Systematic tools for reprogramming plant gene expression in a simple model,Marchantia polymorpha

Abstract

We present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

A simpleAgrobacterium-mediated stable transformation technique for the hornwort modelAnthoceros agrestis

“…The transformation vector contained the hph gene driven by the 35S promoter and terminated with a 35S polyadenylation signal. It also contained a p-35S_s::eGFP-Lti6B transcription unit (same 35S promoter with the one driving GUSPlus in pCAMBIA1305.2 plasmid) terminated by the double nopaline synthase ( Nos ) - 35S polyadenylation signal (Sauret-Güeto et al, 2020) (Fig. 3E) which was fused to the Low Temperature Induced Protein 6B (Lti6B) signal for membrane localization from Arabidopsis thaliana (Arabidopsis) (Cutler et al, 2000).…”

“…high-level expression across almost all tissues and development stages). Commonly used constitutive promoters in other bryophyte model species include the M. polymorpha ELONGATION FACTOR 1 ALPHA ( EF1a ) promoter (Althoff et al, 2014), the rice Actin1 , and the M. polymorpha ubiquitin-conjugating enzyme E2 promoter (Sauret-Güeto et al, 2020). Using the genomic sequence and RNA sequencing data (Li et al, 2020), (Fig.…”

“…The candidate promoter regions were cloned (and if necessary domesticated in order to generate a Loop assembly cloning system compatible DNA part), fused with the eGFP or the mTurquoise2 reporter genes, and terminated with the double Nos - 35S terminator (Sauret-Güeto et al, 2020). The AaEF1a (Fig.…”

“…Putative promoter sequences were amplified from genomic DNA using the KOD Hot start polymerase (#71086-5, Merck Millipore) and cloned into pJET1.2 (#K1231, ThermoFisher) before Sanger sequencing. Loop assembly compatible DNA parts were generated according to (Sauret-Güeto et al, 2020). List of primers can be found in Supplemental Table IV.…”

Comparative analysis of early divergent land plants and construction of DNA tools for hyper-expression in Marchantia chloroplasts

“…Marchantia has a series of characteristics that make it an ideal platform for chloroplast engineering (Boehm et al 2017). It grows rapidly through both asexual and sexual life cycles, has a remarkable regenerative capacity in the absence of phytohormones, the dominant phase of the life cycle is haploid and transplastomic plants can be isolated within 8 weeks (Sauret-Güeto et al 2020). In addition, post-transcriptional regulation of chloroplasts mRNAs in Marchantia is relatively simple compared to vascular plants.…”