Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

T-DNA recombination and replication in maize cells

T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.     

OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice

Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP ( http://rgp.dna.affrc.go.jp ). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3 ; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3 , which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation.     

A rice phenomics study—phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chyr-Guan Chern, Ming-Jen Fan, and Su-May Yu have contributed equally to this work.     

An α-galactosidase with an essential function during leaf development

The putative α-galactosidase gene HvSF11 of barley, previously shown to be expressed during dark induced senescence, is expressed in the growing/elongating zone of primary foliage leaves of barley. The amino acid sequence deduced from the full length HvSF11 cDNA contains a hydrophobic signal sequence at the N-terminus. Phylogenetic relationship of the HvSF11 encoded barley α-galactosidase to other α-galactosidases revealed high homology with the α-galactosidase encoded by the gene At5g08370 from Arabidopsis thaliana. We have isolated two independent heterozygous At5g08370 T-DNA insertion mutants from Arabidopsis thaliana, both of which have a higher number of rosette leaves with a curly surface leaf morphology and delayed flowering time in comparison to wildtype plants. Localization of the Arabidopsis α-galactosidase protein via GUS-tag revealed that the protein is associated with the cell wall. This result was confirmed by immunological detection of the orthologous barley protein in a protein fraction derived from cell walls of barley leaves. It is concluded that the α-galactosidase proteins from barley and Arabidopsis might fulfill an important role in leaf development by functioning in cell wall loosening and cell wall expansion.

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