Optimization of flow cytometry parameters for high-throughput screening of spores of the filamentous fungus Trichoderma reesei.

ABSTRACT

Flow cytometry (FCM) is a powerful technique still little used to study filamentous fungi due to physical constraints the hyphae are too large to enter the FCM fluidic system, unless spores can be analyzed at a very early stage of germination. The technique nevertheless has strong potential for the study of these micro-organisms (spore sorting, viability, characterization etc.). This study focused on the investigation of several parameters, ranging from germination and storage conditions of T. reesei spores through to FCM gating, to detect their fluorescence during the first 24 h of germination. Fluorescent spores were first obtained after aerobic germination at 25 °C and monitored over 24 h using FCM, to screen for nine promoters controlling a green fluorescent protein gene. The fluorescence signal (FL1) was then acquired, in addition to the growth characterization of the spores, based on the size signal or Forward Scatter (FSC). They were combined to identify the best candidate(s) from among the nine promoters for the strongest- and earliest-possible fluorescence emission, which resulted in the following ranking pTEF > pPKI > pGPD > pPDC. There are numerous possible applications of this work, ranging from molecular biology to monitoring fermentation.