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

Tao Zhang, Ping Ren, Magdia de Jesus, Vishnu Chaturvedi, Sudha Chaturvedi

Research output: Contribution to journalArticle

2 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1-10
Number of pages10
JournalMycopathologia
DOIs
StateAccepted/In press - Jul 9 2018
Externally publishedYes

Keywords

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

ASJC Scopus subject areas

  • Microbiology
  • Applied Microbiology and Biotechnology
  • Agronomy and Crop Science
  • veterinary (miscalleneous)

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