title: Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum.
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 10(7) 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.
authors.affiliation: Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY, USA.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Ren
authors.affiliation: Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY, USA; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY, USA.
authors.forename: Vishnu
authors.initials: V
authors.lastname: Chaturvedi
authors.affiliation: Mycology Laboratory, Wadsworth Center, New York State Department of Health, Albany, NY, USA; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY, USA. Electronic address: sudha.chaturvedi@health.ny.gov.
authors.forename: Sudha
authors.initials: S
authors.lastname: Chaturvedi
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000818:Animals; D001203:Ascomycota / Q000235:genetics* / Q000302:isolation & purification; D002685:Chiroptera / Q000382:microbiology; D003080:Cold Temperature; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D012641:Selection, Genetic; D014170:Transformation, Genetic
keywords: Agrobacterium tumefaciens-mediated transformation (ATMT); Homologous recombination; Urease; White-nose syndrome (WNS)