Oryza sativa

What is Tos 17?

Ty-1 copia retrotransposons belong to Long Terminal Repeats retroelements which transpose through a copy and paste mechanism via a RNA intermediate and form the major component of intergenic regions in higher plants (Bennetzen, 2000). Tos17 is a Ty1-copia retrotransposon of rice (Oryza sativa L.) belonging to a 32 member-family, 5 members of which have been found active (Tos10, 17, 19, 25 and 27). Tos17 is the most active among these members, exists in low copy number (1-10) in rice varieties and its transposition is regulated at the transcriptional level during normal culture conditions. During tissue culture, activation is observed and results in an increase of the number of Tos17 copies which is a function of the tissue culture duration (Hirochika et al 1996, Hirochika 2001). Upon regeneration activation stops and the resulting new inserts are stably inherited in the progeny of regenerated plants.

Due to these favourable features, Tos17 has been used for the insertion mutagenesis of the model cereal plant (Hirochika 2001). Nipponbare ,which has been selected as the standard cultivar by the international rice genome sequencing project (IRGSP) carries only two copies per haploid genome located on chromosomes 7 and 10 and an average of 10 transposed copies of Tos17 scattered over the genome are typically observed in plants regenerated from 5 month-old callus cultures. A collection of 51,625 regenerated rice lines carrying 510,000 insertions has been produced at the National Institute for Agrobiological Sciences (NIAS) in Japan (Miyao et al 2003). 20,000 of these insertions have already been characterized by sequencing the genomic region flanking new Tos17 integration sites, allowing to readily search for an insertion in a favourite gene for investigating its function.

How far is the international effort of insertion mutagenesis in rice?

Insertion line libraries are one of the most needed resources for discovering and validating gene function in forward and reverse genetics strategies. The Ac/Ds and En/Spm maize transposon systems, the Tos17 endogenous retrotransposon and the T-DNA are the main rice insertion mutagens. Classical point or deletion mutagenesis populations are also developed in association with TILLING for searching lesions in specific genes. A large international effort of coordination of national mutagenesis initiatives (Korea, Australia, Japan, China, Taiwan, Singapore, USA , The Philippines, France) has been launched through an International Rice Functional Genomics Consortium and an international project of screening for drought tolerance-associated traits of these mutant libraries has been commissioned by the Generation Challenge Program. The well characterized Genoplante insertion line library, which is made accessible to the scientific community as seeds become available, is recognized as a major contributor to this international framework. Like in Arabidopsis it is however very likely that most of the exchanges will be done on the basis of FST search. This is reinforced by the quarantine and biosafety regulations restricting international transfer of rice seeds.

Unlike the Arabidopsis community which now benefits from a coverage of the whole genome with ca 300K public FSTs, the rice community which deals with a 4-fold larger genome, (even though all 3 mutagens have insertional preference in gene-rich regions) is still in the need of more characterized insertion sites (estimated to a current 100,000 FST from 200,000 lines). To facilitate the reverse genetics search, Cirad has developed a GGB based rice genome navigator integrating all the available public FST information (Droc et al 2006).

What is the goal of our project?

In the frame of a multi institutional effort (Cirad, Cnrs, Ird, Inra) supported by Genoplante a library of 30,000 T-DNA insertion lines has been generated through Agrobacterium co-culture of embryogenic calli in the standard cultivar Nipponbare, -the genome of which has been used for the international project of sequencing (IRGSP 2005)-(Sallaud et al 2004). These lines are being field evaluated in a collaborative effort with the international centre CIAT (Centro Internacional de Agricultura Tropical based in Cali, Colombia) for seed amplification and detection of phenotypes while other mutant screens (e.g. pathogens and grain filling) and validation of gene functions are underway. A collection of 20,000 Flanking Sequence Tags has been generated from the left borders of T-DNA inserts in the library.

As the rice transformation procedure includes short duration tissue culture, the 30,000 lines of the Genoplante library contain an estimated total of 90,000 novel Tos17 insertions (3.2 on average per line). A novel method of high-throughput selective amplification of flanking regions of newly transposed Tos17– called TOSTRAP – has been developed. Using the TOSTRAP protocol we amplified flanking sequences from at least one newly-transposed copy of Tos17 in 20,000 lines. We typically generated PCR fragments of size ranging from 300 bp to 2000 bp (average size 700 bp) that contain a TAG sequence that enable the use of one primer to sequence all the generated PCR products. The TOSTRAP protocol enables to amplify multiple PCR products from the same DNA sample in 25% of the analysed rice lines. This implies that 75% of our products are single PCR products and 25% multiple PCR products (in most cases 2 PCR products).

The project aims at sequencing 20,000 Tos17 flanking regions for readily providing the scientific community with additional tags for reverse genetics and improving the insert coverage in the mutant library.

The Genoplante insertion library is used first by both the French and international communities dealing with rice and cereal functional genomics. In France a well structured rice community of ca. 30 scientists (INRA, IRD, CIRAD, CNRS) is concentrated in the Languedoc Roussillon region and has established functional genomics programs on disease response (UMR BGPI 385), grain development and filling and embryogenesis (UMR GDP 5096), plant development in adaptation to stress (UMR PIA 1096). Aside this, the wheat (INRA) and maize (BIOGEMMA) public and private research communities part of the Génoplante consortium are using a comparative genomics approach with pivotal rice genome sequence and associated resources. A growing interest also exists in the Arabidopsis scientific community for validating the function of their favourite genes in rice. The library is also a central resource for international projects such as those of the Generation Challenge Program (CEREALIMMUNITY, and STRESS MUTANT) as well as for projects funded by the European commission (CEDROME). The publicly released Tos17 FST generated will provide helpful additional tags for all these projects and communities.

Contacts: Corinne Cruaud (Genoscope) - Emmanuel Guiderdoni (CIRAD)

Bibliography

Bennetzen JL (2000) Plant Mol Biol 42:251-269

Hirochika H. (2001) Curr Opin Plant Biol , 4:118-122

Hirochika H et al (1996) Proc. Natl. Acad. Sci USA, 93 : 7783- 7788

Miyao A et al (2003) Plant Cell, 15, 1771-1780

Sallaud C et al (2004) Plant J. 39, 450-464