Implementation of robotics and high-throughput imaging to capture growth rate data for Neurospora crassa cultured under different conditions

Implementation of robotics and high-throughput imaging to capture growth rate data for Neurospora crassa cultured under different conditions at  Fungal Genetics Conference, Pacific Grove, CA, Asilomar Conference Grounds

Abstract: 

Growth rate is an important metric for determining the virulence of plant pathogens, the suitability of an organism for biotechnological applications, species roles in ecological niches and many other applications. Traditional methods of determining growth rates on solid media typically capture basal hyphae extension in a single plane over time. These established methods are often laborious and time intensive, and do not typically yield the density or height of the colonies. Working with the NSF-funded Biofoundry for Extreme and Exceptional Fungi, Archaea, and Bacteria (ExFAB) at the University of California, Riverside, we have developed a high-throughput imaging pipeline to determine the growth rate of Neurospora crassa strains, that should be broadly applicable to other filamentous fungi. To establish this method, ~500 knockout mutants lacking genes encoding transcription factors or those implicated in cellular signaling were selected from the N. crassa knockout collection and screened under various conditions. These conditions include several alternative carbon and nitrogen sources and conditions known to induce stress. Utilizing the resources available in the ExFAB facility, we have automated normalization of spore concentration, spore inoculation, and time-lapse imaging of hyphal growth. Imaging analysis was performed utilizing Phenotypic, a software program developed by ExFAB that determines the integrated intensity or area of fungal colonies, which can be extrapolated to growth rate. This pipeline presents a high-throughput method that accounts for multiple colony attributes that may contribute to growth in filamentous fungi.