T-DNA Insertional Mutagenesis Research Articles

Emerging roles in plant defense for cis-jasmone-induced cytochrome P450 CYP81D11

cis-Jasmone is a volatile organic compound emitted constitutively by flowers or leaves of several plant species where it acts as an attractant for pollinators and as a chemical cue for host localisation (or avoidance) for insects.1-3 It is also released by some plant species after feeding damage inflicted by herbivorous insects and in this case might serve as a chemical cue for parasitoids to guide them to their prey (so called “indirect defense”).4,5 Moreover, we have recently shown that plants can perceive cis-jasmone and that it acts as a signaling molecule in A. thaliana, inducing a discrete and distinctive suite of genes, of which a large subset is putatively involved in metabolism and defense responses.6 Cytochrome P450s feature prominently in these functional subsets and of these the highest fold change upon cis-jasmone treatment occurred with the cytochrome CYP81D11 (At3g28740).6 Hence this gene was chosen for a more thorough analysis of the potential biological relevance of the cis-jasmone induced defense response. Although the precise function of CYP81D11 remains to be determined, we could previously demonstrate its involvement in the indirect defense response in Arabidopsis, as plants exposed to cis-jasmone ceased to be attractive to the aphid parasitoid Aphidius ervi when this P450 was inactivated by T-DNA insertion mutagenesis.6 Here we report additional experiments which give further support to a role of CYP81D11 in the direct or indirect defense response of A. thaliana.

Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis.

Verticillium dahliae is the causal agent of vascular wilt in many economically important crops worldwide. Identification of genes that control pathogenicity or virulence may suggest targets for alternative control methods for this fungus. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was applied for insertional mutagenesis of V. dahliae conidia. Southern blot analysis indicated that T-DNAs were inserted randomly into the V. dahliae genome and that 69% of the transformants were the result of single copy T-DNA insertion. DNA sequences flanking T-DNA insertion were isolated through inverse PCR (iPCR), and these sequences were aligned to the genome sequence to identify the genomic position of insertion. V. dahliae mutants of particular interest selected based on culture phenotypes included those that had lost the ability to form microsclerotia and subsequently used for virulence assay. Based on the virulence assay of 181 transformants, we identified several mutant strains of V. dahliae that did not cause symptoms on lettuce plants. Among these mutants, T-DNA was inserted in genes encoding an endoglucanase 1 (VdEg-1), a hydroxyl-methyl glutaryl-CoA synthase (VdHMGS), a major facilitator superfamily 1 (VdMFS1), and a glycosylphosphatidylinositol (GPI) mannosyltransferase 3 (VdGPIM3). These results suggest that ATMT can effectively be used to identify genes associated with pathogenicity and other functions in V. dahliae.

The Hydroxypyruvate-Reducing System in Arabidopsis: Multiple Enzymes for the Same End

Hydroxypyruvate (HP) is an intermediate of the photorespiratory pathway that originates in the oxygenase activity of the key enzyme of photosynthetic CO(2) assimilation, Rubisco. In course of this high-throughput pathway, a peroxisomal transamination reaction converts serine to HP, most of which is subsequently reduced to glycerate by the NADH-dependent peroxisomal enzyme HP reductase (HPR1). In addition, a NADPH-dependent cytosolic HPR2 provides an efficient extraperoxisomal bypass. The combined deletion of these two enzymes, however, does not result in a fully lethal photorespiratory phenotype, indicating even more redundancy in the photorespiratory HP-into-glycerate conversion. Here, we report on a third enzyme, HPR3 (At1g12550), in Arabidopsis (Arabidopsis thaliana), which also reduces HP to glycerate and shows even more activity with glyoxylate, a more upstream intermediate of the photorespiratory cycle. The deletion of HPR3 by T-DNA insertion mutagenesis results in slightly altered leaf concentrations of the photorespiratory intermediates HP, glycerate, and glycine, indicating a disrupted photorespiratory flux, but not in visible alteration of the phenotype. On the other hand, the combined deletion of HPR1, HPR2, and HPR3 causes increased growth retardation, decreased photochemical efficiency, and reduced oxygen-dependent gas exchange in comparison with the hpr1xhpr2 double mutant. Since in silico analysis and proteomic studies from other groups indicate targeting of HPR3 to the chloroplast, this enzyme could provide a compensatory bypass for the reduction of HP and glyoxylate within this compartment.

Towards a novel efficient T-DNA-based mutagenesis and screening system using green fluorescent protein as a vital reporter in the industrially important fungus Trichoderma reesei

Agrobacterium-mediated T-DNA transfer has been proven to be an efficient strategy for insertional mutagenesis and elucidation of gene function in filamentous fungi. The implementation of large-scale T-DNA insertional mutagenesis requires the development of high-efficient transformation and high-throughput screening procedures. Here, using green fluorescent protein (GFP) as a vital marker, a highly efficient T-DNA-based mutagenesis and screening system was developed in Trichoderma reesei. The uridine auxotrophic T. reesei M23 as the host was transformed with A. tumefaciens EH105 strain harboring a binary vector pC-OEP, which beared the pyrG gene for primary selection on minimal medium without uridine and the egfp gene for fluorescence-based rapid screening of the mitotically stable transformants. The efficiency of transformation was up to 10-20 transformants per 10(5) target conidia. Microscopic examination revealed strong GFP expression and fluorescence emission in conidia, growing hyphae and mycelia. An effective and convenient screening procedure using 96-well plates and multilabel counter for fluorescence intensity counting was developed to rapidly identify the T-DNA tagged T. reesei mutants. Furthermore, the presence of T-DNA integration at random sites in the genome was confirmed by Southern blot analysis. This report of the T-DNA-based mutagenesis and rapid screening system using GFP as a vital reporter provides a promising strategy to speeding up the genome-scale T-DNA insertional mutagenesis and functional genomics analysis of this cellulolytic fungus T. reesei.

The polyembryo gene (OsPE) in rice

T-DNA insertional mutagenesis is one of the most important approaches for gene discovery and cloning. A fertile polyembryo mutant generated by T-DNA/Ds insertion in Oryza sativa, cv. Basmati 370 showed twin or triple seedlings at a frequency of 15-20%. T-DNA insertion was confirmed by 950 bp hpt gene amplification in the promoter region of the candidate gene. The annotated protein corresponding to the OsPE candidate gene has been reported as a hypothetical protein in O. sativa. OsPE gene lacked functional homologs in other species. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. RT-PCR showed the expression of OsPE gene in Basmati 370 shoots. Full-length OsPE gene was cloned in Basmati 370. The combined use of Southern blot, genome walking, TAIL-PCR, RT-PCR techniques, and bioinformatics led to the identification of a candidate gene controlling the multiple embryos in rice. There is gain of function, i.e., multiple embryos in the seeds in the knockout mutant OsPE whereas its wild-type allele strictly controls single embryo per seed. The seeds with multiple embryos are distributed at random in the rice mutant panicle. The origin of multiple embryos, whether apomictic, zygotic or both is under investigation.

Arabidopsis glucan synthase-like 10 functions in male gametogenesis

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.

Wax Crystal-Sparse Leaf1 encodes a β–ketoacyl CoA synthase involved in biosynthesis of cuticular waxes on rice leaf

Cuticular waxes, forming the plant/atmosphere interface of plants colonizing the terrestrial environment, are complex mixtures of very-long chain fatty acids (VLCFAs) and their derivatives. In VLCFAs biosynthesis, beta-ketoacyl CoA synthase (E.C.2.3.1.119, KCS) is the key enzyme. Using T-DNA insertional mutagenesis, we identified a cuticle-deficient rice mutant, which displayed a pleiotropic phenotype including reduced growth, leaf fusion, sparse wax crystals, enhanced sensitivity to drought and low fertility. Further analysis indicated that T-DNA was inserted in the 5'-UTR intron of the affected gene, Wax Crystal-Sparse Leaf1 (WSL1), and abnormal transcript caused the loss-of-function of WSL1 gene. Genetic complementation experiment confirmed the function of the candidate gene. WSL1 was predicted to encode a polypeptide containing a conserved FAE1_CUT1_RppA domain typical of the KCS family proteins. Qualitative and quantitative wax composition analyses by gas chromatography-mass spectrometry (GC-MS) demonstrated a marked reduction of total cuticular wax load on wsl1 leaf blades and sheaths, and VLCFA precursors of C20-C24 decreased in both. Moreover, ubiquitous expression of the WSL1 gene gave a hint that WSL1-catalyzed elongation of VLCFAs might participate in a wide range of rice growth and development processes beyond biosynthesis of cuticular waxes.

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

Pollen Lethality: A Phenomenon in Arabidopsis RNA Interference Plants

To study gene function, generation of loss-of-function mutants by RNA interference (RNAi) approaches and T-DNA insertional mutagenesis are widely used in plant science and have proven to be very successful in determining gene functions. AGAMOUS-LIKE18 ( AGL18 ) is a MADS-box gene that is expressed

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.

A Gγ subunit promoter T-DNA insertion mutant—A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

In our previous studies, a nonpathogenic mutant of Magnaporthe grisea, A1-412, which was defective in appressorium formation, penetration and infectious growth, was obtained by T-DNA insertional mutagenesis. Here we reported the identification and characterization of the corresponding gene. Sequence analysis of the genomic DNA franking T-DNA isolated by TAIL-PCR technique showed that T-DNA was inserted into the promoter region of the predicted G protein γ-subunit gene MGG1 (for Magnaporthe grisea G protein Gamma subunit). MGG1 is predicted to encode a 93-aa protein with a typical G-protein gamma like domain (GGL) and C-terminal CAAX box. The amino-acid sequence of MGG1 is highly identical to the Gγ subunits of other filamentous fungi. Further phenotypic investigation of A1-412 showed that exogenous cAMP could induce appressorium formation, although the formed appressoria were abnormal in shape and unable to penetrate onion epidemis or rice leaves. Moreover, few perithecia were observed when A1-412 was crossed with the appropriate mating-type strain. The above phenotypes in A1-412 were partially complemented by reintroduction of the gene MGG1. Our results indicate that the G-protein gamma subunit MGG1 may be involved in regulating morphogenesis, mating and pathogenicity in M. grisea.

Fungi Versus Plants and Mammals

Rice blast is an economically important disease caused by the fungus Magnaporthe grisea , which enters leaves by developing specialized structures called appressoria. Veneault-Fourrey et al . show that during invasion, the fungus undergoes a form of programmed cell death that involves autophagy. Thus, fungal pathogens can use cell death for cellular differentiation and remodeling during host infection. Fungal virulence, the ability of opportunistic fungal pathogens to thrive in mammals, is associated with a transformation from a filamentous, pseudohyphal form that grows at 25°C into a yeast form at 37°C. Using the plant pathogen Agrobacterium tumefaciens as a tool for T-DNA insertional mutagenesis, Nemecek et al. identified mutants that locked the organism in the filamentous form. One mutant that could not make the yeast form also showed defects in cell-wall formation, sporulation, and expression of virulence factors. The defect lay in a gene encoding a histidine kinase, which appeared to be the global regulator for morphological switching and virulence in several species of dimorphic fungi. C. Veneault-Fourrey, M. Barooah, M. Egan, G. Wakley, N. J. Talbot, Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science 312 , 580-583 (2006). [Abstract] [Full Text] J. C. Nemecek, M. Wüthrich, B. S. Klein, Global control of dimorphism and virulence in fungi. Science 312 , 583-588 (2006). [Abstract] [Full Text]

T-DNA mutagenesis in the model plant Medicago truncatula : is it efficient enough for legume molecular genetics?

Abstract The development of efficient transformation protocols in several legume species has opened the possibility to use T-DNA insertional mutagenesis to investigate legume-specific processes such as symbiosis. A T-DNA mutagenesis approach was initiated in the model legume Medicago truncatula by constructing a small collection of 1000 M. truncatula T-DNA lines, using in vitro-mediated transformation-regeneration protocols. In this T-DNA collection, mutant phenotypes were observed but generally did not result from the disruption of genes by the T-DNA but most probably from somaclonal mutations induced by the in vitro regeneration process or by unsuccessful T-DNA integration events resulting in small (undetectable) insertions or deletions in the genome of the regenerated plants. In addition, a promoter trap approach was also not very successful for gene discovery because aberrant gene fusions were observed in this collection. Therefore, the T-DNA mutagenesis approach in M. truncatula seems to be limited by the lack of an efficient in planta transformation protocol that would allow the generation of a large mutant collection suitable for reverse genetic studies. Thus, there is a need for the development of alternative tools for efficient gene disruption in legumes.

Imaging photosynthesis in wounded leaves of Arabidopsis thaliana

Chlorophyll fluorescence imaging provides a non-invasive and non-destructive means with which to measure photosynthesis. This technique has been used, in combination with 14CO2 feeding, to study the spatial and temporal changes in source-sink relationships which occur in mechanically wounded leaves of Arabidopsis thaliana. Twenty-four hours after wounding, cells proximal to the wound margin showed a rapid induction of PhiII upon illumination (a measure of the efficiency of photosystem II photochemistry) whilst cells more distal to the wound margin exhibited a much slower induction of PhiII and a large, transient increase in NPQ (a measure of the rate constant for non-photochemical energy dissipation within the light-harvesting antenna). These results are indicative of an increase in sink strength in the vicinity of the wound and this was confirmed by the retention of 14C photosynthate in this region. It has been hypothesized that wound-induced cell wall (apoplastic) invertase (cwINV) activity plays a central role in generating localized increases in sink strength in stressed plant tissue and that hexose sugars generated by the sucrolytic activity of cwINV may act as a signal regulating gene expression. Enzyme activity measurements, quantitative RT-PCR, and T-DNA insertional mutagenesis have been used to determine that expression of AtcwINV1 is responsible for all induced cwINV activity in mechanically wounded leaves. Whilst inactivation of this gene abolished wound-induced cwINV activity, it did not affect localized alterations in source-sink relationships of wounded leaves or wound-regulated gene expression. The signals that may regulate source-sink relationships and signalling in wounded leaves are discussed.

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

With the availability of complete genome sequences of several organisms, the focus has shifted from structural genomics to functional genomics, specifically in plants where the complete genomic sequences are becoming available i.e., Arabidoposis and rice. Agrobacterium mediated transformation which is exploited for transgenic technology is also being used as an effective mutagen and as a tool for functional genomics in higher plants. Besides the fact that the insertion of T-DNA element into a gene can lead to loss or gain of function, ingenious use of a variety of vectors have led to the identification of genes and regulatory elements in Arabidopsis. In this review, we highlight the progress made in the field of functional genomics of Arabidopsis using T-DNA tagging. Since this strategy has been very successfully employed in Arabidopsis and is now being extended to other plant species, we discuss the various vectors and experimental approaches employed to tag, identify and clone genes and promoter elements in Arabidopsis using T-DNA as a tool. *Corresponding author

Functional analysis of GUS expression patterns and T-DNA integration characteristics in rice enhancer trap lines

Enhancer trapping has been regarded as a powerful tool for functional analysis of novel genes and regulatory elements. In order to evaluate its efficiency in rice genome, we carried out comprehensive analysis on rice T-DNA insertional mutagenesis lines harboring GAL4 /VP16-UAS- GUS enhancer trap system. T 1 seeds of 9120 independent enhancer trap lines were collected for GUS expression pattern analysis on embryo, endosperm and seed capsule. About 40% lines showed positive staining results in one part of the seeds at least, and 212 samples were randomly selected from these lines for further GUS assay on leaves and roots of T 1 seedlings. About 70% seed-positive lines showed seedling-positive GUS staining results. One hundred and twenty-seven T-DNA flanking sequences containing rice homologous sequences were also rescued from 212 selected lines using PCR walking method. Insert locations and integration patterns of the T-DNA insertions were analyzed in detail. Putative genes and promoters were predicted from rice genomic sequences flanking the T-DNA insertion sites, and expression patterns of several candidate promoters were analyzed and compared with those of the original enhancer trap lines. The results confirmed the effectiveness of GAL4 /VP16-UAS- GUS enhancer trap system in rice. Our work provided useful resources for functional analysis of rice enhancer trap lines generated by T-DNA insertion, and indicated that enhancer trapping was an effective strategy for rice functional genomics research.

Genetic analysis and identification of alarge leaf angles (lla) mutant in rice

Mutants are essential genetic materials to elucidation of biological functions of genes involved. Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era. T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species, in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA insertion. About 40% of 620 cloned Arabidopsis genes with mutant phenotypes were determined by T-DNA tagging. In recent years, a large number of T-DNA insertional rice mutant pools were established. Moreover, three genes were successfully isolated from rice. Leaf angles, the angles between main culms and leaves, are one of the important features of the plant types. Investigation on the mechanism of leaf angle formation will probably provide the basic knowledge of plant breeding on plant architectures. Several QTLs of leaf and flag leaf angles in rice were identified, which would provide the useful materials for manipulating these QTLs in a marker-assisted selection program. However, the major genes, which control leaf angles, have not been reported yet and very little is known about the mechanism of leaf angle formation. Based on T-DNA inserted rice (Oryza sativa L. subsp. Japonica cv. Zhonghua11) mutant pool, which contains about 10000 insertional mutant lines, a large leaf angles (lla) mutant T429 was found in T1 lines. At seeding stage, the leaf angles of lla mutant were larger than those of the wild type. In addition, the mutant seedlings showed semi-dwarf. At heading stage, the abnormal phenotypes of lla mutants were observed in plant height, leaf angel, leaf blade length and leaf blade width. The plant of lla was shorter than the wild type (Fig. 1). The length and width of the lla mutant leaves were shorter and wider than those of the wild type (unpublished data). Under natural long day conditions, the heading date of lla was about two weeks later than that of the wild type. Under winter natural short day conditions in greenhouse, the heading date of lla was about one week later than that of the wild type while the plant height and leaf angles remained significantly different from the control. This indicated that the mutant phenotype was slightly influenced by environmental conditions such as photoperiod and temperature. Different concentrations of GA3 were used to treat the plants at both seeding and heading stages; the results showed that the plant heights of wild type and the lla mutant were all sensitive to exogenous GA3 at different concentrations. But plant heights and leaf angles of GA3 treated mutant plants differed significantly from the corresponding wild types. At the same time, leaf angles of GA3 treated wild type and lla did not show any visible differences from the corresponding untreated plants.

The plant exon finder: a tool for precise detection of exons using a T-DNA-based tagging approach

We have developed a new tool, named the plant exon finder (PEF), for identifying exons in plant genome sequences as an applied technique of T-DNA insertional mutagenesis. The T-DNA constructs contain a heat-shock gene promoter or the cauliflower mosaic virus (CaMV) 35S promoter, followed by the first exon of an Arabidopsis gene with its start codon and the intron donor sequence facing the T-DNA left border (LB) in order to trap exons in the genome. The constructs were used to make transgenic Arabidopsis plants. We generated 280 transgenic lines and identified 156 T-DNA-tagged readthrough transcripts by reverse transcriptase-polymerase chain reaction (RT-PCR) using an oligo(dT)-linker primer and a T-DNA-specific primer. Sequence analysis of the RT-PCR products revealed that 18 of them carried cDNAs processed by the use of an intron acceptor sequence adjacent to T-DNA insertion sites and 11 of them were in-frame fusions. In one case, the readthrough transcript trapped an exon located 1.6 kb downstream of the site of the insertion.

High throughput T-DNA insertion mutagenesis in rice: a first step towards in silico reverse genetics.

A library of 29,482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12,707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value < 1.00 e(-05)). To illustrate the in silico reverse genetic approach, identification of 83 T-DNA insertions within genes coding for transcription factors (TF) is presented. Based both on the estimated number of members of several large TF gene families (e.g. Myb, WRKY, HD-ZIP, Zinc-finger) and on the frequency of insertions in chromosome 1 predicted genes, we could extrapolate that 7-10% of the rice gene complement is already tagged by T-DNA insertion in the 6116 independent transformant population. This large resource is of high significance while assisting studies unravelling gene function in rice and cereals, notably through in silico reverse genetics.