The model moss Physcomitrium patens relies heavily on homologous recombination to repair DNA double-strand breaks.

We previously showed that moss (Physcomitrium patens) cells are highly radioresistant and suggested that P. patens uses an efficient mechanism to repair DNA double-strand breaks (DSBs). Homologous recombination (HR), canonical non-homologous end-joining, and alternative end-joining are the major pathways used to repair DSBs.

Establishing CRISPR-Cas9 in the sexually dimorphic moss, Ceratodon purpureus.

The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR-Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock-outs of the APT reporter gene by employing CRISPR-targeted mutagenesis under expression of native U6 snRNA promoters.

The SpRY Cas9 variant release the PAM sequence constraint for genome editing in the model plant Physcomitrium patens.

Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options.

Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens.

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role.

A DELAY OF GERMINATION 1 (DOG1)-like protein regulates spore germination in the moss Physcomitrium patens.

DELAY OF GERMINATION 1 is a key regulator of dormancy in flowering plants before seed germination. Bryophytes develop haploid spores with an analogous function to seeds. Here, we investigate whether DOG1 function during germination is conserved between bryophytes and flowering plants and analyse the underlying mechanism of DOG1 action in the moss Physcomitrium patens.

JGI Plant Gene Atlas: an updateable transcriptome resource to improve functional gene descriptions across the plant kingdom.

Gene functional descriptions offer a crucial line of evidence for candidate genes underlying trait variation. Conversely, plant responses to environmental cues represent important resources to decipher gene function and subsequently provide molecular targets for plant improvement through gene editing. However, biological roles of large proportions of genes across the plant phylogeny are poorly annotated.

Improved prime editing allows for routine predictable gene editing in Physcomitrium patens.

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present an improved methodology that allows for routine prime editing in the model plant Physcomitrium patens, whilst exploring potential new prime editing improvements.

Genome-wide specificity of plant genome editing by both CRISPR-Cas9 and TALEN.

CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR-Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation.

Physcomitrium patens Protoplasting and Protoplast Transfection.

Protoplast production with the moss Physcomitrium (Physcomitrella) patens has a long and successful history. As a tool, it has not only been the base of reverse genetic studies covering research fields as diverse as development, metabolism, or gene network regulation but also allowed its development as a bioengineering platform for protein production. We present here a standardized protocol for protoplast production from Physcomitrium (Physcomitrella) patens protonemata.

Prime Editing in the model plant Physcomitrium patens and its potential in the tetraploid potato.

Since its discovery and first applications for genome editing in plants, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology has revolutionized plant research and precision crop breeding. Although the classical CRISPR-Cas9 system is a highly efficient tool for disruptive targeted mutagenesis, this system is mostly inefficient for the introduction of precise and predictable nucleotide substitutions. Recently, Prime Editing technology has been developed, allowing the simultaneous generation of nucleotide transitions and transversions but also short defined indels.

A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens.

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting Physcomitrium patens that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named Physcomitrium patens amalgavirus 1, or PHPAV1.

The nuclear GUCT domain-containing DEAD-box RNA helicases govern gametophytic and sporophytic development in Physcomitrium patens.

Key Message: In RH1/RH2 are GUCT-domain-containing DEAD-BOX RNA helicases localize to the nucleus. They are implicated in cell and tissue development in all stages of the moss life cycle.

Abstract: The DEAD-box-containing RNA helicase family encompasses a large and functionally important group of enzymes involved in cellular processes committed to the metabolism of RNA, including its transcription, processing, transport, translation and decay.

Single Nucleotide Polymorphism Charting of Reveals Accumulation of Somatic Mutations During Culture on the Scale of Natural Variation by Selfing.

(Hedw.) Mitten (previously known as ) was collected by H.L.

Challenges of protein localization in plants seen through the DEK1 protein lens.

During the last 25 y, fluorescent protein tagging has become a tool of choice to investigate protein function in a cellular context. The information gathered with this approach is not only providing insights into protein subcellular localization but also allows contextualizing protein function in multicellular settings. Here we illustrate the power of this method by commenting on the recent successful localization of the large membrane DEK1 protein during three-dimensional body formation in the moss .

Characterisation of evolutionarily conserved key players affecting eukaryotic flagellar motility and fertility using a moss model.

Defects in flagella/cilia are often associated with infertility and disease. Motile male gametes (sperm cells) are an ancestral eukaryotic trait that has been lost in several lineages like flowering plants. Here, we made use of a phenotypic male fertility difference between two moss (Physcomitrella patens) ecotypes to explore spermatozoid function.

DEK1 displays a strong subcellular polarity during Physcomitrella patens 3D growth.

Defective Kernel 1 (DEK1) is genetically at the nexus of the 3D morphogenesis of land plants. We aimed to localize DEK1 in the moss Physcomitrella patens to decipher its function during this process. To detect DEK1 in vivo, we inserted the tdTomato fluorophore into PpDEK1 gene locus.

PEATmoss (Physcomitrella Expression Atlas Tool): a unified gene expression atlas for the model plant Physcomitrella patens.

Physcomitrella patens is a bryophyte model plant that is often used to study plant evolution and development. Its resources are of great importance for comparative genomics and evo-devo approaches. However, expression data from Physcomitrella patens were so far generated using different gene annotation versions and three different platforms: CombiMatrix and NimbleGen expression microarrays and RNA sequencing.

Analysis of the Localization of Fluorescent PpROP1 and PpROP-GEF4 Fusion Proteins in Moss Protonemata Based on Genomic "Knock-In" and Estradiol-Titratable Expression.

Tip growth of pollen tubes, root hairs, and apical cells of moss protonemata is controlled by ROP (Rho of plants) GTPases, which were shown to accumulate at the apical plasma membrane of these cells. However, most ROP localization patterns reported in the literature are based on fluorescent protein tagging and need to be interpreted with caution, as ROP fusion proteins were generally overexpressed at undefined levels, in many cases without assessing effects on tip growth. ROP-GEFs, important regulators of ROP activity, were also described to accumulate at the apical plasma membrane during tip growth.

The Physcomitrella patens gene atlas project: large-scale RNA-seq based expression data.

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P.

The Physcomitrella patens chromosome-scale assembly reveals moss genome structure and evolution.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle.

Combination of the Endogenous Promoter and Codon Usage Optimization Boosts Protein Expression in the Moss .

The moss is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong expression of genes of interest is important for production of recombinant proteins, e.g.

An intragenic mutagenesis strategy in Physcomitrella patens to preserve intron splicing.

Gene targeting is a powerful reverse genetics technique for site-specific genome modification. Intrinsic homologous recombination in the moss Physcomitrella patens permits highly effective gene targeting, a characteristic that makes this organism a valuable model for functional genetics. Functional characterization of domains located within a multi-domain protein depends on the ability to generate mutants harboring genetic modifications at internal gene positions while maintaining the reading-frames of the flanking exons.

Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins.

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P.

The DEK1 Calpain Linker Functions in Three-Dimensional Body Patterning in Physcomitrella patens.

The DEFECTIVE KERNEL1 (DEK1) calpain is a conserved 240-kD key regulator of three-dimensional body patterning in land plants acting via mitotic cell plane positioning. The activity of the cytosolic C-terminal calpain protease is regulated by the membrane-anchored DEK1 MEM, which is connected to the calpain via the 600-amino acid residue Linker. Similar to the calpain and MEM domains, the Linker is highly conserved in the land plant lineage, the similarity dropping sharply compared with orthologous charophyte sequences.

Endogenous Small-Noncoding RNAs and Potential Functions in Desiccation Tolerance in Physcomitrella Patens.

Early land plants like moss Physcomitrella patens have developed remarkable drought tolerance. Phytohormone abscisic acid (ABA) protects seeds during water stress by activating genes through transcription factors such as ABSCISIC ACID INSENSITIVE (ABI3). Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are key gene regulators in eukaryotes, playing critical roles in stress tolerance in plants.