Tagged Mutagenesis and Gene-trap in the Moss, Physcomitrella patens by Shuttle Mutagenesis

The moss, Physcomitrella patens has been used as a useful materialin many fields, because of its simple body plan, ease of genetargeting, and other reasons. Although many mutants have beenreported, no method to isolate the corresponding genes was reported.We developed a gene tagging and gene-trap system in P. patensby using the shuttle mutagenesis technique, which has been usedin the budding yeast. In 5264 tagged lines, 203 mutants withaltered developmental or morphological phenotypes were obtained.In 129 of 4757 gene-trap lines, ß-glucuronidase (GUS)activity was detected in some tissue. Although multiple copiesof a tag were detected in many tagged lines by Southern analyses,most copies are likely integrated at the same locus accordingto PCR analyses.     

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

Summary In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.     

Preparation and analysis of isogenic mutants in the transferrin receptor protein genes, tbpA and tbpB, from Neisseria meningitidis

Isogenic mutants were constructed in the tbpA and tbpB genes from Neisseria meningitidis strain B16B6, which code for the transferrin receptor proteins, Tbp1 and Tbp2. Insertion mutants of the tbpA and tbpB genes were obtained by shuttle mutagenesis and by in vitro cassette mutagenesis, respectively. The Isogenic mutants were verified by Southern blot and Western blot analysis. Isogenic mutants deficient in Tbp1 or Tbp2 demonstrated a reduced transferrin binding activity in intact cells and total membranes but were incapable of utilizing transferrin iron for growth. Tbp1 could be isolated by affinity methods from the mutant lacking Tbp2 but isolation of Tbp2 from the mutant lacking Tbp1 required the presence of exogenous Tbp1.     

Chromosomal and mutagenic study of the marine macroalga,Gracilaria tenuistipitata

A pressing need of algal aquaculture is the availability of strains with favourable characteristics. Since this goal cannot be achieved easily by traditional breeding methods for Gracilaria tenuistipitata, we developed mutagenesis and selection procedures that bypass the necessity for protoplast isolation and cultivation. A haploid gametophyte was identified through chromosome analysis and used for mutagenesis. Chromosomal analyses showed that the haploid plant contains several layers of mononucleated cells at the surface and many large multinucleated syncytiums in the internal region. Chromosomal division of all nuclei in a syncytium was in synchrony. Mononucleated cells were more sensitive to both UV and DNA insertion mutagenesis. UV treatment generated many cadmium (Cd) resistant mutants, but most mutants exhibited retarded growth rate. However, several stable Cd resistant mutants were obtained through reselection. Plasmid pCaMVCAT could be used for DNA insertion mutagenesis in G. tenuistipitata. Stable mutants with increased resistance to chloramphenicol were selected.     

Embryonic mutants of Arabidopsis thaliana

Genetic analysis of plant em-bryogenesis has been approached in part through the isolation and characterization of recessive embryonic mutants. The most extensive studies have dealt with maize and Arabidopsis. The high frequency of mutants defective in plant embryogenesis is consistent with the presence of many target genes with essential functions at this stage of the life cycle. Some mutants are likely to be defective in genes with general housekeeping functions. Others should facilitate the identification of genes with a more direct role in the regulation of morphogesis. Over 300 embryonic mutants of Arabidopsis isolated following chemical mutagenesis and T-DNA insertional mutagenesis are currently being analyzed. This collection includes embryonic le-thals, defectives, and pattern mutants. Developmental abnormalities include the presence of fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, pattern deletions and duplications, embryos with altered patterns of symmetry, bloated embryos with giant vacuolated cells, reduced hypocotyls that fail to produce roots, and embryos that protrude through the seed coat late in maturation. This review describes the isolation and characterization of embryonic mutants of Arabidopsis and their potential application to plant biology. © 1992 Wiley-Liss, Inc.     

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

Functional analyses of the ABI1-related protein phosphatase type 2C reveal evolutionarily conserved regulation of abscisic acid signaling between Arabidopsis and the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

We employed a comparative genomic approach to understand protein phosphatase 2C (PP2C)-mediated abscisic acid (ABA) signaling in the moss Physcomitrella patens. Ectopic expression of Arabidopsis (Arabidopsis thaliana) abi1-1, a dominant mutant allele of ABI1 encoding a PP2C involved in the negative regulation of ABA signaling, caused ABA insensitivity of P. patens both in gene expression of late embryogenesis abundant (LEA) genes and in ABA-induced protonemal growth inhibition. The transgenic abi1-1 plants showed decreased ABA-induced freezing tolerance, and decreased tolerance to osmotic stress. Analyses of the P. patens genome revealed that only two (PpABI1A and PpABI1B) PP2C genes were related to ABI1. In the ppabi1a null mutants, ABA-induced expression of LEA genes was elevated, and protonemal growth was inhibited with lower ABA concentration compared to the wild type. Moreover, ABA-induced freezing tolerance of the ppabi1a mutants was markedly enhanced. We provide the genetic evidence that PP2C-mediated ABA signaling is evolutionarily conserved between Arabidopsis and P. patens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accession Numbers: PpABI1A-AB369256, PpABI1B-AB369255, pphn39k21-AB369257.     

Isolation of mutants ofClostridium acetobutylicum ATCC 824 deficient in protease activity

In this study we report the isolation of mutants ofClostridium acetobutylicum ATCC 824 that are deficient in protease activity. The mutants were formed by use of Tn916 transposon mutagenesis, and this study further indicates this technique as a viable method for studying the genetics ofC. acetobutylicum. The reduced protease phenotype can be distinguished on milk agar plates and was confirmed by enzyme activity measurements with the fluorescent protease substrate FITC-casein. The protease-deficient strains are capable of normal spore formation and solvent production.     

A CRISPR/LbCas12a‐based method for highly efficient multiplex gene editing in Physcomitrella patens

Due to their high efficiency, specificity, and flexibility, programmable nucleases, such as those of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (Cpf1) system, have greatly expanded the applicability of editing the genomes of various organisms. Genes from different gene families or genes with redundant functions in the same gene family can be examined by assembling multiple CRISPR RNAs (crRNAs) in a single vector. However, the activity and efficiency of CRISPR/Cas12a in the non‐vascular plant Physcomitrella patens are largely unknown. Here, we demonstrate that LbCas12a together with its mature crRNA can target multiple loci simultaneously in P. patens with high efficiency via co‐delivery of LbCas12a and a crRNA expression cassette in vivo. The mutation frequencies induced by CRISPR/LbCas12a at a single locus ranged from 26.5 to 100%, with diverse deletions being the most common type of mutation. Our method expands the repertoire of genome editing tools available for P. patens and facilitates the creation of loss‐of‐function mutants of multiple genes from different gene families.     

A Detached Cotyledon Test for the Isolation of Mutants of Pyrenopeziza brassicae Defective in Pathogenicity Determinants

A detached cotyledon pathogenicity test was devised for the isolation of UV-induced mutants of the hemibiotrophie ascomycete pathogen of Brassica spp., Pyrenopeziza brassicae defective in pathogenicity determinants. At least 95 % of cotyledons of suitable susceptible cultivars of oilseed rape (Brassica napus L. ssp. oleifera cvs Shogun or Bolko) showed symptoms of disease (white spore pustules [conidiomata] on the surface of the cotyledon) within three weeks of inoculation of cotyledons with 10⁴ conidia of P. brassicae. The test allowed rapid screening of UV-induced mutants with a low frequency of false negatives. From 1,700 survivors of UV mutagenesis tested, 20 non-pathogenic mutants were obtained.     

MSH1 maintains organelle genome stability and genetically interacts with RECA and RECG in the moss Physcomitrella patens

Chloroplast and mitochondrial DNA encodes genes that are essential for photosynthesis and respiration, respectively. Thus, loss of integrity of the genomic DNA of organelles leads to a decline in organelle function and alteration of organelle genetic information. RECA (RECA1 and RECA2) and RECG, which are homologs of bacterial homologous recombination repair (HRR) factors RecA and RecG, respectively, play an important role in the maintenance of integrity of the organelle genome by suppressing aberrant recombination between short dispersed repeats (SDRs) in the moss Physcomitrella patens. On the other hand, MutS homolog 1 (MSH1), a plant‐specific MSH with a C‐terminal GIY‐YIG endonuclease domain, is involved in the maintenance of integrity of the organelle genome in the angiosperm Arabidopsis thaliana. Here, we address the role of the duplicated MSH1 genes, MSH1A and MSH1B, in P. patens, in which MSH1A lacks the C‐terminal endonuclease domain. MSH1A and MSH1B localized to both chloroplast and mitochondrial nucleoids in protoplast cells. Single and double knockout (KO) mutants of MSH1A and MSH1B showed no obvious morphological defects; however, MSH1B KO and double KO mutants, as well as MSH1B GIY‐YIG deletion mutants, exhibited genomic instability due to recombination between SDRs in chloroplasts and mitochondria. Creating double KO mutations of each combination of MSH1B, RECA2 and RECG synergistically increased recombination between SDRs in chloroplasts and mitochondria. These results show the role of MSH1 in the maintenance of integrity of the organelle genome and the genetic interaction between MSH1 and homologs of HRR factors in the basal land plant P. patens.     

A fluorescence‐activated cell sorting‐based strategy for rapid isolation of high‐lipid Chlamydomonas mutants

There is significant interest in farming algae for the direct production of biofuels and valuable lipids. Chlamydomonas reinhardtii is the leading model system for studying lipid metabolism in green algae, but current methods for isolating mutants of this organism with a perturbed lipid content are slow and tedious. Here, we present the Chlamydomonas high‐lipid sorting (CHiLiS) strategy, which enables enrichment of high‐lipid mutants by fluorescence‐activated cell sorting (FACS) of pooled mutants stained with the lipid‐sensitive dye Nile Red. This method only takes 5 weeks from mutagenesis to mutant isolation. We developed a staining protocol that allows quantification of lipid content while preserving cell viability. We improved separation of high‐lipid mutants from the wild type by using each cell's chlorophyll fluorescence as an internal control. We initially demonstrated 20‐fold enrichment of the known high‐lipid mutant sta1 from a mixture of sta1 and wild‐type cells. We then applied CHiLiS to sort thousands of high‐lipid cells from a pool of about 60 000 mutants. Flow cytometry analysis of 24 individual mutants isolated by this approach revealed that about 50% showed a reproducible high‐lipid phenotype. We further characterized nine of the mutants with the highest lipid content by flame ionization detection and mass spectrometry lipidomics. All mutants analyzed had a higher triacylglycerol content and perturbed whole‐cell fatty acid composition. One arbitrarily chosen mutant was evaluated by microscopy, revealing larger lipid droplets than the wild type. The unprecedented throughput of CHiLiS opens the door to a systems‐level understanding of green algal lipid biology by enabling genome‐saturating isolation of mutants in key genes.     

The d-mannose/l-galactose pathway is the dominant ascorbate biosynthetic route in the moss Physcomitrium patens

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d -mannose/l -galactose (d -Man/l -Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d -galacturonate (d -GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d -Man/l -Gal pathway and an orthologous gene encoding aldonolactonase (ALase) – a key enzyme for the d -GalUA pathway – Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l -galactonic acid to the final precursor l -galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l -Gal phosphorylase in the d -Man/l -Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d -Man/l -Gal pathway, not the d -GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.     

Analysis of gametophytic development in the moss,Physcomitrella patens,using auxin and cytokinin resistant mutants

Mutants altered in their response to auxins and cytokinins have been isolated in the moss Physcomitrella patens either by screening clones from mutagenized spores for growth on high concentrations of cytokinin or auxin, in which case mutants showing altered sensitivities can be recognized 3–4 weeks later, or by non-selective isolation of morphologically abnormal mutants, some of which are found to have altered sensitivities. Most of the mutants obtained selectively are also morphologically abnormal. The mutants are heterogeneous in their responses to auxin and cytokinin, and the behaviour of some is consistent with their being unable to make auxin, while that of others may be due to their being unable to synthesize cytokinin. Physiological analysis of the mutants has shown that both endogenous auxin and cytokinin are likely to play important and interdependent roles in several steps of gametophytic development. Although their morphological abnormalities lead to sterility, genetic analysis of some of the mutants has been possible by polyethyleneglycol induced protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthalene acetic acid - 2,4D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAP 6-( ²isopentenyl) aminopurine - NAR NAA resistant mutants - BAR BAP resistant mutants     

The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens

Summary Eighteen nutritional mutants have been isolated in the haploid, monoecious moss, Physcomitrella patens: five nicotinic acid auxotrophs, four p-aminobenzoic acid auxotrophs, four adenine auxotrophs, two amino acid requiring mutants and three nitrate non-utilising mutants. Seventeen of them were obtained using total isolation; one was isolated selectively. Strains resistant to the amino acid analogues, D-serine and p-fluorophenyl-alanine, and the purine analogue, 8-azaguanine, have been selected. Many of the auxotrophs are self-sterile. Crosses between auxotrophic strains have been effected and the progeny analysed. No linkage has been detected. Nicotinic acid auxotrophy has resulted from mutation in at least two genes. Self-sterility segregates as a pleiotropic effect of four mutations which produce nutritional dependence. A diploid strain has been obtained by aposporus regeneration from a hybrid sporophyte and the phenotypes of progeny resulting from the self-fertilisation of this strain have been analysed.     

Identification of a hotspot for transformation of Neisseria meningitidis by shuttle mutagenesis using signature-tagged transposons

Shuttle mutagenesis using signature-tagged transposons was employed to generate a library of individually tagged mutants of the Neisseria meningitidis strain B1940, which belongs to serogroup B. The use of tagged transposons allowed us to monitor for enrichment for single mutants during the process of shuttle mutagenesis, by amplification of the tags and subsequent sequence determination. Enrichment of a single clone occurred during the transformation of the meningococci with transposon-containing plasmid DNA. Sequence determination around the site of transposon insertion revealed that the transposon had mutagenized a previously unknown locus, which was designated hrtA (high rate of transformation). hrtA-mediated transformation was independent of TnMax5 and tag sequences, and it most probably involved recombination events. The hrtA locus is restricted to meningococci and gonococci and is present in few apathogenic neisserial species. Chromosomal mapping of hrtA and six further hrt sites revealed a random distribution of highly transforming DNA fragments on the meningococcal chromosome. In conclusion, our data demonstrate that shuttle mutagenesis of naturally competent bacteria using signature-tagged transposons allows the isolation of chromosomal DNA fragments, which exhibit a high transformation efficiency, and which, therefore, are likely to be involved in horizontal gene transfer. Received: 12 January 1998 / Accepted: 30 April 1998     

Characterization and Molecular Genetic Complementation of Mutants Affecting Dimorphism in the FungusUstilago maydis

Ustilago maydis,the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase,uac1.Secondary mutagenesis of auac1disruption strain allowed the isolation of a large number of suppressor mutants, termedubc,forUstilagobypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of theubc1gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three newubcgenes, termedubc2, ubc3,andubc4.We also describe the morphology of theubc2, ubc3,andubc4mutants in auac1⁻background as well as in a background with a functionaluac1gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additionalubcgenes.     

小立碗藓GPX家族的功能分化与催化特性研究

谷胱甘肽过氧化物酶(GPX)在植物抵抗氧化胁迫中发挥重要作用。该研究从小立碗藓(Physcomitrella patens)基因组中挖掘到3个GPX基因,分别命名为PpGPX1、PpGPX2和PpGPX3。其中PpGPX1和PpGPX3只含有1个外显子,而PpGPX2含有6个外显子。表达模式分析发现PpGPX1和PpGPX2在检测的所有条件下均表达,而PpGPX3在检测的所有条件下均不表达。蛋白亚细胞定位分析发现,PpGPX1蛋白定位在细胞质,而PpGPX2蛋白定位在叶绿体。在大肠杆菌中表达并纯化了PpGPX1和PpGPX2蛋白,酶学性质分析发现,PpGPX1和PpGPX2蛋白均只能利用Trx电子供体系统,而不能利用GSH电子供体系统;PpGPX2蛋白对过氧化物底物的催化活性和催化效率均高于PpGPX1。基因结构、表达模式、亚细胞定位和蛋白酶学性质的差异预示小立碗藓GPX基因家族成员发生了功能分化,将PpGPX2蛋白的Pro158、Phe167和Phe172氨基酸残基均突变为Ala,发现突变体蛋白对底物催化活性降低,说明这3个氨基酸位点对PpGPX2蛋白具有重要催化活性。