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Arabidopsis Gene Knockout: Phenotypes Wanted Nicolas Bouché* and David Bouchez†

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Arabidopsis Gene Knockout: Phenotypes Wanted Nicolas Bouché* and David Bouchez† 111 Arabidopsis gene knockout: phenotypes wanted Nicolas Bouché* and David Bouchez† Gene knockout is considered to be a major component of the paradigm (i.e. finding biological functions behind genes) functional genomics toolbox, and is aimed at revealing the [2]. The challenge now is to attach functions to an function of genes discovered through large-scale sequencing unprecedented number of genes: out of more than programs. In the past few years, several Arabidopsis 25,000 genes identified in Arabidopsis, the function of only populations mutagenized with insertion elements, such as the a few thousand can be defined with great confidence. T-DNA of Agrobacterium or transposons, have been produced. These large populations are routinely screened for insertions As part of the toolbox for functional genomics, reverse into specific genes, allowing mass-isolation of knockout lines. genetics comprises a set of methods devised to interfere Although many Arabidopsis knockouts have already been with proper gene activity, in particular by abolishing gene obtained, few of them have been reported to present function (i.e. by knockout) [2]. Homologous recom- informative phenotypes that provide a direct clue to gene bination allows gene replacement in vivo, substituting a function. Although functional redundancy explains the lack of wild-type gene for a copy that has been modified in vitro. phenotypical alterations in some cases, it also appears that This approach is widely used in yeast genetics to assess or many mutations are conditional and/or do not alter plant modify gene function and hundreds of knockouts have morphology even in the presence of severe physiological been obtained in mice. Homologous recombination allows defects. Consequently, gene knockout per se is not sufficient construction of lines carrying not only null mutations, but to assess gene function and must be integrated into a more also site-specific mutations such as amino-acid replace- global approach for determining biological functions. ments, insertions, deletions and modification of regulatory elements. As far as plants are concerned, homologous Addresses recombination is only available in the moss Physcomitrella INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 patens [3]. Until now, and despite considerable efforts, targeted Versailles, France disruption by homologous recombination has been mostly *Present address: Centre for Plant Sciences, School of Biology, unsuccessful in higher plants, or at least not feasible in University of Leeds, Leeds LS2 9JT, UK †e-mail: [email protected] routine experiments [4]. Current Opinion in Plant Biology 2001, 4:111–117 For several plant species in which homologous recombi- 1369-5266/01/$ — see front matter nation is not available, gene-disruption methods have © 2001 Elsevier Science Ltd. All rights reserved. been designed by making use of large populations mutag- Abbreviations enized with an insertion element – transposons or the AKT1 Arabidopsis K+ TRANSPORTER1 T-DNA of Agrobacterium [5–11]. These methods rely on CCA1 CIRCADIAN-CLOCK ASSOCIATED 1 the use of a polymerase chain reaction (PCR) to screen ORF open reading frame DNA pools prepared from the population for the desired PCR polymerase chain reaction insertions. PCR primers specific for both the target gene SEP1–3 SEPALLATA1–3 SHP1 SHATTERPROOF1 and the insertion element are used. The large populations SKOR Stellar outward-rectifying K+ channel (Table 1), DNA pools and protocols currently available already allow the isolation of knockout lines for virtually Introduction every Arabidopsis gene [12••]. Surprisingly, although The sequencing of the genome of the higher plant PCR-based reverse genetics screens have been available Arabidopsis thaliana is now completed [1••], and large col- to Arabidopsis researchers for several years, relatively few lections of expressed sequence tags are available for a informative knockouts have been reported in the litera- number of other plants. Such a large amount of plant ture to date. In fact, the vast majority of the several sequencing data is expected to provide new ways of hundred knockouts obtained in Arabidopsis do not give addressing biological questions from an integrated per- rise to visible, directly informative phenotypes, and they spective, ranging from molecular genetics to evolution. By await further analysis. Several limitations are already themselves, however, DNA or protein sequences fail to apparent from the experience gained in the past few provide much biologically relevant information for the vast years. They essentially concern the difficulty in finding majority of genes that have been identified through large- and studying informative phenotypes, and in dealing with scale sequencing programs. Although more efficient the large amount of structural redundancy unravelled by bioinformatic tools are becoming available for the annotation the genomic sequence of Arabidopsis. Indeed, although of genome sequences most, if not all, such information Arabidopsis, Drosophila and Caenorhabditis elegans share a must be regarded as hypothetical in the absence of exper- similar number of gene types (about 11,000 in imental evidence. Thus, the previous paradigm of Arabidopsis), multigene families, present as clustered molecular genetics (i.e. finding genes behind biological and/or dispersed copies, are particularly frequent in this functions) is gradually being reversed into a post-genomic simple plant genome [1••]. 112 Genome studies and molecular genetics Table 1 Populations available for reverse genetics in Arabidopsis thaliana. Type of element Average copy Element mobility* Reporter gene/ Lines available from Reference(s) number per element design the stock centers genome Two component transposon 1–3 Stable (transposase 47,450 ‘SLAT’ lines (50% are [7] removed system based on removed by negative are available as DNA pools)† En/Spm selection) Two component transposon 1 Stable (transposase GUS reporter (gene 931 ‘IMA’ lines† [6] system based on Ac/Ds removed by negative trap and enhancer trap) selection) Two component transposon 20–25 Stable (transposase ‘ITS’ lines available available [5] system based on En/Spm removed by genetic on request from A Pereira† segregation) T-DNA 1–3 Stable 15,400 [11] T-DNA 1–3 Stable GUS reporter 9,800 [43,44] T-DNA No data Stable GUS reporter 17,600 [9] (enhancer/promotor trap) En-1 (En/Spm family) 6 Mobile [10] autonomous transposon T-DNA No data Stable Activation tag 22,672 [8] composed by multi- merized 35S enhancers Populations and DNA pools are available from Arabidopsis stock (URL http://nasc.nott.ac.uk/) and through the recently developed centers: URLs http://nasc.nott.ac.uk/ and http://aims.cps.msu.edu/aims/ Arabidopsis transposon insertion database (URL http://formaggio.cshl.org/ *Different trap systems developed in plants were recently reviewed [15•]. ~h-liu/attdb/index.html). Ac, Activator element; Ds, Dissociation † Systematic sequencing of insertion sites (‘flanking sequence tags’ element; En, Enhancer; GUS, β-glucuronidase; IMA, Institute of [FSTs]) was performed for several of these lines. Sequences are Molecular Agrobiology, Singapore; ITS, Inhibitor-tagged site; SLAT, available at the Nottingham Arabidopsis stock centre Sainsbury Laboratory Arabidopsis thaliana; Spm, Suppressor-mutator. Several recent reviews describe various resources available based on homologous recombination. Modified copies of for gene knockout in Arabidopsis [12••,13,14•,15•]. In this genes under study can, however, be re-introduced into null review, we will give an update on the outcome of reverse lines for fine structure–function analyses. genetics approaches in this species so far, and discuss some limitations and perspectives that have emerged from a few Phenotypic characterisation of knockouts years experience. Since the first disruption of actin genes in Arabidopsis [17], only a few of the several hundred knockouts obtained in Isolation of knockouts in Arabidopsis different laboratories have been reported to exhibit infor- It is expected that about 150,000–200,000 insertions are neces- mative phenotypes (Table 2). Even fewer display sary to obtain a reasonable knockout saturation of the genome. morphological changes that provide a direct clue to gene To date, several hundred thousand T-DNA and transposon function. Out of about 200 knockout lines isolated by our insertion lines have been generated in many laboratories and, group, fewer than 2% display significant morphological at least in part, have gradually become available to the com- alterations. Nevertheless, unexpected phenotypes can be munity. Recently, several groups have initiated programs for found upon careful examination of mutant plants. DAG1 is the systematic sequencing of insertion sites in various popula- known to encode a transcription factor, but the sequence tions (Table 1) [5–7]. In the next few years, it can be expected and mRNA expression pattern of this gene do not give any that the combined size of these populations will allow the clue to its function at the whole-plant level. Analysis of a systematic disruption of all Arabidopsis genes, and that the knockout line, however, showed that DAG1 is involved in search for knockout mutants will be greatly facilitated by the the control of seed dormancy [18••]; dag1 plants have dis- use of knockout databases. In addition, recent reports on the torted siliques, with seeds that germinate earlier than double-stranded RNA-mediated interference (RNAi) approach
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