RT Journal Article SR Electronic T1 Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa JF bioRxiv FD Cold Spring Harbor Laboratory SP 2022.09.12.507692 DO 10.1101/2022.09.12.507692 A1 Ziyan Wang A1 Bradley M. Bartholomai A1 Jennifer J. Loros A1 Jay C. Dunlap YR 2022 UL http://biorxiv.org/content/early/2022/09/12/2022.09.12.507692.abstract AB Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The efficacy of ten different fluorescent protein tags were compared in a constant context of genomic and intracellular location; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.Competing Interest StatementThe authors have declared no competing interest.