Ac/Ds transposon mutagenesis in Arabidopsis thaliana: mutant spectrum and frequency of Ds insertion mutants

Using a two-component Ac/Ds system consisting of a stabilized Ac element (Ac_c1) and a non-autonomous element (Ds_A), 650 families of plants carrying independent germinal Ds_A excisions/transpositions were isolated. Progenies of 559 of these Ac_c1/Ds_A families, together with 43 families of plants selected for excision/transposition of wild-type (wt)Ac, were subjected to a broad screening program for mutants exhibiting visible alterations. This resulted in the identification of 48 mutants showing a wide variety of mutant phenotypes, including embryo lethality (24 mutants), chlorophyll defects (5 mutants), defective seedlings (2 mutants), reduced fertility (5 mutants), reduced size (3 mutants), altered leaf morphology (2 mutants), dark green, unexpanded rosette leaves (3 mutants), and aberrant flower or shoot morphology (4 mutants). To test whether these mutants were due to transposon insertions, a series of Southern blot experiments was performed on 28 families, comparing in each case several mutant plants with others showing the wild-type phenotype. A preliminary analysis revealed in 4 of the 28 families analyzed a common, novel Ds_A fragment in all mutant plants, which was present only in heterozygous plants with wt phenotype, as expected for Ds_A insertion mutations. These four mutants included two showing embryo lethality, one with dark green, unexpanded rosette leaves and stunted inflorescences, and one with curly growth of stems, leaves and siliques. Further evidence for Ds_A insertion mutations was obtained for one embryo lethal mutant and for the stunted mutant, while in case of the second embryo lethal mutant, the Ds_A insertion could be separated from the mutant locus by genetic recombination.     

Probing the evolution of senescence inDrosophila melanogaster withP-element tagging

Natural populations host a wealth of genetic variation in longevity and age-specific schedules of reproduction. This variation provides critical information for inferring the evolutionary origin of senescence. Patterns of mutational effects on age-specific fecundity and survival provide additional insight to distinguish alternative models of senescence. In this study,P-elements bearing thewhite minigene were inserted at random into a common genetic background, generating lines ofD. melanogaster with single, stable transposon inserts. A series of 48 single-P-element lines revealed statistically significant heterogeneity in both longevity and fecundity. Longevity and early fecundity were only weakly positively correlated (r=0.286,P=0.0398). Both the pooled sample and 30 of the individual lines exhibited a leveling of age-specific mortality at advanced ages, in opposition to the classical demographic models. To the extent that these mutational effects are representative of naturally-occurring mutations in heterogeneous populations, this result presents a problem for the evolutionary theory of senescence. Natural selection is inefficient at removing deleterious mutations that are expressed only at late ages, and selection may not differentiate between mutations whose effects on longevity are post-reproductive. A leveling of the mortality rate would also be seen if mutations whose expression is delayed until very late simply do not occur. A simulation of mutation-selection balance among the 48P-element tagged lines shows that the mean longevity declines monotonically with increasing mutation rate, consistent with the mutation-accumulation model.     

<Emphasis Type="Italic">Ds</Emphasis> insertion mutagenesis as an efficient tool to produce diverse variations for rice breeding

The availability of diversified germplasm resources is the most important for developing improved rice varieties with higher seed yield or tolerance to various biotic or abiotic stresses. Here we report an efficient tool to create increased variations in rice by maize Ac/Ds transposon (a gene trap system) insertion mutagenesis. We have generated around 20,000 Ds insertion rice lines of which majority are homozygous for Ds element. We subjected these lines to phenotypic and abiotic stress screens and evaluated these lines with respect to their seed yields and other agronomic traits as well as their tolerance to drought, salinity and cold. Based on this evaluation, we observed that random Ds insertions into rice genome have led to diverse variations including a range of morphological and conditional phenotypes. Such differences in phenotype among these lines were accompanied by differential gene expression revealed by GUS histochemical staining of gene trapped lines. Among the various phenotypes identified, some Ds lines showed significantly higher grain yield compared to wild-type plants under normal growth conditions indicating that rice could be improved in grain yield by disrupting certain endogenous genes. In addition, several 1,000s of Ds lines were subjected to abiotic stresses to identify conditional mutants. Subsequent to these screens, over 800 lines responsive to drought, salinity or cold stress were obtained, suggesting that rice has the genetic potential to survive under abiotic stresses when appropriate endogenous genes were suppressed. The mutant lines that have higher seed yielding potential or display higher tolerance to abiotic stresses may be used for rice breeding by conventional backcrossing combining with molecular marker-assisted selection. In addition, by exploiting the behavior of Ds to leave footprints upon remobilization, we have shown an alternative strategy to develop new rice varieties without foreign DNA sequences in their genome. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Behavioral analysis ofDrosophila mutants displaying abnormal male courtship

We describe six recessive autosomal male sterile mutations inDrosophila, generated by mobilization of single P-elements, exhibiting abnormal male courtship behavior. Detailed analysis of courtship behavior elicited by virgin wild type females indicated that five of the six mutants are affected in the early steps of courtship. The sixth mutant is blocked at the step of attempted copulation which occurs later in the courtship sequence. All of the mutants have normal olfactory responses and normal locomotor activity. No defect in the visual modality has been observed for the five mutants affected in the initiation of courtship. The mutant blocked at attempted copulation lacks the ‘on’ and ‘off’ transients, but this appears to be due to genetic background rather than the mutation itself. Abnormal spermatogenesis was observed in five of the mutants. Spermatogenic defects vary and include lesions in the proliferation of the germline, in meiosis, and in the differentiation and maturation of the spermatids into motile sperm.     

Influence of the<Emphasis Type="Italic">SMT2</Emphasis> knock-out on hypocotyl elongation in<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Inhibitors are very important in the study of hormone function. Brasinazole (Brz) is a specific inhibitor of brassinosteroids (BRs) biosynthesis. To expand our knowledge of the molecular mechanisms of plant steroid signaling, we performed genetic screening using medium containing Brz under dark conditions. Mutants insensitive to Brz developlonger hypocotyls than their wild type counterparts. We isolatedabz453 as a Brz insensitive mutant. TAIL-PCR and the segregation ratio of T2 plants indicated a single T-DNA insertion at the 24-Sterol C-methyltransferase (SMT2) gene in theabz453 mutant. Recapitulation for putative FCP serine phosphatase (FSP), the gene neighboringSMT2, indicated no significant phenotypes, but theSMT2 anti-sense (SMT2-AS) line developed longer hypocotyls than the wild type in medium containing Brz. Additionally, theSMT2-AS line displayed similar phenotypes to theabz453 line in soil including enhanced growth and smaller silique. Theabz453 andSMT2-AS mutants showed phenotypes similar to those of wild type in medium containing benzylaminopurine, pacrobutrazol and ACC (precursor for ethylene) under dark conditions. However, when brassinolide (BL) dose response was observed, theabz453 andSMT2-AS lines showed higher sensitivity than wild type. Theabz453/det2 andabz453/bri1-119 double mutants showed enhanced growth compared to thedet2 andbri1-119 line under both dark and light conditions. Specially, in dark conditions double mutants displayed nearly 2- and 1.5-fold longer hypocotyls thandet2 andbri1-119 plants. Brz insensitivity to theSMT2 knock-out mutant and phenotypes of double mutants indicate that not only do BRI1 and DET2 influence the BRs response, as evidenced by hypocotyl elongation, but another sterol derived signals may also be affected in mutants, suggesting that another pathway is involved in hypocotyl elongation due to SMT2.     

茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究

[目的]劳尔氏菌（Ralstonia solanacearum）在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。[方法]利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。[结果]以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基（nuoF）中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。[结论]NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R.solanacearum生物膜形成、细胞运动和致病性也有重要作用。     

Rapid selection of mutants of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> for carbohydrate and fatty acid metabolism by fourier transform infrared spectroscopy and gas chromatography combined with multivariate analysis

Transgene-tagged mutants of Chlamydomonas reinhardtii were generated by random insertional mutagenesis for screening of mutants of carbohydrate and fatty acid metabolism. Approximately 2,500 insertion mutants tagged with the aph7″ gene were produced from one mutagenesis in three weeks. To establish a rapid screening system for numerous insertional lines, whole cell extracts of 100 insertional lines were subjected to Fourier transform infrared spectroscopy (FT-IR) and gas chromatography (GC) analysis combined with multivariate analysis. Mutant lines 28, 67, and 90 showed dramatic differences in the carbohydrate (1,000∼1,200 cm⁻¹) and amide (1,500∼1,700 cm⁻¹) regions of the FT-IR spectrum compared to wild type strain CC-124. Separate GC analysis also showed that 16:0 iso, palmitic acid (16:0), and oleic acid (18:1) were the major fatty acids in the wild type strain. In mutant 80, the relative content ratio of 16:0 iso in total fatty acids was significantly lower than in wild type, whereas the ratios of palmitic acid and oleic acid to 16:0 iso were higher. In mutant 95, the ratio of 16:0 iso to total fatty acids was increased, whereas ratios of palmitic acid and oleic acid to 16:0 iso were decreased. In particular, mutant 57 showed remarkably different fatty acid patterns with novel peaks of long-chain fatty acids having more than 20 carbon atoms. The results of this study show that FT-IR and GC combined with multivariate analysis enable rapid selection of mutants of carbohydrate and fatty acid metabolism in C. reinhardtii.     

A multiplexed,three-dimensional pooling and next-generation sequencing strategy for creating barcoded mutant arrays: construction of a Schizosaccharomyces pombe transposon insertion library

Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.     

Mutations affecting the pattern of the larval cuticle inDrosophila melanogaster

Summary The present report describes the recovery and genetic characterization of mutant alleles at zygotic loci on the third chromosome ofDrosophila melanogaster which alter the morphology of the larval cuticle. We derived 12600 single lines from ethyl methane sulfonate (EMS)-treatedst e orrucuca chromosomes and assayed them for embryonic lethal mutations by estimating hatch rates of egg collections. About 7100 of these lines yielded at least a quarter of unhatched eggs and were then scored for embryonic phenotypes. Through microscopic examination of unhatched eggs 1772 lines corresponding to 24% of all lethal hits were classified as embryonic lethal. In 198 lines (2.7% of all lethal hits), mutant embryos showed distinct abnormalities of the larval cuticle. These embryonic visible mutants define 45 loci by complementation analysis. For 32 loci, more than one mutant allele was recovered, with an average of 5.8 alleles per locus. Complementation of all other mutants was shown by 13 mutants. The genes were localized on the genetic map by recombination analysis, as well as cytologically by complementation analysis with deficiencies. They appear to be randomly distributed along the chromosome. Allele frequencies and comparisons with deficiency phenotypes indicate that the 45 loci represent most, if not all, zygotic loci on the third chromosome, where lack of function recognizably affects the morphology of the larval cuticle.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

Induction and characterization of pigmentation mutants in Porphyra yezoensis (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda conchospore germlings (1–4-cell stages) were treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) for inducing mutations. Three kinds of color-mutated gametophytic blades, which were composed of the mutated cells wholly, sectorially or spottedly, were obtained; and most of them were sectorially variegated blades. The highest frequency of these mutated blades was 1.3%. Four different pigmentation mutant strains were obtained by regenerating single cells and protoplasts that were enzymatically isolated from the mutated sectors of the sectorially variegated blades. The mutants were relatively stable in color in both gametophytic blade and conchocelis phases. In the two phases, each mutant strain showed characteristic differences in the in vivo absorption spectra, and had different pigment contents of major photosynthetic pigments (chlorophyll a, phycoerythrin and phycocyanin) as compared with the wild-type and with each other. The gametophytic blades from the four mutant lines showed significant differences in growth and photosynthetic rates, when they were cultured in the same conditions. By crossing the mutant with the wild-type, it was found that the color phenotypes of two mutants reported above, were resulted from two mutations in different genes, respectively. This revised version was published online in August 2006 with corrections to the Cover Date.     

Novel<Emphasis Type="Italic"> eceriferum</Emphasis> mutants in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

We conducted a novel non-visual screen for cuticular wax mutants in Arabidopsis thaliana (L.) Heynh. Using gas chromatography we screened over 1,200 ethyl methane sulfonate (EMS)-mutagenized lines for alterations in the major A. thaliana wild-type stem cuticular chemicals. Five lines showed distinct differences from the wild type and were further analyzed by gas chromatography and scanning electron microscopy. The five mutants were mapped to specific chromosome locations and tested for allelism with other wax mutant loci mapping to the same region. Toward this end, the mapping of the cuticular wax (cer) mutants cer10 to cer20 was conducted to allow more efficient allelism tests with newly identified lines. From these five lines, we have identified three mutants defining novel genes that have been designated CER22, CER23, and CER24. Detailed stem and leaf chemistry has allowed us to place these novel mutants in specific steps of the cuticular wax biosynthetic pathway and to make hypotheses about the function of their gene products.Abbreviations EMS Ethyl methane sulfonate - SEM Scanning electron microscopy - SSLP Simple sequence length polymorphism - WT Wild type     

The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana

Thirty-six phytohormone-affected mutants of Arabidopsis thaliana (L.) Heynh. and their parental ecotypes were tested for resistance/susceptibility to Botrytis cinerea Pers.; Fr. and ability to develop Trichoderma-mediated induced systemic resistance (ISR). Ecotype Colombia-0 (Col-0) was relatively resistant to B. cinerea, and Trichoderma harzianum Rifai T39 application at sites spatially separated (roots) from the B. cinerea inoculation (leaves) resulted in reduction of grey mold symptoms. Ecotypes Wassilewskija-4, Nossen-0 and Landsberg-0 had low levels of basal resistance to B. cinerea and were unable to express ISR. Mutants derived from ISR-non-inducible ecotypes displayed ISR-non-inducible phenotypes, whereas the ISR inducibility of mutants derived from the ISR-inducible genotype Col-0 varied according to the type of mutant. Thus, salicylic acid (SA)-impaired mutants derived from Col-0 were ISR-inducible, while ethylene/jasmonic acid (ethylene/JA)-impaired mutants of the same origin were ISR-non-inducible. SA-impaired mutants retained basal level of resistance to B. cinerea, while most ethylene/JA-impaired mutants were highly susceptible. Abscisic acid- and gibberellin-impaired mutants were highly susceptible to B. cinerea and showed ISR-non-inducible phenotypes irrespective of their lines of origin. Auxin-resistant mutants derived from Col-0 were ISR-inducible; mutant originating from Landsberg-0 and mutants which were resistant to both auxin and ethylene were ISR-non-inducible. Most of the arabidopsis genotypes which were unable to express Trichoderma-mediated ISR against B. cinerea exhibited enhanced susceptibility to this pathogen. T. harzianum treatments enhanced the growth of arabidopsis plants regardless of genotype or ISR inducibility.     

Analysis of pathogenicity mutants of a bacteriocin producing Xanthomonas perforans

Since the initial discovery of Xanthomonas perforans on tomato in 1991, it has completely displaced Xanthomonas euvesicatoria as the bacterial spot of tomato pathogen in Florida. Previous research has shown that X. perforans produces at least three different bacteriocin-like compounds (BcnA, BcnB, BcnC) antagonistic toward X. euvesicatoria strains. In this study pathogenicity-attenuated, bacteriocin-producing mutants of X. perforans were created to determine their potential as biological control agents for control of X. euvesicatoria. Several candidate genes were chosen based on previous studies in which mutant phenotypes exhibited reduced virulence in either X. perforans (OpgH_Xcv) or the closely related X. euvesicatoria strain 85-10 (hpaB, hpaC, xopA, xopD, avrBs2 and gumD). Each candidate gene in X. perforans was amplified and PCR-assisted deletion mutagenesis was performed in the wild-type (wt) X. perforans strain to create potential attenuation mutants. Each mutant was tested for growth rate, disease severity and antagonism toward X. euvesicatoria strains. Three mutants, XopA, opgH, and gumD were significantly less pathogenic than the wild-type strain with the opgH mutant reaching significantly lower internal populations than all other mutants except hpaC. The opgH-strain was the most affected in its ability to grow internally in plant tissue while inhibiting X. euvesicatoria populations equal to or more than the other mutant strains. This mutant strain could potentially be used as part of an effective biological control strategy.     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

Characterization of three male-sterile mutants of Arabidopsis thaliana exhibiting alterations in meiosis

Male-sterile mutants are being studied to deepen our understanding of the complex processes of microsporogenesis and microgametogenesis. Due to difficulties associated with isolating the mutated gene, there is currently very little molecular information on the defects responsible for male sterility. As a first step in utilizing male-sterile mutants to better understand the bio-chemical and molecular processes that control pollen development, we have characterized a number of Arabidopsis thaliana lines that were generated by seed transformation and exhibit male sterility. We report here the identification and characterization of three male-sterile A. thaliana lines, all of which are tagged with T-DNA and show aberrant meiosis. A detailed cytochemical study was conducted on these lines to better understand the timing and nature of each mutation and to investigate how these mutations affect subsequent steps of pollen development. All three mutants undergo apparently normal morphogenesis until the onset of meiosis. In one line (6492) the mutation is most notable at the tetrad stage when up to eight microspores can be seen in each callose-encased tetrad. The resulting mutant microspores are of variable sizes and contain different amounts of DNA. Two other mutants (7219 and 7593) possess many common features, including variable developmental pathways, failure to produce callose, production of vacuolate, coenocytic (multi-nucleate) cells that are surrounded by persistent microsporocyte walls, and asynchronous patterns of development. Unlike the situation in wild-type plants, where developmental stages are correlated with bud length, such correlations are almost impossible with these two mutants. The sporogenous tissue within all three of these mutant lines collapses prior to anthesis.     

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and β-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Suppressible P-element alleles of the vestigial locus in Drosophila melanogaster

Summary A series of P-element insertion mutations at one site in the vestigial (vg) locus was tested for cytotype dependent effects on vg expression. The mutant phenotypes for four P-element vg alleles were suppressed when the alleles were stabilized in the P-cytotype. The suppression was observed whenever repressor-producing P-elements were present in the genome. Genetic and molecular analysis indicated that the suppression is not due to excision or other irreversible alterations of the inserts. The results are consistent with a model in which somatic P-element repressor binding to the ends of P-element inserts can modify the effects of these inserts on target gene expression.