Linked and Unlinked Transposition of a Genetically Marked Dissociation Element in Transgenic Tomato

We have introduced a genetically marked Dissociation transposable element (Ds(neo)) into tomato. In the presence of Ac transposase, Ds(neo) excised from an integrated T-DNA and reinserted at numerous new sites in the tomato genome. The marker genes of Ds(neo) (NPTII) and the T-DNA (HPT) facilitated identification of plants bearing transposon excisions and insertions. To explore the feasibility of gene tagging strategies in tomato using Ds(neo), we examined the genomic distribution of Ds(neo) receptor sites, relative to the location of the donor T-DNA locus. Restriction fragment length polymorphism mapping of transposed Ds(neo) elements was conducted in two tomato families, derived from independent primary transformants each bearing Ds(neo) within a T-DNA at a unique position in the genome. Transposition of Ds(neo) generated clusters of insertions that were positioned on several different tomato chromosomes. Ds(neo) insertions were often located on the same chromosome as the T-DNA donor site. However, no insertion showed tight linkage to the T-DNA. We consider the frequency and distance of Ds(neo) transposition observed in tomato to be well suited for transposon mutagenesis. Our study made use of a novel, stable allele of Ac (Ac3) that we discovered in transgenic tomato. We determined that the Ac3 element bears a deletion of the outermost 5 base pairs of the 5'-terminal inverted repeat. Though incapable of transposition itself, Ac3 retained the ability to mobilize Ds(neo). We conclude that a dual element system, composed of the stable Ac3 trans-activator in combination with Ds(neo), is an effective tool for transposon tagging experiments in tomato.     

Heterologous transposon tagging of the DRL1 locus in Arabidopsis.

The development of heterologous transposon tagging systems has been an important objective for many laboratories. Here, we demonstrate the use of a Dissociation (Ds) derivative of the maize transposable element Activator (Ac) to tag the DRL1 locus of Arabidopsis. The drl1 mutant shows highly abnormal development with stunted roots, few root hairs, lanceolate leaves, and a highly enlarged, disorganized shoot apex that does not produce an inflorescence. The mutation was shown to be tightly linked to a transposed Ds, and somatic instability was observed in the presence of the transposase source. Some plants showing somatic reversion flowered and produced large numbers of wild-type progeny. These revertant progeny always inherited a DRL1 allele from which Ds had excised. Analysis of the changes in DNA sequence induced by the insertion and excision of the Ds element showed that they were typical of those induced by Ac and Ds in maize.     

A four-element based transposon system for allele specific tagging in plants—Theoretical considerations

The two-element transposon constructs, utilizing either Ac/Ds or Spm/dSpm, allow random tagging of genes in heterologous model species, but are inadequate for directed tagging of specific alleles of agronomic importance. We propose the use of Ac/Ds in conjunction with Spm/dSpm to develop a four-element system for directed tagging of crop-specific alleles. The four-element based construct would include both Ds and dSpm along with relevant marker genes and would function in two steps. In the first step dSpm(Ds) stocks (a minimum of two) would be crossed to a line containing transposases of Spm and unlinked integrations would be selected from segregating population by the use of a negative selection marker to develop stocks representing integration of dSpm(Ds) at a large number of locations in the genome. Selections would be made for a line in which dSpm(Ds) shows partial or complete linkage to the allele of interest. In the second step selected line would be crossed to a line containing Ac transposase to induce transpositions of Ds element to linked sites thereby exploiting the natural tendency of Ds element to jump to linked sites. Unlinked jumps of dSpm(Ds) and linked jumps of Ds could be monitored by appropriate marker genes. The proposed model would allow tagging of allele of interest in chromosome addition lines and also help in the efficient use of genic male sterility systems for hybrid seed production by tightly marking the fertility restorer gene with a negative selection marker.     

Somatic Variegation and Germinal Mutability Reflect the Position of Transposable Element Dissociation within the Maize R Gene

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.     

The utility of transposable elements as tools for the isolation of plant genes

Transposable elements are segments of DNA which have the unique capability of being able to excise from one site in the genome and reintegrate into new, different sites elsewhere in the genome. When transposition takes place and integration occurs within a gene locus, mutations are frequently generated producing variegated or recessive phenotypes. This ability of transposable elements to act as mutagenic agents through their association with particular gene sequences has lead to the development of the procedure of transposon tagging or gene tagging in higher plants. Through this technique, transposable elements can be used to clone and isolate genes of interest for which little or nothing is known about the final product (i.e., polypeptide). This offers tremendous potential for the isolation of a variety of agronomically important genes, which are virtually impossible to recover by other currently available gene cloning methodologies. To date, the technique has been used successfully to isolate genes from corn and snapdragon. Using gene transfer technologies, the potential now exists to extend this approach to clone genes from other plant species. Advantages and limitations of transposon tagging for isolating plant genes will be discussed.     

Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast terminal oxidase associated with phytoene desaturation.

The immutans (im) mutant of Arabidopsis shows a variegated phenotype comprising albino and green somatic sectors. We have cloned the IM gene by transposon tagging and show that even stable null alleles give rise to a variegated phenotype. The gene product has amino acid similarity to the mitochondrial alternative oxidase. We show that the IM protein is synthesized as a precursor polypeptide that is imported into chloroplasts and inserted into the thylakoid membrane. The albino sectors of im plants contain reduced levels of carotenoids and increased levels of the carotenoid precursor phytoene. The data presented here are consistent with a role for the IM protein as a cofactor for carotenoid desaturation. The suggested terminal oxidase function of IM appears to be essential to prevent photooxidative damage during early steps of chloroplast formation. We propose a model in which IM function is linked to phytoene desaturation and, possibly, to the respiratory activity of the chloroplast.     

Selection of independent Ds transposon insertions in somatic tissue of potato by protoplast regeneration

Potato is an autotetraploid crop plant that is not very amenable to the deployment of transposon tagging for gene cloning and gene identification. After diploidisation it is possible to get potato genotypes that grow well, but they are self-incompatible. This prevents the production of selfed progeny that are normally used in gene tagging approaches to select for parental lines with the target gene to be tagged in a homozygous stage. We describe here an alternative selection method for directed transposon tagging for a gene of interest in a heterozygous background. Diploid potato plants with a Ds transposon linked to the desired gene of interest (the Phytophthora infestans R1 resistance locus) in a heterozygous stage were used for the development of this directed transposon tagging strategy. After crossing to a diploid Ac transposon-containing genotype, 22 ’interesting’ seedlings (R1Ds/r–; Ac/–) were selected that showed active Ds transposition as displayed by DNA blot hybridisation, empty donor site PCR and sequencing. Protoplast isolation and the use of the hygromycin gene as a cell-specific selection marker of Ds excision enabled the direct selection of Ds excision sectors in these highly chimaeric seedlings. This somatic selection of Ds transpositions and the regeneration through protoplasts resulted in the development of a large population of almost 2000 hygromycin-resistant plants. Southern blot analysis confirmed the insertion of Ds at independent positions in the genome. Every selected plant displayed independent Ds excisions and re-insertions due to the expression of the Ac transposase throughout development. This population, which is developed from seedlings with the desired R1 gene in a heterozygous stage, is directly useful for searching for transposon-tagged R1 mutants. In general, this approach for selecting for somatic transpositions is particularly suitable for the molecular isolation of genes in a heterozygous crop like potato. Received: 29 November 1999 / Accepted: 30 December 1999     

Assignment of 44 Ds Insertions to the Linkage Map of Arabidopsis

Transposon tagging is a useful tool for biological studies. Transposon insertions have been used to obtain new mutants which are extremely helpful in understanding gene function. These insertions immediately provide a means to isolate the corresponding genes. Transposon tagging has also been used to clone genes previously defined by point mutations. In addition, transposon insertions into cloned genes that lack mutations can be generated to facilitate functional analysis. The maize Ac/Ds transposon elements are known to transpose to local sites with high frequencies and have been shown to function in several dicots. To generate a collection of Ds elements for the purpose of targeted insertional mutagenesis of mapped genes in Arabidopsis, we have mapped 44 Ds insertions by simple sequence length polymorphism (SSLP). Because the Arabidopsis genome project is advancing rapidly, many genes will be discovered whose functions are unknown. The mapped 44 Ds insertions will be a useful resource for post-genome analysis of gene functions in Arabidopsis.     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.     

The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis

Variegated leaves are often caused by a nuclear recessive mutation in higher plants. Characterization of the gene responsible for variegation has shown to provide several pathways involved in plastid differentiation. Here we describe an Arabidopsis variegated mutant isolated by T-DNA tagging. The mutant displayed green and yellow sectors in all green tissues except for cotyledons. Cells in the yellow sector of the mutant contained both normal-appearing and mutant chloroplasts. The isolated mutant was shown to be an allele of the previously reported mutant, yellow variegated (var2). Cloning and molecular characterization of the VAR2 locus revealed that it potentially encodes a chloroplastic homologue of FtsH, an ATP-dependent metalloprotease that belongs to a large protein family involved in various cellular functions. ftsH-like genes appear to comprise a small gene family in Arabidopsis genome, since at least six homologues were found in addition to VAR2. Dispensability of VAR2 was therefore explained by the redundancy of genes coding for FstHs. In the yellow regions of the mutant leaves, accumulation of photosynthetic protein components in the thylakoid membrane appeared to be impaired. Based on the role of FtsH in a protein degradation pathway in plastids, we propose a possibility that VAR2 is required for plastid differentiation by avoiding partial photooxidation of developing chloroplasts.     

Localization of Ds-transposon containing T-DNA inserts in the diploid transgenic potato: linkage to the R1 resistance gene against Phytophthora infestans (Mont.) de Bary.

The Dissociation transposable element (Ds) of maize containing NPTII was introduced into the diploid potato (Solanum tuberosum) clone J91-6400-A16 through Agrobacterium tumefaciens mediated transformation. Genomic DNA sequences flanking the T-DNAs from 312 transformants were obtained with inverse polymerase chain reaction or plasmid rescue techniques and used as probes for RFLP linkage analysis. The RFLP map location of 60 T-DNAs carrying Ds-NPTII was determined. The T-DNA distribution per chromosome and the relative distance between them appeared to be random. All 12 chromosomes have been covered with Ds-containing T-DNAs, potentially enabling tagging of any gene in the potato genome. The T-DNA insertions of two transformants, BET92-Ds-A16-259 and BET92-Ds-A16-416, were linked in repulsion to the position of the resistance gene R1 against Phytophthora infestans. After crossing BET92-Ds-A16-416 with a susceptible parent, 4 desired recombinants (Ds carrying T-DNA linked in coupling phase with the R1 gene) were discovered. These will be used for tagging the R1 gene. The efficiency of the pathway from the introduction to localization of T-DNAs is discussed. Key words : Solanum tuberosum, Phytophthora infestans, Ds element, transposon tagging, R genes, euchromatin.     

The maize high-lysine mutant opaque7 is defective in an acyl-CoA synthetase-like protein

Maize (Zea mays) has a large class of seed mutants with opaque or nonvitreous endosperms that could improve the nutritional quality of our food supply. The phenotype of some of them appears to be linked to the improper formation of protein bodies (PBs) where zein storage proteins are deposited. Although a number of genes affecting endosperm vitreousness have been isolated, it has been difficult to clone opaque7 (o7), mainly because of its low penetrance in many genetic backgrounds. The o7-reference (o7-ref) mutant arose spontaneously in a W22 inbred, but is poorly expressed in other lines. We report here the isolation of o7 with a combination of map-based cloning and transposon tagging. We first identified an o7 candidate gene by map-based cloning. The putative o7-ref allele has a 12-bp in-frame deletion of codons 350-353 in a 528-codon-long acyl-CoA synthetase-like gene (ACS). We then confirmed this candidate gene by generating another mutant allele from a transposon-tagging experiment using the Activator/Dissociation (Ac/Ds) system in a W22 background. The second allele, isolated from ～1 million gametes, presented a 2-kb Ds insertion that resembles the single Ds component of double-Ds, McClintock's original Dissociation element, at codon 496 of the ACS gene. PBs exhibited striking membrane invaginations in the o7-ref allele and a severe number reduction in the Ds-insertion mutant, respectively. We propose a model in which the ACS enzyme plays a key role in membrane biogenesis, by taking part in protein acylation, and that altered PBs render the seed nonvitreous.     

Excision of transposable elements from the chalcone isomerase and dihydroflavonol 4-reductase genes may contribute to the variegation of the yellow-flowered carnation (Dianthus caryophyllus)

In the "Rhapsody" cultivar of the carnation, which bears white flowers variegated with red flecks and sectors, a transposable element, dTdic1, belonging to the Ac/Ds superfamily, was found within the dihydroflavonol 4-reductase (DFR) gene. The red flecks and sectors of "Rhapsody" may be attributable to a reversion to DFR activity after the excision of dTdic1. The yellow color of the carnation petals is attributed to the synthesis and accumulation of chalcone 2'-glucoside. In several of the carnation cultivars that bear yellow flowers variegated with white flecks and sectors, both the chalcone isomerase (CHI) and DFR genes are disrupted by dTdic1.