Labeling of Peroxisomes for Live Cell Imaging in the Filamentous Fungus Ustilago maydis

Abstract

SummaryThe basidiomycete fungus Ustilago maydis has emerged as a powerful model organism to study fundamental biological processes. U. maydis shares many important features with human cells but provides the technical advantages of yeast. Recently, U. maydis has also been used to investigate fundamental processes in peroxisome biology. Here, we present an efficient yeast recombination-based cloning method to construct and express fluorescent fusion proteins (or conditional mutant protein alleles) which target peroxisomes in the fungus U. maydis. In vivo analysis is pivotal for understanding the underlying mechanisms of organelle motility. We focus on the in vivo labelling of peroxisomes in U. maydis and present approaches to analyze peroxisomal motility.

New insights into the functions of ACBD4/5-like proteins using a combined phylogenetic and experimental approach across model organisms

“…All plasmids were generated by yeast recombination-based cloning (YRBC) in S. cerevisiae strain DS94 (MATα, ura3-52, trp1-1, leu2-3, his3-111, and lys2-801 ), which enables assembly of multiple overlapping DNA fragments in a single-cloning step (Kilaru and Steinberg, 2015). Next, plasmids were transformed into U. maydis strains AB33 or FB1 (Guimarães et al, 2017). Briefly, protoplasts were transformed with linearized plasmids and subsequently plated on regeneration agar plates with selectable antibiotic.…”

“…For daily use, U. maydis strains were maintained on agar plates (1% agar (w/v), 1% glucose (w/v) in complete medium (CM; see for full composition (Holliday, 1974; Guimarães et al, 2017)). From this, liquid U. maydis cultures were grown overnight at 28°C in CM with 1% glucose (w/v), shaking at 200 rpm.…”