An oligo selection procedure for identification of sequence-specific DNA-binding activities associated with the plant defence response

Sequence-specific DNA-binding (SSDB) factors play central roles in transcri ption, DNA replication, recombination and repair. This report describes a s imple procedure for high-throughput identification of SSDB activities witho ut prior knowledge of their target genes or binding sequences. The procedur e starts with a population of completely random oligo(nucleotide) sequences and selects for those oligo molecules that specifically bind to cellular S SDB factors by use of common gel-retardation assays and PCR. Amplification and subsequent cloning of these selected oligo molecules result in the esta blishment of oligo DNA libraries enriched in DNA molecules containing speci fic sequences recognized by SSDB factors. These oligo libraries can be rapi dly screened to identify a large number of SSDB activities, including those that are differentially regulated by developmental and environmental signa ls. With identified oligo DNA as probes, the corresponding SSDB factors can be isolated and analysed with respect to their structures, regulation and functions. Using this procedure, we have identified approximately 100 SSDB activities from tobacco leaves, including seven that are differentially reg ulated during the tobacco mosaic virus-induced hypersensitive response in r esistant tobacco plants.