Nalayeni
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  5. Amalraj Ramesh Sundar
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  7. Rasappa Viswanathan
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Contributions

V. N. A., N. M. R. A. and A. R. S. conceived and designed the experiments. V. N. A. performed the experiments and wrote the first draft of the manuscript. N. M. R. A., R. T. V. and K. N. are involved in analyzing the results and in revising the manuscript. A. R. S., N. M. R. A., P. M. and R. V. shared their expertise in execution of experiments, and were involved in revising and improving the intellectual content of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Amalraj Ramesh Sundar.

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Agisha, V.N., Ashwin, N.M.R., Vinodhini, R.T. et al. Protoplast-mediated transformation in Sporisorium scitamineum facilitates visualization of in planta developmental stages in sugarcane. Mol Biol Rep 48, 7921–7932 (2021). https://doi.org/10.1007/s11033-021-06823-x

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