Conditional Gene Expression/Deletion Systems forMarchantia polymorphaUsing its Own Heat-Shock Promoter and Cre/loxP-Mediated Site-Specific Recombination

Abstract

The liverwort Marchantia polymorpha is an emerging model plant suitable for addressing, using genetic approaches, various evolutionary questions in the land plant lineage. Haploid dominancy in its life cycle facilitates genetic analyses, but conversely limits the ability to isolate mutants of essential genes. To overcome this issue and to be employed in cell lineage, mosaic and cell autonomy analyses, we developed a system that allows conditional gene expression and deletion using a promoter of a heat-shock protein (HSP) gene and the Cre/loxP site-specific recombination system. Because the widely used promoter of the Arabidopsis HSP18.2 gene did not operate in M. polymorpha, we identified a promoter of an endogenous HSP gene, MpHSP17.8A1, which exhibited a highly inducible transient expression level upon heat shock with a low basal activity level. Reporter genes fused to this promoter were induced globally in thalli under whole-plant heat treatment and also locally using a laser-assisted targeted heating technique. By expressing Cre fused to the glucocorticoid receptor under the control of the MpHSP17.8A1 promoter, a low background, sufficiently inducible control for loxP-mediated recombination could be achieved in M. polymorpha. Based on these findings, we developed a Gateway technology-based binary vector for the conditional induction of gene deletions.

Design principles of a minimal auxin response system

“…We therefore generated Cre/ lox P-mediated conditional knock-out sectors (Mp arf2-1 cko and Mp arf2-2 cko ; Ref. 22,23) through heat-shock and DEX-inducible deletion of a CRISPR-resistant Mp ARF2 gene in a background with a CRISPR/Cas9-induced mutation in the endogenous Mp ARF2 gene ( Fig. S8a,b ).…”

Phytochrome Signaling Is Mediated by PHYTOCHROME INTERACTING FACTOR in the Liverwort Marchantia polymorpha

“…The liverwort Marchantia polymorpha is an emerging model plant among the basal land plants, owing to several advantages for genetic studies: a low-genetic redundancy, a haploid-dominant life cycle, and tractable genetic tools, such as high-efficiency transformation methods and genetic technologies (Ishizaki et al, , 2016Kubota et al, 2013;Sugano et al, 2014;Eklund et al, 2015;Kato et al, 2015;Flores-Sandoval et al, 2016;Nishihama et al, 2016). Since liverworts represent an early divergent lineage of extant land plants (Qiu et al, 2006), investigations using liverworts are of particular interest in terms of land plant evolution.…”

Droplet-based microfluidic analysis and screening of single plant cells

“…For this purpose, transgenic PMpHSP17.8 lines of M. polymorpha were generated, which expressed mVenus yellow fluorescent protein (YFP) 40 under control the endogenous heat-responsive MpHSP17.8 promoter. It was previously shown that incubation of transgenic M. polymorpha at 37°C for 1 h induced a P MpHSP17.8 -controlled targeted gene by approximately 700-fold 42 .…”