A Comprehensive Toolkit for Quick and Easy Visualization of Marker Proteins, Protein-Protein Interactions and Cell Morphology in Marchantia polymorpha.

Even though stable genomic transformation of sporelings and thalli of Marchantia polymorpha is straightforward and efficient, numerous problems can arise during critical phases of the process such as efficient spore production, poor selection capacity of antibiotics or low transformation efficiency. It is therefore also desirable to establish quick methods not relying on stable transgenics to analyze the localization, interactions and functions of proteins of interest. The introduction of foreign DNA into living cells via biolistic mechanisms has been first reported roughly 30 years ago and has been commonly exploited in established plant model species such as Arabidopsis thaliana or Nicotiana benthamiana. Here, we report the fast and reliable transient biolistic transformation of Marchantia thallus epidermal cells using fluorescent protein fusions. We present a catalog of fluorescent markers which can be readily used for tagging of a variety of subcellular compartments. Moreover, we report the functionality of the bimolecular fluorescence complementation (BiFC) in M. polymorpha with the example of the p-body markers MpDCP1/2. Finally, we provide standard staining procedures for live cell imaging in M. polymorpha, applicable to visualize cell boundaries or cellular structures, to complement or support protein localizations and to understand how results gained by transient transformations can be embedded in cell architecture and dynamics. Taken together, we offer a set of easy and quick tools for experiments that aim at understanding subcellular localization, protein-protein interactions and thus functions of proteins of interest in the emerging early diverging land plant model M. polymorpha.

An Evolutionarily Conserved Receptor-like Kinases Signaling Module Controls Cell Wall Integrity During Tip Growth.

Rooting cells and pollen tubes-key adaptative innovations that evolved during the colonization and subsequent radiation of plants on land-expand by tip growth. Tip growth relies on a tight coordination between the protoplast growth and the synthesis/remodeling of the external cell wall. In root hairs and pollen tubes of the seed plant Arabidopsis thaliana, cell wall integrity (CWI) mechanisms monitor this coordination through the Malectin-like receptor kinases (MLRs), such as AtANXUR1 and AtFERONIA, that act upstream of the AtMARIS PTI1-like kinase. Here, we show that rhizoid growth in the early diverging plant, Marchantia polymorpha, is also controlled by an MLR and PTI1-like signaling module. Rhizoids, root hairs, and pollen tubes respond similarly to disruption of MLR and PTI1-like encoding genes. Thus, the MLR and PTI1-like signaling module that controls CWI during tip growth is conserved between M. polymorpha and A. thaliana, suggesting that it was active in the common ancestor of land plants.

The Protein Phosphatases ATUNIS1 and ATUNIS2 Regulate Cell Wall Integrity in Tip-Growing Cells.

Fast tip-growing plant cells such as pollen tubes (PTs) and root hairs (RHs) require a robust coordination between their internal growth machinery and modifications of their extracellular rigid, yet extensible, cell wall (CW). Part of this essential coordination is governed by members of the Catharanthus roseus receptor-like kinase1-like (CrRLK1L) subfamily of RLKs with FERONIA (FER) and its closest homologs, ANXUR1 (ANX1) and ANX2, controlling CW integrity during RH and PT growth, respectively. Recently, Leucine-Rich Repeat Extensin 8 (LRX8) to LRX11 were also shown to be important for CW integrity in PTs. We previously reported an anx1 anx2 suppressor screen in Arabidopsis thaliana that revealed MARIS (MRI) as a positive regulator of both FER- and ANX1/2-dependent CW integrity pathways. Here, we characterize a suppressor that exhibits a weak rescue of the anx1 anx2 PT bursting phenotype and a short RH phenotype. The corresponding suppressor mutation causes a D94N substitution in a Type One Protein Phosphatase we named ATUNIS1 (AUN1). We show that AUN1 and its closest homolog, AUN2, are nucleocytoplasmic negative regulators of tip growth. Moreover, we demonstrate that AUN1D94N and AUN1H127A harboring mutations in key amino acids of the conserved catalytic site of phosphoprotein phosphatases function as dominant amorphic variants that repress PT growth. Finally, genetic interaction studies using the hypermorph MRIR240C and amorph AUN1D94N dominant variants indicate that LRX8-11 and ANX1/2 function in distinct but converging pathways to fine-tune CW integrity during tip growth.