Selection of a histidine auxotrophic <i>Marchantia polymorpha</i> strain with an auxotrophic selective marker

Abstract

Marchantia polymorpha has emerged as a model liverwort species, with molecular tools increasingly available.In the present study, we developed an auxotrophic strain of M. polymorpha and an auxotrophic selective marker gene as new experimental tools for this valuable model system. Using CRISPR (clustered regularly interspaced palindromic repeats)/Cas9-mediated genome editing, we mutated the genomic region for IMIDAZOLEGLYCEROL-PHOSPHATE DEHYDRATASE (IGPD) in M. polymorpha to disrupt the biosynthesis of histidine (igpd). We modified an IGPD gene (IGPDm) with silent mutations, generating a histidine auxotrophic selective marker gene that was not a target of our CRISPR/Cas9-mediated genome editing. The M. polymorpha igpd mutant was a histidine auxotrophic strain, growing only on medium containing histidine. The igpd mutant could be complemented by transformation with the IGPDm gene, indicating that this gene could be used as an auxotrophic selective marker. Using the IGPDm marker in the igpd mutant background, we produced transgenic lines without the need for antibiotic selection. The histidine auxotrophic strain igpd and auxotrophic selective marker IGPDm represent new molecular tools for M. polymorpha research.

Targeting imidazole-glycerol phosphate dehydratase in plants: novel approach for structural and functional studies, and inhibitor blueprinting

“…The advent of new molecular tools, e.g., CRISPR-Cas9 (clustered regularly interspaced palindromic repeats)/Cas9-mediated genome editing), has proved that the lack of auxotrophic mutants is no longer a problem. Recently, a model liverworth, Marchantia polymorpha, was established as a eukaryotic his auxotrophic system for studying biocontainment and transformant selection without the need for antibiotics (Fukushima and Kodama, 2022). For many years, crystallography has been the only way to study the structures of plant HBP enzymes in detail (Ruszkowski andDauter, 2016, Ruszkowski andDauter, 2017;Ruszkowski, 2018;Witek et al, 2021;Rutkiewicz et al, 2023).…”