Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modifiedDs transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.Equal contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this work     

The Chloroplast Function Database: a large‐scale collection of Arabidopsis Ds/Spm‐ or T‐DNA‐tagged homozygous lines for nuclear‐encoded chloroplast proteins,and their systematic phenotype analysis

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post‐translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor–mutator) transposon‐ or T‐DNA‐tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein‐coding regions. We systematically observed 3‐week‐old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild‐type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear‐encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/ . The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.     

Frequency and distance of transposition of a modifiedDissociation element in transgenic tobacco

Effective transposon tagging with theAc/Ds system in heterologous plant species relies on the accomplishment of a potentially high transposon-induced mutation frequency. The primary parameters that determine the mutation frequency include the transposition frequency and the transposition distance. In addition, the development of a generally applicable transposon tagging strategy requires predictable transposition behaviour. We systematically analysedDs transposition frequencies andDs transposition distances in tobacco. An artificialDs element was engineered with reporter genes that allowed transposon excision and integration to be monitored visually. To analyse the variability ofDs transposition between different tobacco lines, eight single copy T-DNA transformants were selected. Fortrans-activation of theDs elements, differentAc lines were used carrying an unmodifiedAc ⁺ element, an immobilizedsAc element and a stableAc element under the control of a heterologous chalcone synthas (chsA) promoter. With allAc elements, eachDs line showed characteristic and heritable variegation patterns at the seedling level. SimilarDs line-specificity was observed for the frequency by whichDs transpositions were germinally transmitted, as well as for the distances of theDs transpositions. ThesAc element induced transposition ofDs late in plant development, resulting in low germinal transposition frequencies (0.37%) and high incidences of independent transposition (83%). The majority of theseDs elements (58%) transposed to genetically closed linked sites (10 cM).     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation(Ds) transposon lines with insertions on all 5 Arabidopsischromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsisgenome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsislines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Chromosomal translocations are a common phenomenon in Arabidopsis thaliana T-DNA insertion lines

Ordered collections of Arabidopsis thaliana lines containing mapped T-DNA insertions have become an important resource for plant scientists performing genetic studies. Previous reports have indicated that T-DNA insertion lines can have chromosomal translocations associated with the T-DNA insertion site, but the prevalence of these rearrangements has not been well documented. To determine the frequency with which translocations are present in a widely-used collection of T-DNA insertion lines, we analyzed 64 independent lines from the Salk T-DNA mutant collection. Chromosomal translocations were detected in 12 of the 64 lines surveyed (19%). Two assays were used to screen the T-DNA lines for translocations: pollen viability and genome-wide genetic mapping. Although the measurement of pollen viability is an indirect screen for the presence of a translocation, all 11 of the T-DNA lines showing an abnormal pollen phenotype were found to contain a translocation when analyzed using genetic mapping. A normal pollen phenotype does not, however, guarantee the absence of a translocation. We observed one T-DNA line with normal pollen that nevertheless had a translocation based on genetic mapping results. One additional phenomenon that we observed through our genetic mapping experiments was that the T-DNA junctions on the 5'- and 3'-sides of a targeted gene can genetically separate from each other in some cases. Two of the lines in our survey displayed this 'T-DNA borders separate' phenomenon. Experimental procedures for efficiently screening T-DNA lines for the presence of chromosomal abnormalities are presented and discussed.     

Establishment of a gene tagging system in Arabidopsis thaliana based on the maize transposable element Ac

Summary An Ac-derived, two-component transposable element system has been developed and analyzed with respect to its use in Arabidopsis thaliana. This system consists of an immobilized Ac element (Ac clipped wing, Ac_cl) as the source of transactivating transposase and a nonautonomous Ds element, Ds_A, which is inserted into a chimaeric neomycinphosphotransferase gene used as excision marker. After separate introduction of Ac_c1 and Ds_A into Arabidopsis thaliana, progeny analysis of crosses between five different Ac_cl lines and seven different Ds_A lines shows that: (1) different Ac_cl lines differ greatly in their capacity to transactivate Ds_A; (2) different Ds_A lines do not differ significantly with respect to Ds_A transactivation by one Ac_cl line; (3) reintegration of excised Ds_A elements, both at (genetically) linked and unlinked sites, occurs in about 50% of the excision events; and (4) plants with a high rate of somatic excisions can be used as source of new Ds_A transpositions, allowing the creation of a large number of independent Ds_A insertions.     

Factors affecting the excision frequency of the maize transposable element Ds in Arabidopsis thaliana

A two-element transposon system based on the maize elements Ac and Ds is currently being used for insertional mutagenesis in Arabidopsis. With the aim of making this system as efficient as possible we have continued to analyse several parameters which affect Ds activity in Arabidopsis. The influence of genomic position on Ds excision has been analysed in five lines carrying Ds integrated in different genomic locations. Differences in both somatic and germinal excision were observed between the different lines. The relationship between somatic and germinal excision, the timing of excision events and environmental influences on transposition frequency have been investigated. The effect of varying dosage of the different elements was also analysed. A strong positive dosage effect was observed for the transposase source, but not for the Ds element. Analysis of germinal excision events showed that the majority of them occurred very late in the development of the plant, resulting in the majority of Ds transpositions being independent events.     

Extensive Phenotypic Variation among Allelic T-DNA Inserts in Arabidopsis thaliana

T-DNA insertion mutants are a tool used widely in Arabidopsis thaliana to disrupt gene function. We phenotyped multiple homozygous T-DNA A. thaliana mutants at each of two loci (AT1G11060 and AT4G00210). We measured life history traits, including germination, size at reproduction and fruit production. Allelic T-DNA lines differed for most traits at AT1G11060 but not at AT4G00210. However, insertions in exons differed from other insertion positions in AT4G00210 but not in AT1G11060. We found evidence for additional insertions in approximately half of the lines, but found few phenotypic consequences. In general, our results suggest that a cautious interpretation of T-DNA phenotypes is warranted.     

Deviating T-DNA transfer fromAgrobacterium tumefaciens to plants

We analyzed 29 T-DNA inserts in transgenicArabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA. DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data. Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences. Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region. On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.     

<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.     

A transiently expressed transposase system to generate <Emphasis Type="Italic">Ds</Emphasis>-tagged mutants for functional genomics in sorghum

Six T-DNA/Ds launch pad lines (T₀) previously generated by Agrobacterium-mediated transformation of M 35-1 genotype of sorghum were confirmed by PCR. T₁ plants of all six lines showed 3:1 segregation when sprayed with 12 ppm Basta herbicide, indicating single copy insertion, which was also confirmed by left border flanking sequence tag. Calli derived from pNU435-T₀(1) primary transformant was co-infected with Agrobacterium-carrying iAc construct for transient expression of transposase to generate stable Ds-tagged mutants in the T₀ generation. All nine regenerants were PCR-positive for Ds. However, four contained intact T-DNA/Ds launch pad, while five plants carried empty launch pad, indicating transposition of the Ds. One of these plants, IDs-T₀(8), was negative for iAc PCR, indicating that it was a stable Ds-tagged mutant. Of the four plants with intact T-DNA/Ds, IDs-T₀(5) carrying iAc was a double transformant and mutagenic, which can generate mutants in the subsequent generation. Hence, the transient expression of transposase system in sorghum reported here can be employed for high throughput mutagenesis.     

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.     

An inducible targeted tagging system for localized saturation mutagenesis in Arabidopsis

We describe a system of inducible insertional mutagenesis based on the Ac-Ds family of transposons for targeted tagging in Arabidopsis (Arabidopsis thaliana). In this system, the Ac and Ds elements are carried within the same T-DNA and a heat shock-inducible transposase fusion is utilized to control the levels of transposase gene expression, generating transpositions that can be subsequently stabilized without requiring crossing or segregation. We have mapped 40 single-copy lines by thermal asymmetric interlaced-PCR, which can be used as potential launch pads for heat shock mutagenesis. Using a starter line selected for detailed analysis, the efficiency of tagging over a 50-kb region in the genome was examined. Hits were obtained in the targeted genes with multiple alleles for most genes, with approximately equal numbers of hits detected in genes on either side of the T-DNA. These results establish the feasibility of our approach for localized saturation mutagenesis in Arabidopsis. This system is very efficient and much less laborious as compared to conventional crossing schemes and may be generally applicable to other plant species for which large-scale T-DNA tagging is not currently feasible.     

Molecular analysis of <Emphasis Type="Italic">Agrobacterium</Emphasis> T-DNA integration in tomato reveals a role for left border sequence homology in most integration events

Studies in several plants have shown that Agrobacterium tumefaciens T-DNA can integrate into plant chromosomal DNA by different mechanisms involving single-stranded (ss) or double-stranded (ds) forms. One mechanism requires sequence homology between plant target and ssT-DNA border sequences and another double-strand-break repair in which preexisting chromosomal DSBs “capture” dsT-DNAs. To learn more about T-DNA integration in Solanum lycopersicum we characterised 98 T-DNA/plant DNA junction sequences and show that T-DNA left border (LB) and right border transfer is much more variable than previously reported in Arabidopsis thaliana and Populus tremula. The analysis of seven plant target sequences showed that regions of homology between the T-DNA LB and plant chromosomal DNA plays an important role in T-DNA integration. One T-DNA insertion generated a target sequence duplication that resulted from nucleolytic processing of a LB/plant DNA heteroduplex that generated a DSB in plant chromosomal DNA. One broken end contained a captured T-DNA that served as a template for DNA repair synthesis. We propose that most T-DNA integrations in tomato require sequence homology between the ssT-DNA LB and plant target DNA which results in the generation of DSBs in plant chromosomal DNA.     

An improved method to identify the T-DNA insertion site in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> genome

An improved method to identify the T-DNA insertion site in transgenic Arabidopsis thaliana (Columbia ecotype) genome was presented. Firstly, the pre-adaptor was amplified by PCR from the plasmid pLASC11.12.8 and digested by HindIII to produce the adaptor. After treated with calf intestine alkaline phosphatase, the adaptor was ligated to the genomic restriction digested fragment with the same restriction endonucleases. Then two rounds of PCR (nested-PCR) were carried out and an unknown sequence between the T-DNA and the adaptor was amplified. Further analysis would reveal the accurate site of T-DNA insertion into transgenic A. thaliana genome. This text was submitted by the authors in English.     

Influence of the Nature of the T-DNA Insertion Region on Transgene Expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In the experiment reported here, effect of the nature of T-DNA integration region on the activity of the transgenes was studied by using a color marker gene in Arabidopsis thaliana. For this purpose, a pale homozygous ch-42 mutant was transformed with the wild-type copy of the gene (CH-42) using kanamycin resistance gene as a selectable marker. Two independent lines were identified in which CH-42 transgene was inactive. The T-DNA flanking sequences were recovered from these inactive and two active lines. These flanking sequences were used to examine copy number and DNA methylation of the T-DNA insertion site in active and inactive lines. Southern blots produced by using MspI/HpaII digested genomic DNA showed signs of methylation in both inactive lines. Furthermore, in one of the inactive line, the T-DNA flanking sequence probe hybridized to highly repetitive sequence. The results suggest some correlation between silencing of the transgene and methylation of its insertion region.     

Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns

 In comparison with the production of transgenic plants, the generation of hairy roots has the advantage that more independent transgenic lines can be produced in a shorter period of time. Therefore, we wanted to combine this approach with the promoter-trapping strategy to identify nematode-induced plant promoters. For the efficient production and culture of transgenic hairy root lines of Arabidopsis thaliana, the standard Agrobacterium rhizogenes transformation procedure was modified to avoid rapid callusing of the hairy roots. An average of 0.72 independent kanamycin-resistant (Km^R) roots were obtained per leaf piece. However, a much lower frequency of reporter gene activation was obtained than expected from experiments with the same vectors in Agrobacterium tumefaciens: of more than 700 independent Km^R hairy roots tested, only 8 were β-glucuronidase (GUS) positive. DNA hybridization was done on ten hairy root lines, of which one had a single truncated T-DNA and the others multiple copies of T-DNA that led to complex hybridization patterns. In a parallel analysis of A. thaliana plants transformed with the same vectors using A. tumefaciens, relatively simple T-DNA integration patterns were obtained. The low occurrence of GUS-positive hairy root lines in our experiments could be explained by the multiple T-DNA copies, especially in inverted array, that result in high frequencies of gene inactivation. Received: 11 August 1998 / Revision received: 17 February 1999 / Accepted: 18 March 1999     

Collection of Arabidopsis thalianaMorphological Insertion Mutants

A collection of transgenic Arabidopsis thalianaplants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12–18% of the total number of A. thalianagenes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.