Conditional Gene Expression/Deletion Systems for<i>Marchantia polymorpha</i>Using its Own Heat-Shock Promoter and Cre/<i>lox</i>P-Mediated Site-Specific Recombination

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.