Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum

Tao Zhang, Ping Ren, Vishnu Chaturvedi, Sudha Chaturvedi

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The mechanisms of cold adaptation by fungi remain unknown. This topic is of high interest due to the emergence of white-nose syndrome (WNS), a skin infection of hibernating bats caused by Pseudogymnoascus destructans (Pd). Recent studies indicated that apart from Pd, there is an abundance of other Pseudogymnoascus species in the hibernacula soil. We developed an Agrobacterium tumefaciens-mediated transformation (ATMT) system for Pd and a related fungus Pseudogymnoascus pannorum (Pp) to advance experimental studies. URE1 gene encoding the enzyme urease was used as an easy to screen marker to facilitate molecular genetic analyses. A Uracil-Specific Excision Reagent (USER) Friendly pRF-HU2 vector containing Pd or Pp ure1::hygromycin (HYG) disruption cassette was introduced into A. tumefaciens AGL-1 cells by electroporation and the resulting strains were co-cultivated with conidia of Pd or Pp for various durations and temperatures to optimize the ATMT system. Overall, 680 Pd (0.006%) and 1800 Pp (0.018%) transformants were obtained from plating of 107 conidia; their recoveries were strongly correlated with the length of the incubation period (96h for Pd; 72h for Pp) and with temperature (15-18°C for Pd; 25°C for Pp). The homologous recombination in transformants was 3.1% for Pd and 16.7% for Pp. The availability of a standardized ATMT system would allow future molecular genetic analyses of Pd and related cold-adapted fungi.

Original languageEnglish (US)
Pages (from-to)73-81
Number of pages9
JournalFungal Genetics and Biology
Volume81
DOIs
StatePublished - Aug 1 2015
Externally publishedYes

Fingerprint

Agrobacterium tumefaciens
Agrobacterium
Fungi
Fungal Spores
Molecular Biology
Temperature
Electroporation
Uracil
Urease
Homologous Recombination
Nose
Soil
Skin
Enzymes
Infection
Genes

Keywords

  • Agrobacterium tumefaciens-mediated transformation (ATMT)
  • Homologous recombination
  • Urease
  • White-nose syndrome (WNS)

ASJC Scopus subject areas

  • Microbiology
  • Genetics

Cite this

Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum. / Zhang, Tao; Ren, Ping; Chaturvedi, Vishnu; Chaturvedi, Sudha.

In: Fungal Genetics and Biology, Vol. 81, 01.08.2015, p. 73-81.

Research output: Contribution to journalArticle

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