Induction of chromosomal inversion by integration of T-DNA in the rice genome

Transfer DNA(T-DNA) of Agrobacterium tumefaciens integration in the plant genome may lead to rearrangements of host plant chromosomal fragments,including inversions.However,there is very little information concerning the inversion.The present study reports a transgenic rice line selected from a T-DNA tagged population,which displays a semi-dwarf phenotype.Molecular analysis of this mutant indicated an insertion of two tandem copies of T-DNA into a locus on the rice genome in a head to tail mode.This inserti...     

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.     

Molecular analysis of rice plants harboring a multi-functional T-DNA tagging system

About 25,000 rice T-DNA insertional mutant lines were generated using the vector pCAS04 which has both promoter-trapping and activation-tagging function. Southern blot analysis revealed that about 40% of these mutants were single copy integration and the average T-DNA insertion number was 2.28. By extensive phenotyping in the field, quite a number of agronomically important mutants were obtained. Histochemical GUS assay with 4,310 primary mutants revealed that the GUS-staining frequency was higher than that of the previous reports in various tissues and especially high in flowers. The T-DNA flanking sequences of some mutants were isolated and the T-DNA insertion sites were mapped to the rice genome. The flanking sequence analysis demonstrated the different integration pattern of the right border and left border into rice genome. Compared with Arabidopsis and poplar, it is much varied in the T-DNA border junctions in rice.     

Systematic reverse genetic screening of T-DNA tagged genes in rice for functional genomic analyses: MADS-box genes as a test case

We have generated 47 DNA pools and 235 subpools from 21,049 T-DNA insertion lines of rice. DNA pools of 500-1,000 lines were adequate for screening a T-DNA insertion within a 2-kb region. To examine the efficacy of the DNA pools, we selected MADS-box genes, which play an important role in controlling various aspects of plant development. A total of 34 MIKC-type MADS-box genes have now been identified from rice sequence databases. Our PCR screening for T-DNA insertions within 12 MADS-box genes resulted in the identification of five insertions in four different genes. These DNA pools will be valuable when isolating T-DNA insertional mutants in various rice genes. The DNA pool screening service and the mutant seeds are available upon request to genean@postech.ac.kr.     

Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library

We isolated 13 804 T-DNA flanking sequence tags (FSTs) from a T-DNA insertion library of rice. A comprehensive analysis of the 13 804 FSTs revealed a number of features demonstrating a highly non-random distribution of the T-DNA insertions in the rice genome: T-DNA insertions were biased towards large chromosomes, not only in the absolute number of insertions but also in the relative density; within chromosomes the insertions occurred more densely in the distal ends, and less densely in the centromeric regions; the distribution of the T-DNA insertions was highly correlated with that of full-length cDNAs, but the correlations were highly heterogeneous among the chromosomes; T-DNA insertions strongly disfavored transposable element (TE)-related sequences, but favored genic sequences with a strong bias toward the 5' upstream and 3' downstream regions of the genes; T-DNA insertions preferentially occurred among the various classes of functional genes, such that the numbers of insertions were in excess in certain functional categories but were deficient in other categories. The analysis of DNA sequence compositions around the T-DNA insertion sites also revealed several prominent features, including an elevated bendability from -200 to 200 bp relative to the insertion sites, an inverse relationship between the GC and TA skews, and reversed GC and TA skews in sequences upstream and downstream of the insertion sites, with both GC and TA skews equal to zero at the insertion sites. It was estimated that 365 380 insertions are needed to saturate the genome with P = 0.95, and that the 45 441 FSTs that have been isolated so far by various groups tagged 14 287 of the 42 653 non-TE related genes.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

A chromosomal paracentric inversion associated with T-DNA integration in Arabidopsis

T-DNA integration in the nuclear plant genome may lead to rearrangements of the plant target site. Here we present evidence for a chromosomal inversion of 26 cM bordered by two T-DNAs in direct orientation, which is linked to the mgoun2 mutation. The integration sites of the T-DNAs map at positions 80 and 106 of chromosome I and we show that each T-DNA is bordered by plant sequences from positions 80 and 106, respectively. Although the T-DNAs are physically distant, they are genetically closely linked. In addition, three markers located on the chromosome segment between the two T-DNA integration sites show no recombination with the mgo2 mutation. We show that the inversion cannot be a consequence of a recombination event between the two T-DNAs, but that the integration of the T-DNAs and the inversion were two temporally linked events. T-DNA integration mechanisms that could have led to this inversion are discussed.     

利用Inverse PCR快速筛选单个T-DNA拷贝转基因水稻植株的方法

为了获得单个T-DNA插入拷贝的植株, 我们建立了一套利用Inverse PCR(IPCR)快速检测转基因水稻中T-DNA拷贝数的方法。用IPCR的方法可以扩增出与已知T-DNA序列相邻的水稻基因组DNA未知序列,由此推测转基因水稻植株中T-DNA的拷贝数。我们共对15个转化株系20棵不同植株的DNA进行了IPCR检测。其中12株表现为T-DNA单拷贝插入,3株为双拷贝插入,1株为三拷贝插入。另外4株未检测到T-DNA插入拷贝。IPCR分析结果经过Southern杂交和测序的验证。     

T-DNA Integration Category and Mechanism in Rice Genome

T-DNA integration is a key step in the process of plant transformation, which is proven to be important for analyzing T-DNA integration mechanism. The structures of T-DNA right borders inserted into the rice (Oryza sativa L.) genome and their flanking sequences were analyzed. It was found that the integrated ends of the T-DNA right border occurred mainly on five nucleotides "TGACA" in inverse repeat (IR)sequence of 25 bp, especially on the third base "A". However, the integrated ends would sometimes lie inward of the IR sequence, which caused the IR sequence to be lost completely. Sometimes the right integrated ends appeared on the vector sequences rightward of the T-DNA right border, which made the TDNA, carrying vector sequences, integrated into the rice genome. These results seemingly suggest that the IR sequence of the right border plays an important role in the process of T-DNA integration into the rice genome, but is not an essential element. The appearance of vector sequences neighboring the T-DNA right border suggested that before being transferred into the plant cell from Agrobacterium, the entire T-DNA possibly began from the left border in synthesis and then read through at the right border. Several nucleotides in the T-DNA right border homologous with plant DNA and filler DNAs were frequently discovered in the integrated position ofT-DNA. Some small regions in the right border could match with the plant sequence, or form better matches, accompanied by the occurrence of filler DNA, through mutual twisting, and then the TDNA was integrated into plant chromosome through a partially homologous recombination mechanism. The appearance of filler DNA would facilitate T-DNA integration. The fragments flanking the T-DNA right border in transformed rice plants could derive from different parts of the inner T-DNA region; that is, disruption and recombination could occur at arbitrary positions in the entire T-DNA, in which the homologous area was comparatively easier to be disrupted. The structure of flanking sequences of T-DNA integrated in the rice chromosome presented various complexities. These complexities were probably a result of different patterns of recombination in the integrating process. Some types of possible integrating mechanism are detailed.     

利用Inverse PCR快速筛选单个T—DNA拷贝转基因水稻植株的方法

In order to obtain single T-DNA copy transgenic rice, we have established a quick method to estimate the T-DNA copy number in transgenic rice using inverse PCR (IPCR). IPCR was used to amplify junction fragments, i.e. plant genomic DNA sequences flanking the known T-DNA sequences, which will help to estimate the T-DNA copy number in transgenic rice. We have analyzed 20 transgenic plants of 15 transgenic lines. Most plants (12) contain one integrated T-DNA copy per genome, 3 plants contain two and 1 plant contains 3 copies. In 4 transgenic plants no T-DNA copies could be detected using this method. The IPCR results were further tested by Southern analysis and sequence analysis.     

T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites

A statistical analysis of 9000 flanking sequence tags characterizing transferred DNA (T-DNA) transformants in Arabidopsis sheds new light on T-DNA insertion by illegitimate recombination. T-DNA integration is favoured in plant DNA regions with an A-T-rich content. The formation of a short DNA duplex between the host DNA and the left end of the T-DNA sets the frame for the recombination. The sequence immediately downstream of the plant A-T-rich region is the master element for setting up the DNA duplex, and deletions into the left end of the integrated T-DNA depend on the location of a complementary sequence on the T-DNA. Recombination at the right end of the T-DNA with the host DNA involves another DNA duplex, 2–3 base pairs long, that preferentially includes a G close to the right end of the T-DNA.     

Generation and flanking sequence analysis of a rice T-DNA tagged population

Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice (Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T₁ generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by D. Mackill     

水稻T-DNA插入突变体库的构建及突变类型的分析

利用农杆菌介导的转化系统转化中花11成熟胚愈伤组织,获得1489个独立转化的T-DNA插入再生株系。PCR和Southern杂交的结果表明,69．8％转化株系被整合了T-DNA,通过Tail-PCR也从转化植株中扩增出T-DNA侧翼序列。同时对1066个T1转化株系的抽穗期、株高、单株穗数的调查结果表明,不同株系中分离出了突变植株。     

Generation and analysis of end sequence database for T-DNA tagging lines in rice

We analyzed 6749 lines tagged by the gene trap vector pGA2707. This resulted in the isolation of 3793 genomic sequences flanking the T-DNA. Among the insertions, 1846 T-DNAs were integrated into genic regions, and 1864 were located in intergenic regions. Frequencies were also higher at the beginning and end of the coding regions and upstream near the ATG start codon. The overall GC content at the insertion sites was close to that measured from the entire rice (Oryza sativa) genome. Functional classification of these 1846 tagged genes showed a distribution similar to that observed for all the genes in the rice chromosomes. This indicates that T-DNA insertion is not biased toward a particular class of genes. There were 764, 327, and 346 T-DNA insertions in chromosomes 1, 4 and 10, respectively. Insertions were not evenly distributed; frequencies were higher at the ends of the chromosomes and lower near the centromere. At certain sites, the frequency was higher than in the surrounding regions. This sequence database will be valuable in identifying knockout mutants for elucidating gene function in rice. This resource is available to the scientific community at http://www.postech.ac.kr/life/pfg/risd.     

Transgene repeats in aspen: molecular characterisation suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome

Rearrangements of T-DNAs during genetic transformation of plants can result in the insertion of transgenes in the form of repeats into the host genome and frequently lead to loss of transgene expression. To obtain insight into the mechanism of repeat formation we screened 45 transgenic lines of aspen and hybrid aspen transformed with six different gene constructs. The frequency of T-DNA repeat formation among randomly screened transgenic lines was found to be about 21%. In ten transgenic lines direct repeats were detected. An inverted repeat was found in one other transgenic line. Sequencing of the junctions between the T-DNA inserts revealed identical residual right-border repeat sequences at the repeat junctions in all ten transgenic lines that had direct repeats. Formation of "precise" junctions based on short regions of sequence similarity between recombining strands was observed in three transgenic lines transformed with the same plasmid. Additional DNA sequences termed filler DNAs were found to be inserted between the T-DNA repeats at eight junctions where there was no similarity between recombining ends. The length of the filler DNAs varied from 4 to almost 300 bp. Small filler DNAs--a few base pairs long--were in most cases copied from T-DNA near the break points. The large filler sequences of about 300 bp in two transgenic lines were found to be of host plant origin, suggesting that transgene repeat formation occurred as a result of the simultaneous invasion of a receptive site in the host genome by two independent T-DNA strands. On the basis of the results obtained, and in the light of previous reports on T-DNA/plant DNA junctions in aspen and other crop plants, a mechanistic model for transgene rearrangement and filler formation is suggested.     

A Non-canonical Transferred DNA Insertion at the BRI 1 Locus in Arabidopsis thaliana

Agrobacterium-mediated transformation is widely used in transgenic plant englnserlng and has been proven to be a powerful tool for insertional mutagenesis of the plant genome.The transferred DNA (T-DNA) from Agrobacterlum is Integrated into the plant genome through illegitimate recombination between the T-DNA and the plant DNA.Contrasting to the canonical insertion,here we report on a locus showing a complex mutation associated with T-DNA insertion at the BRI 1 gene in Arabidopsis thaliana.We obtained a mutant line,named salade for its phenotype of dwarf stature and proliferating rosette,Molecular charactedzation of this mutant revealed that in addition to T-DNA a non.T.DNA-Iocalized transposon from bacteda was inserted in the Arabidopsis genome and that a region of more than 11.5 kb of the Arebidopsis genome was deleted at the insertion site.The deleted region contains the brassinosteroid receptor gene BRI 1 and the transcdption factor gene WRKY13.Our finding reveals non-canonical T-DNA insertion,implicating horizontal gene transfer and cautioning the use of T-DNA as mutagen in transgenic research.