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Finding novel patterning genes

A number of regulatory genes remain unidentified in the plant genome. However it is not easy to find those genes by conventional mutant screening. This is mainly due to funtional reduandacy between genes and/or pathways. We have developed a novel screening method that allows one to identify novel patterning genes, regardless of the existence of funtionally overlapping genes and/or pathways.

Many of well-known key regulatory genes had been identified in plants before the year 2000, when the first complete genomic sequences of higher plants was determied for Arabidopsis. These genes could be identified via forward genetic screening of corresponding mutants either because the absence of functionally overlapping genes, or fortuitous isolation of dominant alleles that rendered gene functions.

Plant morphology and physiological responses are determined by about 30,000 genes present in the genome. After the completion of the Arabidopsis genome project in 2000, it has been expected that the functions of all Arabidopsis genes might have been determined in ten years. By a reverse genetic approah, it is theoretically possible to disrupt or knock-down every gene of Arabidopsis.

In contrast with the genome projects, which can be performed by technical staffs, functional characterization of genes is driven by the interests (i.e. curiosity) of biologists. For example, developental biologists like to see morphological consequence of gene disruption, and hence are not eager to disrupt hundreds of genes to find one or two of interesting ones.

 

As a means to selectively identify novel developmental regulators, we developed a novel screening system. In this system host plants expressing a synthetic transcription factor GAL4:VP16 (GV) throughout the root tissues, are transformed with a T-DNA containing 5xUAS, the binding sequence for GV. If the 5xUAS is inserted upstream of a patterning gene (which is normally expressed in a limited tissue), its expression is forced to expand throughout the root tissues. This leads to a conspicuous morphological phenotype. By determining insertion position of the 5xUAS in such mutant plants, one can identify responsible genes relatively easily.

This type of screening, called "activation tagging", had already been used for more than a decade in plants. Nevertheless, our system is favorable to isolated developmental regulators, as it restricts the gene activation to take place only in the root, and hence does not lead to lethality or infertility. We could indeed identify important regulators of embryogenesis and root development, which will be introduced in the following pages.
 
Reference:
Waki T. et al., (2013) "A GAL4-based targeted activation tagging system in Arabidopsis thaliana." Plant J. 73, 357-367.PubMedPublisher