A Next-Generation Sequencing Approach to Study the Transcriptomic Changes during the Differentiation of Physarum at the Single-Cell Level

Abstract

Physarum polycephalum is a unicellular eukaryote belonging to the amoebozoa group of organisms. The complex life cycle involves various cell types that differ in morphology, function, and biochemical composition. Sporulation, one step in the life cycle, is a stimulus-controlled differentiation response of macroscopic plasmodial cells that develop into fruiting bodies. Well-established Mendelian genetics and the occurrence of macroscopic cells with a naturally synchronous population of nuclei as source of homogeneous cell material for biochemical analyses make Physarum an attractive model organism for studying the regulatory control of cell differentiation. Here, we develop an approach using RNA-sequencing (RNA-seq), without needing to rely on a genome sequence as a reference, for studying the transcriptomic changes during stimulus-triggered commitment to sporulation in individual plasmodial cells. The approach is validated through the obtained expression patterns and annotations, and particularly the results from up- and downregulated genes, which correlate well with previous studies.

Quantifying 35 transcripts in a single tube: model-based calibration of the GeXP multiplex RT-PCR assay

“…The cellular commitment to sporulation is associated to the differential expression of developmentally regulated genes [ 13 ]. Based on transcriptome data [ 13 , 14 ] we have previously designed gene specific primers for the quantification of the transcripts of a set of 35 genes with a single GeXP RT-PCR reaction. The concentrations of the gene-specific reverse primers were attenuated (adjusted) so that amplification of each of the 35 cDNAs yields a sufficient amount of each fragment to be resolved side-by-side during electrophoretic separation (Fig.…”

Expansion and transformation of the minor spliceosomal system in the slime mold Physarum polycephalum

“…Reannotation of the P. polycephalum genome We downloaded the Physarum polycephalum genome assembly and annotation from https://www.regulationsbiologie.ovgu.de/ Downloads/Physarum+polycephalum+Genome+Resource.html, and RNA-seq from NCBI's SRA database (accession numbers DRR047256, ERR089824-ERR089827, and ERR557103-ERR557120). 16,17,45,46 To reannotate the genome, we combined several de novo and reference-based approaches. First, we generated a de novo transcriptome from the aggregate RNA-seq data using Trinity 47 (v2.5.1).…”

Developmental switching inPhysarum polycephalum: Petri net analysis of single cell trajectories of gene expression indicates responsiveness and genetic plasticity of the Waddington quasipotential landscape

“…Sporulation is associated with a cascade of differentially expressed genes [19][20][21][22]. By repeatedly taking samples at different time points after triggering sporulation with a far-red light pulse, we have shown that individual plasmodial cells proceed to sporulation along variable pathways that involve qualitatively different correlation patterns of differentially expressed genes [23].…”