Green Fluorescent Protein Expression in Pseudogymnoascus destructans to Study Its Abiotic and Biotic Lifestyles

Abstract

Pseudogymnoascus destructans (Pd) is the etiologic agent of bat White-nose syndrome, a disease that has caused the unprecedented reduction in the hibernating bat populations across eastern North America. The Pd pathogenesis appears to be a complex adaptation of fungus in its abiotic (caves and mines) and biotic (bats) environments. There is a general lack of experimental tools for the study of Pd biology. We described the successful expression of codon-optimized synthetic green fluorescent protein sGFP in Pd. The sGFP(S65T) gene was first fused in frame with the Aspergillus nidulans promoter in the tumor-inducing plasmid pRF-HUE, and the resulting plasmid pHUE-sGFP(S65T) was transformed into Pd by Agrobacterium tumefaciens-mediated transformation system. The integration of sGFP(S65T) in Pd genome was analyzed by PCR, and single integration frequency of approximately 66% was confirmed by Southern hybridization. Fluorescent microscopy and flow cytometric analyses of two randomly selected transformants with single integration revealed high expression of sGFP in both spores and hyphal structures. The biology of mutants as judged by sporulation, growth rate, and urease production was not altered indicating sGFP is not toxic to Pd. Thus, we have generated a valuable tool that will facilitate the elucidation of Pd biology, ecology, and pathogenicity in real time.

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Acknowledgements

This study was partly supported with funds from the National Science Foundation (16-0039-01). Dr. Rasmus JN Frandsen (Frederiksberg, Denmark) is thanked for the generous gift of plasmid pRF-HUE. We also thank Renjie Song for assistance with flow cytometry.

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Correspondence to Vishnu Chaturvedi or Sudha Chaturvedi.

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Handling Editor: J.-P. Bouchara.

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Supplementary Figure 1. Screening of hyg+ transformants. Twelve hyg+ transformants were randomly picked, gDNA extracted, and subjected to PCR. All the transformants produced 700-bp amplicon against hyg gene (A) and 400-bp amplicon against sGFP(S65T) gene (B). The pHUE-sGFP(S65T) served as a positive control, while non-template DNA control and the DNA from the WT Pd strain served as negative controls. Supplementary Figure 2. Southern blot analysis of hyg+ transformants. DNA extracted from hyg+ transformants were digested with NdeI. Following electrophoresis and transfer to a nylon membrane, they were probed with 585-bp hyg gene. A representative blot with 5 transformants with single (lane 1, 2, 5), double (lane 3) and triple integration (lane 4) events of T-DNA is shown (DOCX 486 kb).

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Zhang, T., Ren, P., De Jesus, M. et al. Green Fluorescent Protein Expression in Pseudogymnoascus destructans to Study Its Abiotic and Biotic Lifestyles. Mycopathologia 183, 805–814 (2018). https://doi.org/10.1007/s11046-018-0285-2

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Keywords

  • Pseudogymnoascus destructans
  • Green fluorescent protein (GFP)
  • White-nose syndrome (WNS)
  • ATMT
  • Confocal microscopy
  • Flow cytometry