48. Tagged Transcriptome Display (TTD): using Ac elements to identify transcribed sequences in indica rice
  A. KOHLI 1, J. XIONG 1, R. GRECO 2, P. CHRISTOU 1 and A. PEREIRA 2

1) Molecular Biotechnology Unit, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK
2) Plant Genomics Unit, Plant Research International, PO Box 16, 6700 AA, Wageningen, The Netherlands

Transposon mutagenesis in rice has recently emerged as a model system for the study of plant functional genomics (Enoki et al. 1999, Chin et al. 1999, Greco et al. 2000). Sorting genes from the background of non-expressed genomic DNA remains one of the greatest challenges in this area, and a number of techniques based on the high-throughput detection and mapping of expressed DNA sequences have been used, such as exon trapping, cDNA selection, reverse blotting with complex cDNA probes and the random cloning and mapping of expressed sequence tags. We have devised an approach called Tagged Transcriptome Display (TTD), in which a population of rice plants carrying the maize autonomous transposable element Ac is screened using a combination of transposon insertion display and macroarray-based functional genomics to selectively detect transposon insertions in transcribed sequences (Yephremov and Saedler 2000). Uniquely, we have applied this strategy to an indica rice population, whereas previous reports have exclusively studied japonica cultivars.

We generated a population of indica rice plants carrying the Ac transposon by introducing the transposon into rice embryos by particle bombardment. The transposon was flanked by the maize ubiquitin-1 promoter and the gene for green fluorescent protein (GFP). In this way, active excision of the element could be monitored by assaying for the onset of GFP expression (Fig. 1; Greco et al. 2000). We found that the behavior of Ac elements in rice differed to that in maize. For example, we found that transposition occurred very early in development, so that about 40% of the callus derived from bombarded explants expressed GFP universally. We also found that the element could reinsert a great distance from its original location. In maize, the Ac element usually inserts only a few centiMorgans away from the original site, whereas we frequently observed genetic segregation of Ac and the gfp marker in rice indicating long-distance transposition.

Three to four weeks after transformation, we assayed the callus for GFP activity. Initially, we divided the callus into three groups: a) callus with no GFP activity; b) callus with mosaic GFP activity; and c) callus with GFP activity in all cells. PCR analysis of these tissues was then carried out using primers spanning the junction between the promoter and the Ac element, primers spanning the junction between the Ac element and the gfp gene, and primers delineating just the Ac element itself (Fig. 1). If there has been no Ac excision, the junction-spanning primers generate a left and right full donor site (FDS) product. However, if the Ac element has excised, the external primers generate a single empty donor site (EDS) product (Fig. 1).

Of the callus with no GFP activity, 90% generated FDS and Ac products, but no EDS product, showing there had been no Ac excision. The remaining 10% failed to generate any PCR products at all, indicating the cassette had been disrupted upon integration. Most of the callus with mosaic GFP expression generated both FDS and EDS products, indicating mosaic Ac excision or perhaps the presence of multiple Ac elements, only a proportion of which had excised. Other callus in this category generated only the EDS product, indicating universal non-mosaic excision of the Ac element. The callus with universal GFP activity generated only Ac and EDS products. These results were confirmed by Southern blot hybridization to genomic DNA.

To identify transcribed sequences flanking transposon insertion sites, genomic DNA

was digested with SalI, which does not cut within the Ac element. Each Ac element would thus be present embedded within a larger genomic DNA fragment defined by SalI sites. The products were self-ligated and amplified by PCR using inverse primers annealing to the Ac element. Using a three-tier nested PCR strategy, we selectively amplified only those genomic fragments directly linked to an Ac insertion. The tertiary PCR gel, containing the third-round nested PCR products was blotted and probed with total rice cDNA. This identified 41 out of approximately 200 products corresponding to rice genes. All these PCR products were subcloned in the TOPO-TA vector and then sequenced. The sequences were compared to those in the NCBI database revealing 41 hits on previously isolated rice and Arabidopsis genes and ESTs. The fact that approximately one quarter of the genomic sequences flanking the transposon insertion sites turned out to be transcribed sequences indicates that transposition events may not be entirely random and that there is preferential transposition into transcribed sequences.

The rice genome sequencing project is expected to uncover up to 30,000 genes, half of which will have no known function. Transposon mutagenesis using knock-out and gene detection insertions will be a very important tool to discover these gene functions. However, even with a small genome such as that of Arabidopsis, knocking out every gene would entail 100,000 random insertion events. For rice, approximately four times that number would be required. Multiple independent inserts per plant, averaging four in many of the Ac lines, will bring down the required number of plants. The insertional preference of Ac for genes as described here, will reduce the required number further by a factor of 3-5. A population of about 20-30,000 Ac plants would therefore be sufficient to recover knock-out mutants in most genes. These populations can be produced and then made available to research programs worldwide involved in rice functional genomics.

References

Enoki, H., T. Izawa, M. Kawahara, M. Komatsu, S. Koh, J. Kyozuka and K. Shimamoto, 1999. Ac as a tool for the functional genomics of rice. Plant J. 19: 605-613.

Chin, H.G., M.S. Choe, S.H. Lee, S.H. Park, S.H. Park, J.C. Koo, N.Y. Kim, J.J. Lee, B.G. Oh, G.H. Yi, S.C. Kim, H.C. Choi, M.J. Cho and C.D. Han, 1999. Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system. Plant J. 19: 615-623.

Greco, R., P.B.F. Ouwerkerk, A.J.C. Taal, C. Favalli, T. Beguiristain, P. Puigdomenech, L. Colombo, J.H.C. Hoge and A. Pereira, 2000. Early and multiple Ac transpositions in rice generated by an adjacent strong enhancer. Plant Mol. Biol. (in press).

Yephremov, A. and H. Saedler, 2000. Display and isolation of transposon-flanking sequences starting from genomic DNA or RNA. Plant J. 21: 495-505.