A Microplate Reader-Based System for Visualizing Transcriptional Activity During in vivo Microbial Interactions in Space and Time.
Here, we report the development of a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. A bacterium expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. This novel technique enables real-time live imaging of samples by time-series multi-channel automatic recordings using a microplate reader as both an incubator and image recorder of general use to researchers. The technique can aid in deciding when to destructively sample for other methods e.g. transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during the interaction.
Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, 1871, Denmark.
Rosanna C
RC
Hennessy
Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, 1871, Denmark.
Peter
P
Stougaard
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 35002, China. stefan@olssonstefan.com.
Stefan
S
Olsson
D019677:Artificial Gene Fusion; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D056265:Microbial Interactions; D008828:Microbiological Techniques / Q000379:methods*; D011401:Promoter Regions, Genetic; D062211:Spatio-Temporal Analysis; D059008:Time-Lapse Imaging; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein