Critical Reviews in Plant Sciences, 21(3):143165 (2002)

T-DNA Tagging in a Genomics Era

Richard Walden
Department of Plant Breeding and Biotechnology, Horticulture Research International, East Malling, West Malling, Kent, ME19 6BJ England U.K.
Referee: Dr. Tatsuo Kakimoto, Department of Biology, Graduate School of Science, Osaka University, Machiikaneyama 1-1, Toyonaka, Osaka 560-0043 Japan

Address for correspondence: Tel: 0044 (0) 1732 843833, Fax: 0044 (0) 1732 521557, E-mail: rick.walden@hri.ac.uk

ABSTRACT: From a relatively simple start, T-DNA tagging has emerged to become an important tool in plant physiology and molecular biology. Initially, it was primarily used as a means of gene isolation. A T-DNA insertion might result in an obvious, changed phenotype in the host plant and the sequence of the tag provided a landmark allowing its isolation along with the mutated gene. However, more recently, with plant genome sequences becoming available along with transgenic plant populations where collectively T-DNA insertions may saturate the genome, T-DNA insertion is being used increasingly to determine gene function. A variety of T-DNA gene tags are available. Many integrate within transcriptional units so that promoters, enhancers, exons, and introns can be tagged. In such cases if the tag contains marker genes endogenous patterns of expression of tagged sequences can be monitored. Tags containing transcriptional enhancers can be used to create gain of function mutations by the activation of gene expression. In the foreseeable future we can anticipate that with the model plant Arabidopsis libraries will become available where accessions will represent the knockout and/or activation of every gene coding region as defined by sequencing. Increasingly, T-DNA tagging is also being used to characterize genes in other plant species. Thus, it may be anticipated that this method of gene tagging will play an important role in investigating gene function in plant developmental processes that are not easily accessible with Arabidopsis. KEY WORDS: Agrobacterium, plant transformation, T-DNA tagging, Arabidopsis.

I. INTRODUCTION The touchstone of functional genomics is linking gene function to gene sequence. This is best achieved by modifying gene sequences in situ followed by observing the effect of the modification on the host organism. The foundations of this concept were laid using plants as model systems. Mutant generation and analysis has played an important role in both breeding and plant biology in general (Allard, 1960; Vose and Blixt; 1984, Hayward et al., 1993). Often, however, it has been difficult to link an observable phenotype arising from a mutation with the physical basis of the genetic modification. One method of resolving this problem capitalized on the generation of mutable alleles resulting from the activity of plant transposons (McClintock, 1950; Wessler, 1988). Molecular characterization of
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transposons provided an opportunity to link the physical basis of the mutation with the observed phenotype (Burr and Burr, 1982; MullerNeumann et al., 1984., Dring and Starlinger, 1984). Once the sequence of a transposon was known ways and means could be developed for the isolation not only of the transposon, but also the affected gene (Fedoroff et al., 1994; Sutton, et al., 1984). Gene tagging in plants had come of age. Initially, transposon tagging was limited to plants such as maize and snapdragon where the endogenous transposons involved were relatively well characterized (Balcells et al., 1991). Subsequently, the observation that transposons were active in heterologous hosts extended their use in other plants, most notably Arabidopsis (Baker et al., 1986; Long et al., 1993; Aarts et al., 1993). At about the same time it was realized that the T-DNA of Agrobacterium, generally used as a

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vector in genetic transformation, could also be considered as a insertional mutagen (Koncz et al., 1989) and with its sequence characterized T-DNA could also be used as a tag (Koncz et al., 1990). The relative merits of transposons and T-DNA as gene tags have been described extensively elsewhere (Balcells et al., 1991; Feldmann, 1991; Walden et al., 1991; Koncz et al., 1992; Walbot, 1992; Dilkes and Feldmann, 1998; Pereira and Aarts, 1998; Martienssen, 1998). Together transposons and T-DNA provide the researcher with a means to carry out a wide range of experiments. Many of the concepts of tagging developed with one system have been applied with success in the other. Initially, gene tagging relied on the insertion of a well-defined DNA into the genome, an ability to detect a modified phenotype resulting from the insertion, a means of demonstrating genetic linkage between the insertion, and an observed phenotype as well as a way of isolating the tagged genes themselves. The approach of initiating gene isolation by changed phenotype has come to be known as forward genetics. More recently, progress in genome sequencing, coupled with access to large populations of transformed plants, has allowed the isolation of mutations in specific genes, a process known as reverse genetics. Gene tagging, whether with transposons or T-DNA, has been largely limited to model plant systems such as maize, snapdragon, and Arabidopsis. However, increasingly gene tagging, in the context of T-DNA tagging, can be applied to other plants to address specific questions of cell biochemistry and development. With the completion of sequencing the first plant genome, it seems appropriate to take stock of the methods available to turn this vast source of new genetic information into functional relevance. With this in mind, this review focuses on T-DNA tagging and how it can be applied to studying gene function. In the spirit of the title of this journal I aim to cast a critical view of current techniques, results and available resources and provide suggestions of where this form of tagging might contribute further to our understanding of plant gene function.

A. Agrobacterium-Based Genetic Transformation the Practicalities The molecular basis of transfer of T-DNA from the Ti plasmid of Agrobacterium to the genome of the host plant cell has been studied in detail and reviewed regularly and extensively (Gelvin, 2000; Zhu et al., 2000; Zupan et al., 2000; Chilton, 2001). For the purpose of this review, there are several salient points to consider using T-DNA as a gene tag: T-DNA is flanked by border sequences of imperfect 25-bp repeats (Zambryski et al., 1982; Yadav et al., 1982). The left and right border sequences define the sites of single-strand nuclease digestion initiating release of the single stranded T strand from the Ti plasmid. Nicking is carried out by the virD1/D2 products encoded by the virulence (vir) region of the Ti plasmid and is apparently initiated at the right border repeat (Yanofsky et al., 1986; Stachel et al., 1987; Scheiffele et al., 1995). Maximal T-strand formation may require using the same Ti plasmid as a source of vir and border regions. Reduced efficiency of nicking at the left border prior to release of the T-strand may result in the creation of inserts in the plant genome of regions of the Ti plasmid additional to the T-DNA (Konov et al., 1997). During the natural infection process Agrobacterium attaches to the cell wall of a wounded plant. Attachment is apparently a twostep process involving binding via a cell-associated acetylated polysaccharide (Reuhs et al., 1997) followed by the formation of cellulose fibrils anchoring bacteria at the wound surface (Matthysse et al., 1995). Optimal transformation occurs at about 19 to 22C, the temperature at which Agrobacterium form the pili associated with transfer of the T-DNA to the plant cell (Fullner et al., 1996; Dillen et al., 1997). Wounding and/or cell division appears to be a prerequisite for optimal rates of transformation (Sangwan et al., 1991; Villemont et al., 1997), supporting the notion that open chromatin associated with DNA replication or transcription provides both access and the enzymatic mechanisms to assist T-DNA insertion. However, it

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has been reported that Agrobacterium can infect cells in the absence of wounding (Escudero and Hohn, 1997), as presumably they do in the case of in planta transformation (see later). The T-strand is transferred from the infecting bacteria to the host by a process analogous to bacterial conjugation (Lessl and Lanka, 1994; Zupan et al., 2000). The DNA is coated by a single strand DNA binding protein, virE2 (Christie et al., 1988; Citovsky et al., 1988) and has virD2 covalently bound to the 5' end (Ward and Barnes, 1988; Durrenberger et al., 1989). Both may attach to the T-strand within the bacteria, although virE2 can be independently transported into the infected plant cell, where binding can also occur (Sundberg et al., 1996; Gelvin, 1988; Lee et al., 1999; Chen et al., 2000; Vergunst et al., 2000). VirE2 and D2 have nuclear targeting properties. Thus, they are thought to both protect the single-stranded DNA from nuclease action and target the DNA/protein complex to the plant nucleus (Durrenberger et al., 1989; Citovsky et al., 1992; Howard et al., 1992; Shurvinton et al., 1992; Tinland et al., 1992; Ziemienowitcz et al., 2001). T-DNA transfer to the host cell can take place within 2 to 6 h of infection (Virts and Gelvin, 1985). Expression of T-DNA can be observed from 18 h after infection, although this is transient declining after 36 h (Narashimhulu et al., 1996) and probably represents expression of double-stranded DNA prior to integration. Integration of the T-DNA into the genome is thought to be one of the limiting steps in Agrobacterium-based transformation (Nam et al., 1997) although T-DNA transfer itself is also an important factor (De Buck et al., 2000). The mechanism of integration of the T-strand into the plant nuclear genome is not completely understood, although it is thought to involve a process of illegitimate recombination (Gheyssen et al., 1991; Mayerhofer et al., 1991; Tinland, 1996; De Buck et al., 1999). T-DNA may integrate via strand invasion of single-stranded DNA followed by second strand DNA synthesis or the T-strand may be converted to double stranded DNA prior to integration (De Neve et al., 1997). Being covalently attached to the 5' end of the T-strand, virD2 is probably in-

volved in the integration process and indeed virD2 has ligase activity in vitro (Pansegrau et al., 1993). It is notable that integration of the 5' end of the T-DNA (the right border) into the plant genome is usually precise with few nucleotides being lost on integration (Tinland et al., 1995). In contrast, integration of the 3' (left border) sequence is not so precise with loss of up to several hundred bases of T-DNA being possible (Azpiroz-Leehan and Feldmann, 1997). This may result from loss of virE2 from the T-strand during transfer in the plant cell exposing the T-strand to nuclease digestion (Rossi et al., 1986). Based on the comparison of the activation of expression of promoterless marker genes contained on T-DNAs transferred to different host plants (see later) integration of the T-DNA apparently preferentially occurs into transcribed regions of the genome (Koncz et al., 1989; Topping and Lindsey, 1995). Thus, the types of genes that are transcriptionally active at the time of transformation may mirror those that are tagged (Topping and Lindsey 1995, Martirani et al., 1999). When individual genes are independently tagged inserts are likely to occur throughout the gene (Winkler et al., 1998; Gottwald et al., 2000). Preliminary analysis of T-DNA inserts in the Arabidopsis genome suggests that these are randomly sited (AzpirozLeehan and Feldmann, 1997), although a definitive result possibly requires positioning of more inserts on the Arabidopsis genome. Direct or inverted repeats of T-DNA at insert sites in the plant genome may be obtained. This may reflect end to end ligation of right T-DNA borders, or illegitimate recombination at left border sequences prior to integration (De Buck et al., 1999). Orientation of T-DNA insert can vary depending on the method of transformation. For example, when transforming root explants of Arabidopsis the majority of insertions were single copy, whereas multicopy inserts were obtained using leaf discs (Grevelding et al., 1993). It is hard to distinguish whether this results from differences in the target tissue or the tissue culture conditions used. However, the notion of tissue specificity in susceptibility to transformation has support from the observation

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that Arabidopsis ecotypes and mutants recalcitrant to transformation via root explants can be transformed by vacuum infiltration (Mysore et al., 2000a). T-DNA can insert into the genome without the target plant DNA being modified extensively (Gheysen et al., 1991; Matasumoto et al., 1990; Meyerhofer et al., 1991). However, varying degrees of modification of host sequences at the insertion site have also been reported. They may be small involving base pair additions or deletions (Gheysen et al., 1987; Gheysen et al., 1991; Mayerhofer et al., 1991; Ohba et al., 1995; Negruk, et al., 1996; Noguchi, et al., 1999). On the other hand, they may be large and include chromosomal translocations (Castle et al., 1993; Nacry et al., 1998; Laufs et al., 1999). Modifications of host DNA sequences on T-DNA insertion can complicate analysis of flanking plant sequences, for example, in reverse genetic screening (see later, Tax and Vernon, 2001). In addition, host genomic DNA might be modified as a result of abortive insertion of the T-DNA. This could result in mutations occurring that are not physically linked to T-DNA insertion. This can occur up to 60% of the time in some transgenic populations (Dilkes and Feldmann, 1998). Once integrated into the plant genome single T-DNA insertions are transmitted as single Mendelian characters. Expression of genes carried by the T-DNA can be stable for long periods, for example, in perennial crops (James et al., 1996), but inactivation of expression of T-DNA genes as a result of methylation has been observed (Finnegan et al., 1998; Fagard and Vaucheret, 2000).

II. T-DNA TAGGING VECTORS Generally, T-DNA tagging vectors have been derived from vectors developed for plant transformation. Indeed, it is perhaps ironic that one of the most extensively applied T-DNA tags used in the pioneering work of Feldmann and co-workers (Feldmann, 1991; Dilkes and Feldmann, 1998) in Arabidopsis is based on one of the earliest plant

gene vectors pGV3850 (Velten and Schell 1985; Zambryski et al., 1983). These general transformation vectors have been described in detail by a number of authors (Walden et al., 1990; White, 1993; McCormac et al., 1999; Mozo and Hooykaas, 1992; Hellens et al., 2000) and will not be described in detail here. However, there are some features of these vectors that are relevant to the process of gene tagging. Two types of T-DNA-based vector have been developed, co-integrative and binary vectors. Cointegrative vectors are based on Ti plasmids in which the T-DNA has been modified for purposes of insertion of foreign DNA. Being based on Ti plasmids these vectors are relatively stable in Agrobacterium and do not require the presence of antibiotic selection for maintenance. This may have been a contributing factor the initial success of using them in in planta transformation (see later) although this technique does not require cointegrative vectors per se (Bechtold and Pelletier, 1998; Clough and Bent, 1998). Binary vectors are plasmids able to replicate in both E. coli and Agrobacterium. They are engineered to contain the left and right T-DNA border sequences flanking DNA to be transferred to the plant genome. They are used in an Agrobacterium that contains a Ti plasmid retaining a functional vir region but from which the T-DNA has been deleted. Although binary vectors are usually based on plasmids containing a broad host range origin of replication, they can be unstable in Agrobacterium and require antibiotic selection for their maintenance. The design of many T-DNA tagging vectors capitalizes on the precise integration of DNA flanked by T-DNA borders into the plant genome with integration at the right border sequence being relatively precise. This allows the design of tagging vectors to achieve a variety of functions. Two general types of tagging vector based on T-DNA have been developed (Figure 1). Those where the aim is to achieve gene knockout and thus recessive mutations and activation tags where overexpression of genes is sought to produce a dominant mutation. The nature of the mutation (recessive or dominant) has a profound effect on the tagging experiment and the procedures used to carry out selection of tagged individuals. In the former a modified phenotype will only be ob-

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FIGURE 1. Differing types of T-DNA tagging. A target gene is shown (exons as hatched boxes) with its promoter (open box) directing production of a transcript (grey arrow) (A). Enhancer trapping involves insertion of a T-DNA containing a marker gene (stippled box) linked to a minimal promoter (filled box). The enhancer triggers expression from the minimal promoter to produce a transcript (black arrow) allowing screening for marker gene activity (B). Promoter trapping uses a T-DNA containing a marker gene (stippled box) either lacking a promoter or linked to a minimal promoter that is unable to direct gene expression by itself. Expression of the marker gene carried by the T-DNA can occur if the T-DNA inserts upstream of the coding region when lacking a promoter or downstream or within the coding sequence of the target gene when containing a minimal promoter (C). Intron trapping uses a T-DNA with a marker gene linked to splice junction sites so that the marker gene forms a translational fusion with the target gene following splicing (D). Gene trapping occurs when the T-DNA with marker gene (stippled box) inserts and forms a translational fusion with an exon (E). Activation tagging involves the use of T-DNA containing multiple transcriptional enhancers to activate expression of a tagged gene. An activation tag can insert upstream or downstream of a target gene and still activate gene expression (F).

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served in a proportion of the offspring from primary transformants, whereas in the latter an observable phenotype may be observed in primary tansformants.

A. Promoter Tagging Earliest attempts of using T-DNA as a tag involved linking a promoterless marker gene to the border of the T-DNA and selecting for transgenics expressing the marker with the aim of tagging promoter sequences. For example, a vector containing a promoterless NPTII gene 30 base pairs from the right border sequence used in protoplast transformation resulted in kanamycin-resistant calli arising at an order less than using a vector containing the NPTII gene linked to a constitutive promoter (Andre et al., 1986). When linked to the left border sequence at least 35% of the transformants express the marker gene, although when located within the T-DNA this frequency drops to 1% as the activation of the gene only occurs after truncation of the T-DNA (Herman et al., 1990). Similar experiments resulted in the tagging of promoters producing a variety of expression patterns (Teeri et al., 1986). This work demonstrated the feasibility of the approach and more recently this type of tagging has been used to generate a number of gene fusions in Arabidopsis (Babichuk et al., 1997). Here the vector contained an NPTII gene flanked by the Arabidopsis arp gene with the aim of detecting gene targeting to the host arp gene by homologous recombination. Gene targeting was not observed, but 50% of the kanamycin-resistant individuals recovered resulted from the T-DNA inserting in a manner allowing processing of chimeric introns with the remainder resulting from exon fusion events. However, a concern with this type tagging is that only gene fusions expressed at the time of selection may be recovered and that T-DNA insertions can occur at high copy number, possibly as a result of the selection pressure applied (Koncz et al., 1989, 1992). Subsequent promoter tagging work has focused on preliminary selection for transformants being based on constitutive expression of a marker gene followed by screening for expression of a

promoterless marker gene to identify events where a promoter has been tagged. For example, the selection for the presence of a hygromycin resistance gene followed by screening for activity of a promoterless NPTII gene (Koncz et al., 1989). With such an approach up to 30% of transformants expressed reporter gene fusions. In addition, it was shown that the frequency of promoter tagging was remarkably similar in tobacco and Arabidopsis. Assuming one, two, or more T-DNA inserts occurred in a 1:1:1 ratio and considering the difference in genome size, transcript complexity, and the relative amounts of repeat sequence in the two plant genomes, this result led to the notion that T-DNA preferentially inserts into potentially transcribed regions of the genome (Koncz et al., 1989). This has also been observed by other workers (Topping and Lindsey, 1995). Subsequently, other promoterless marker genes have been adopted that are easier to screen for activity of tagged promoters such as -glucuronidase (GUS) and luciferase (LUX) (Koncz et al., 1989; Goldsborough, et al., 1991; Fobert et al., 1991; Topping et al., 1991; Lindsey et al., 1991). These have been particularly useful in tagging promoter sequences because of the ease of viewing enzyme activity by histochemical analysis. Detailed analysis of tobacco, Arabidopsis, and potato transformants containing a promoterless GUS gene confirmed that the frequency of achieving active fusions were similar in each plant (Topping et al., 1991; Topping and Lindsey, 1995). Intriguingly, however, there appeared to be a bias of developmental expression patterns seen between differing species. For example, root-specific expression occurred in 73% of the transgenic tobacco lines, 30% of Arabidopsis, and 9% of potato. This may be linked with the chromatin confirmation at the time of transformation (i.e., allowing T-DNA integration), which in turn might reflect on the tissue and the culture conditions used in the transformation. Vectors can be constructed so that marker gene expression depends on the formation of transcriptional or translational fusions. A comparison of the frequency of these events in Arabidopsis revealed that 54% of the transgenics contained a transcriptional fusion, whereas only 1.6% of these with a potential for translational fusions expressed

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GUS activity (Kertbundit et al., 1991). Several factors are likely to account for this difference such as the relative precision of insertion with regard to the target gene required to produce a fused open reading frame as well as the ability of the GUS protein to tolerate fusions and retain its enzymatic activity. An additional factor, however, is the potential occurrence of cyptic promoters in the plant genome. Tagging using a promoterless GUS gene has revealed sequences in both tobacco and Arabidopsis able to act in transcriptional fusions as promoters functioning in either a tissue-specific (Fobert et al., 1994; Plesch et al., 2000) or constitutive manner (Foster et al., 1999). Such promoters are not linked to obvious open readings frames, are AT rich, and apparently not transcribed in nontransformed tissues. Although it is hard to exclude that in these cases enhancer sequences are promoting expression over long distances, the work suggests that intergenic regions of the genome may contain elements able to transcribe novel coding regions that might in turn have an impact on the evolution of the plant genome. The strength in the use of histochemical markers in promoter tagging is that it allows screening for the activity of potentially tagged promoters in a heterozygotic population of plants. Hence, screening can be carried out for the activation of promoters as a result of differing environmental stimuli, such heat shock (Mandal et al., 1993) or at specific developmental phases (Campisi et al., 1999). One approach has been to screen populations of promoterless GUS tagged Arabidopsis lines following infection with nematodes for those displaying GUS activity at the site of infection (Goddijn et al., 1993). This type of study has revealed a number of promoters activated at the site of infection, including one linked to a gene that may be involved in the pentose phosphate pathway (Bartels et al., 1997; Favery et al., 1998). It is not hard to imagine that a variety of genes, other than marker genes, could be used in potential fusion experiments, such as those resulting in cell death or changing patterns of cell growth (Koncz et al., 1992). The difficulty of this type of experiment, however, remains in tagging promoters that are strictly tissue specific in their expression and quantifying the effects of differing levels

of expression in differing cell types. An example of this latter problem is demonstrated by using a cytokinin synthesising ipt gene as a marker in promoter tagging experiments (Hewelt et al., 1994). A variety of novel developmentally regulated phenotypic changes typical of enhanced cytokinin production were obtained, but information concerning the tagged promoters and their patterns of expression was relatively limited. On the other hand, this type of experiment in some plants might produce individuals with novel characteristics that may be of potential economic value, for example, with ornamentals in horticulture.

B. Activation Tagging The creation and the analysis of the effects of gene knockouts is a very effective method in assigning gene function. However, there are certain types of genes that by their very nature may not yield functional information after knockout. For example, those with products carrying out important functions in the life cycle of the plant so that when the gene is disrupted there is a lethal effect, or genes that are functionally redundant. Gain of function mutations may overcome these difficulties as well as shed new light on the involvement of target genes in biochemical or developmental processes. The physical basis of gain of function mutations can vary. They can result from the coding region of the gene being altered so that the activity of the resultant protein becomes deregulated, or that the expression of the gene becomes up-regulated and/or occurs in a novel tissue. An example of the former case is provided by the constitutive activity of an ethylene response mutant (Chang et al., 1993). However, these types of mutations are relatively rare. The potential of the latter type of mutation is demonstrated by experiments where the transcription factors R and C1 from maize were linked to the CaMV 35S RNA promoter and found to induce anthocyanin synthesis in transgenic Arabidopsis and tobacco (Lloyd et al., 1992). Gain of function mutations can be obtained by activation tagging. In the earliest experiments a T-DNA tag was designed to contain four transcriptional enhancer sequences derived from the

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CaMV 35S RNA promoter (Odell et al., 1985) close to the right border sequence (Walden et al., 1991; Walden et al., 1994; Kakimoto, 1994). The underlying notion was based on the observation that transcriptional enhancers had been found to be active in up-regulating gene expression at long distances in transgenic tobacco (Kay et al., 1987) and functioned in an orientation-independent manner with respect to the target gene (Fang et al., 1989). Similar vectors have been constructed by others (Huang et al., 2001), and the original modified to contain a herbicide-resistance gene suited for use in Arabidopsis (Weigel et al., 2000). An analogous vector has been developed containing a GUS marker gene (Van der Fits et al., 2000). Originally, activation T-DNA tagging was carried out using tobacco protoplast cocultivation with the aim of isolating mutant cell lines changed in their hormonal requirments for callus formation. However, much of this work is disputed (Galbraith et al., 1983; Carle et al., 1998; Schell et al., 1999). Another application of activation tagging involved selection for shoot formation from callus formed on Arabidopsis hypocotyls in vitro (Kakimoto, 1996). Under normal conditions cytokinins are required in the culture media to allow callus formed from Arabidopsis hypocotyls to green, rapidly proliferate and undergo shoot formation. In an experimental tour de force Kakimoto carried out transformation with an activation T-DNA tag on calli formed from 50,000 hyocotyl segments followed by transfer to solid media lacking cytokinin. Five calli were found that turned green, proliferated rapidly, and formed shoots. In four of the mutants the tag had inserted near the same gene CKI1. This is a dramatic demonstration of the power of the technique. Sequence analysis of CKI1 showed that it encoded a protein with similarity to histidine kinase and receiver domains of two component receptors. However, whether CKI1 is indeed a cytokinin receptor is yet to be demonstrated directly. A variation of activation tagging is to make the activation of expression conditional on an external stimulus. An example of this is provided by linking a heat shock promoter to the left border sequence to direct expression following heat shock at 37C (Matsuhara et al., 2000).

Large populations of Arabidopsis containing activation T-DNA tags have been created and are becoming available to the research community (Weigel et al., 2000, www.biotech.wisc.edu/ Arabidopsis). To date, this population has yielded a variety of novel mutants and tagged genes, including a floral inducer (Kardailsky et al., 1999), a flavin monoxygenase-like gene implicated in auxin biosynthesis (Zhao et al., 2001), and MYB factor that regulates phenyl propenoid biosynthesis (Borevitz et al., 2001). Initial results from these experiments allow some preliminary conclusions to be drawn concerning activation tagging in general (Weigel et al., 2000). First, genes tagged giving a novel phenotype appear to be relatively close to the enhancer sequences (380 bp to 3.6 kb). Second, the enhancers might be inserted upstream or downstream of the transcriptional start site of the tagged gene. Third, tagged genes might not be constitutively overexpressed. Fourth, mutations are apparently dominant or semidominant, although the degree of phenotypic change that results might not be as pronounced as with the tagged gene cloned directly behind the CaMV 35S RNA promoter. Finally, in some cases the novel phenotype arising from tagging may be unstable over time. It is possible that this results from the methylation of the T-DNA insert and/or flanking DNA (Finnegan et al., 1998; Fagard and Vaucheret, 2000). Indeed, this may well explain the instability of phenotype observed in the original experiments using activation tagging with tobacco protoplasts.

III. MAKING T-DNA TAGGED POPULATIONS There are a number of practical prerequisites to the successful application of T-DNA tagging. As we have seen, bacterial/plant cell contact, optimal temperature, and culture conditions and a suitable target cell type as well as a plant genotype susceptible to transformation are important criteria in achieving maximal rates of transformation. However, a means of creating a large number of transformed individual cells, or plants, from which tagged mutants can be isolated is essential. A variety of transformation methods are available

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(Hansen and Wright, 1999) and are adaptable to the type of tagging experiment envisaged. Nevertheless, the limiting factor resides in the amount of labor, and/or resource required to create large enough populations of plants or cells for mutant screening.

A. Explant Inoculation This method is the simplest and most versatile means of creating transgenic plants. Generally, aseptic tissue explants are inoculated with Agrobacterium containing the tagging vector. Following an incubation period allowing transformation of wounded cells on the periphery of the explant, the explants are washed to remove excess bacteria and placed on media to induce callus formation. The media also contains antibiotics to stop further bacterial growth and selection for growth of transgenic cells. Callus is then excised and transferred to new media to induce shoot formation. Shoots are subsequently excised and placed on new media to induce root formation. The selection for growth of transgenic tissue can be applied at the stage of callus and/or shoot formation, but this technique is extremely labor intensive when applied to creating gene tagged populations. For example, from 110 transgenic lines arising from Arabidopsis leaf disc inoculation, 7 mutants were found, one of which cosegregated with the T-DNA (Van Lijsebettens et al., 1991). Although labour intensive, for many plants explant inoculation, in one form or another, may be the only convenient means of creating transgenic populations. For example, rice scutellum-derived embryonic calli have been co-cultivated with Agrobacterium to produce tagged plant populations (Jeon et al., 2000). In this instance 20 to 40% of the calli tested were resistant to the marker contained in the T-DNA and of these frequencies of plant regeneration varied from 50 to 85%. In total, 22,090 transgenic lines were recovered. In a sample of 34 studied in detail 35% contained single T-DNA inserts. Explant inoculation has been very successful where direct selection can be applied for a desired phenotype, for example, in activation tagging. We

have already seen how this approach has been applied to callus formed on the hypocotyls of Arabidopsis (Kakimoto, 1996). Another innovative approach involved callus formed on leaf discs of the resurrection plant Craterostigma plantagineum (Furini et al., 1997). This desiccation tolerant plant has the interesting feature that calli derived from it are only desiccation tolerant when treated with ABA prior to desiccation. With this in mind, activation tagging was carried out using 25,000 calli derived from leaf tissue. Selection was based on screening for tolerance to hygromycin (the resistance gene carried by the T-DNA) followed by desiccation in the absence of ABA treatment. One callus surviving under these conditions was recovered and plants regenerated. Regenerated plants had no obvious phenotypic changes compared with untransformed plants, although callus reisolated from them resembled ABA treated callus from untransformed plants on desiccation, including the expression of ABA responsive genes. The tagged gene CDT-1 when linked to a constitutive promoter also confers this phenotype when retransferred to Craterostigma. However, intriguingly the CDT-1 sequence contains no long open reading frame. Currently, the mode of action of CDT-1 is unclear, although it might act as regulatory RNA or a small peptide. Variations of explant inoculation have been applied to studying the determinants of nitrogen fixing nodule formation in legumes. Two T-DNA tagged mutants arrested in the infection process were identified in progeny from 1112 primary transformants following the transformation of Lotus japonicus (Schauser et al., 1998). Here transformation was carried out using hypocotyl explants and the transgenics were screened for the ability to form effective nodules with Mesorhizobium loti on nitrogen-free media. Another approach has used transformation with the hairy root inducing Agrobacterium rhizogenes (Martirani et al., 1999). In this case wounded roots of L. japonicus were infected and transgenic roots, each thought to represent an independent transformation event, form at the wound site. Up to 10 hairy roots can be obtained per infection site, allowing the production of large numbers of transformants. In transformants, T-DNA copy numbers correspond to 37% single, 25% double, and 37% more than two inserts. Using this transformation system with a promoterless GUS

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gene, GUS positive transgenic lines were generated with differing expression patterns. However, it has been argued that the A. rhizogenes approach of producing promoter fusions might be less efficient when compared with A. tumefaciens because of multiple T-DNA insertions having the potential to produce gene inactivation (Karimi et al., 1999).

B. Protoplast Co-Cultivation Using Agrobacterium to transform protoplast populations by co-cultivation (Marton et al., 1979; Depicker et al., 1987) allows the creation of extremely high numbers of transformed cells from which putative tagged mutants can be recovered. Protoplasts are released from plant tissue or suspension cells by enzymatic digestion of the cell wall. They are co-cultivated with Agrobacterium containing the tagging vector for periods up to 2 days, washed to remove excess bacteria, and the cells then cultured further to divide and form callus. If required, shoot and root formation can be induced and transgenic plants recovered. The feasibility of producing large numbers of transformed Arabidopsis lines has been demonstrated by the transformation of cell suspensions (Mathur et al., 1998). In this scheme up to 50% of microcalli were judged as transformed based on screening for GUS activity and the ability to grow on selective levels of hygromycin. This, coupled with a regeneration frequency of greater than 90% for microcalli, provides an opportunity to recover large numbers of transgenic individuals. While effective in producing large numbers of transformed cells, this technique remains a labor-intensive method of creating large populations of tagged plants. An imaginative use of activation tagging with isolated cell suspensions has been used to isolate a transcriptional regulator of plant primary and secondary metabolism (Van der Fits and Memelink, 2000). Suspension cells of Catharanthus roseus were transformed with an activation tagging vector and cells selected by their abilitiy to grow in the presence of 4-methyltryptophan (4-mT). 4-mT can be dedoxyfied by trypophan decarboxylase (tdc) so that cells recovered are likely to be upregulated by tdc activity. Screening 20 cell lines with increased

tdc expression revealed six with increased levels of a second gene involved in the production of terpenoid indole alkaloid secondary metabolites, suggesting that the gene tagged might be involved in regulating genes encoding this biochemical pathway. Plasmid rescue recovered a gene encoding a transcription factor approximately 600 base pairs from the T-DNA. Reintroduction of the tagged gene into Catheranthus linked to the 35S RNA promoter resulted in the activation of expression of several primary and secondary metabolite biosynthetic genes and the accumulation of terpenoid indole alkaloids. This is an excellent example of how T-DNA tagging can be used to isolate components of a complex biochemical pathway from a non-model plant. The isolation of genes involved in terpenoid biosynthesis has obvious pharmaceutical application, and this work demonstrates how knowledge of a biochemical pathway and an appropriate inhibitor can be used with molecular genetics to isolate genes with products of biochemical importance.

C. In planta transformation The most widely adopted means of creating T-DNA tagged populations of plants has been in planta transformation of Arabidopsis, and this has been reviewed recently (Bent, 2000). The technique developed initially from the observation of Feldmann and Marks that transgenic Arabidopsis plants could be created by the simple expedient of imbibing seeds in a solution of Agrobacterium (Feldman and Marks, 1987). The treated seedlings were grown to maturity, selfed, and up to 0.32% were found to contain T-DNA inserts, as judged by resistance to antibiotic encoded by the T-DNA. At a stroke this provided a non-tissue culture approach to produce transgenic Arabidopsis. The technique also lent itself to scaling up so that it could be used to generate populations of T-DNA mutagenised plants. The significance of this was not lost on Feldmann and co-workers and rounds of transformation experiments were used to create populations of plants for screening for obvious phenotypic changes and now seeds from these populations are available to the research community (Feldmann et al., 1989;

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Feldmann, 1991; Azpiroz-Leehan and Feldmann, 1997; Dilkes and Feldmann, 1998). The obvious advantages of this type of approach spurred a number of workers to modify the technique to increase transformation efficiency and create protocols generally applicable for a variety of experiments. A further development of the technique involved severing apical shoots from Arabidopsis plants, applying Agrobacterium and generating shoots from the severed site. With this approach, on average 5.5% of newly formed shoots produced transgenic progeny of which 33% were single insert (Chang et al., 1994; Katavic et al., 1994). One of the most widely applied variants of in planta transformation has been the approach of vacuum infiltration (Bechtold et al., 1993; Bechtold and Pelletier, 1998). The idea was based on the observation that transformants arising from in planta transformation were hemizygous for T-DNA inserts. This led to the notion that transformation occurred late in the plants life cycle either at the end of gametogenesis or at the zygote stage. The technique is based on submerging young plants undergoing floral initiation in a fresh suspension of Agrobacterium and subjecting them to a vacuum for up to 20 min. Following treatment the plants are dried, replanted, and allowed to set seed. Bulked populations of seed are screened for the presence of T-DNA inserts based on antibiotic resistance. With this approach 10 to 15 transformants have been reported recovered from each treated plant. Greater than 50% of the transformants have a T-DNA insert at a single locus with 70% of these being tandem inserts (Bechtold et al., 1993; Bechtold and Pelletier, 1998). Vacuum infiltration is particularly suited to gene tagging. It has the potential to be scaled up to allow the production of large populations of transformed individuals with relative ease. Moreover, adoption of a herbicide-resistance gene in the tag itself allows direct selection for transformants by spraying seedling progenies. Indeed, the method has been simplified further in the approach of the floral dip (Clough and Bent, 1998). Here floral tissues, in particular immature floral buds, are simply dipped in a solution of Agrobacterium, sucrose, and a surfacant. Repeated

dipping over a period of 5 to 6 days results in transformation frequencies of up to 3% being obtained. This technique has obvious advantages of speed and ease compared with vacuum infiltration and indeed can be simplified further by simply spraying solutions of Agrobacterium to flowering plants (Chung et al., 2000). A series of experiments involving outcrossing of treated plants as the pollen donor or recipient and using GUS expression to investigate sites of DNA uptake suggest that the developing ovule is the site of transformation during in planta transformation (Ye et al., 1999; Desfeux et al., 2000). These observations have been confirmed by genetic linkage analysis that indicates that the majority of the transformation events are linked with maternally derived chromosomes (Bechtold et al., 2000). Obviously, such a facile method of producing transgenic plants begs the question of whether the in planta method of transformation is generally applicable to other plants. Recently, the successful transformation by vacuum infiltration of M. truncatula has been reported (Trieu et al., 2000). In contrast to Arabidopsis, transformants homozygous for the transgene were observed, suggesting that the transformation event occurred at an earlier stage of development. In addition, pakchoi has been transformed by vacuum infiltration (Qing et al., 2000). Hence, the approach may be generally applicable to differing plant species. However, there is a potential limitation to a widescale adoption of this technique: low frequencies of transformation and the potential to detect transformants. Where transformation frequencies are low, there will be a need to recover a large number of seeds for screening. In the case of pakchoi for example, two positives were detected out of approximately 200,000 seeds. On the other hand, this may not be a general problem. In the case of M. truncatula, 33 seeds were collected and a remarkable 76% were transgenic.

IV. RECOVERY AND FUNCTIONAL ANALYSIS OF TAGGED PLANT SEQUENCES The ultimate goal of gene tagging is recovering the plant gene tagged by T-DNA. Thus, a

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ways and means is required to rescue plant DNA flanking the insertion from the genome, establishing the physical basis of the mutation and confirming which candidate gene may produce a specific phenotype. Until recently, this was a process of forward genetics; genetically linking an observable phenotype with a T-DNA insertion and devising a means isolating the plant DNA linked to the tag. However, with a knowledge of the target gene sequence reverse genetics is feasible using PCR-based techniques to identify individuals containing T-DNA in defined sequences. In forward genetics it is essential to establish that the T-DNA is genetically linked to an observed phenotype. This is carried out on the basis of segregation analysis described in detail elsewhere (Walbot, 1992; Dilkes and Feldmann, 1998). With T-DNA tags containing dominant selectable markers this is a relatively straightforward process of assessing the co-segregation of the marker and an observable phenotype in progeny on selective media. A central element of this is the 100% co-segregation of T-DNA marker and phenotype as a result of a homozygote back crossed to wild type. In the case of activation tagging unexpected dominant mutations unlinked to the T-DNA are likely to be rare hence in largescale programs of activation tagging sequencing of flanking sequences can be initiated while genetic analysis is underway with relative confidence that the tagged gene is nearby the T-DNA insert. Once linkage of a tag to a phenotype is established, the tag becomes the landmark by which it and flanking plant sequences can be identified. A variety of ways have been adopted to achieve this.

nomic library made from the mutant and used as probes to isolate the wild-type sequence. Nested deletions of the clones of wild-type DNA were reintroduced into the gli mutant and tested for complementation, in this case of the glabrous phenotype. It was found that GL1 is a myb-like DNA binding protein and that the T-DNA had inserted 730 bp downstream of the myb coding region.

B. Plasmid Rescue Plasmid rescue relies on the T-DNA tag containing a plasmid origin of replication and a resistance gene functional in E.coli. Genomic DNA is isolated from the mutant and digested to completion with a restriction enzyme. DNA fragments are ligated in a large volume to avoid cloning inserts of noncolinear plant DNA, and transformed into E.coli. Bacteria are plated onto selective antibiotics and plasmid DNA analyzed for the presence of T-DNA and flanking plant sequences. Enzymes cutting outside of the T-DNA can be used to recover T-DNA and plant DNA either side of the insert or cutting with an enzyme cleaving within the DNA can be used to recover plant DNA flanking either the left of right border separately (Figure 2). This is particularly useful when the T-DNA has inserted as a multimer. This approach has been used extensively to recover tagged plant genes, for example, from the Feldmann mutant collections (Dilkes and Feldmann, 1998). It is particularly suitable when the T-DNA insert is single copy and nonrearranged.

C. PCR-Based Approaches A. Library Screening Recovery of T-DNA and flanking plant sequences from complete or partial genomic libraries is an option when the T-DNA insert is a complex one hindering isolation by other means. For example, for the isolation of GL1 from Arabidopsis, physical mapping revealed four tandem T-DNAs integrated at the same locus (Herman and Marks 1989; Marks and Feldman, 1989). DNA fragments flanking the T-DNA were isolated from a geLong range inverse PCR (LR-iPCR) involves cleavage of the transgenic plant DNA with a restriction enzyme cutting within the T-DNA. Following ligation of the fragments, PCR is carried out using nested primers derived from the TDNA to amplify flanking plant DNA, which can then be sequenced (Figure 2). This approach has been used to characterize genes flanking T-DNA inserts in plants regenerated from populations of transformed Arabidopsis suspension cells (Mathur et al., 1998).

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FIGURE 2. Recovery of T-DNA tagged plant genomic sequences. A single T-DNA insert is shown flanked by left and right borders (L.B. and R.B.) inserted into the plant genome (stippled boxes). The T-DNA contains a bacterial origin of replication (ori) and an ampicillin resistance gene (Amp) functioning in bacteria. Plasmid rescue can be achieved by cutting genomic DNA outside of the T-DNA with a restriction enzyme, religating and transforming E. coli. Plasmids containing the ori and Amp-resistance gene contained within the T-DNA will be recovered (A). Plasmids recovered by the above approach but lacking the ori and/or Amp resistance gene can, following ligation, be subjected to PCR using primers based on the T-DNA sequences to amplify flanking plant sequences (B). Interlaced PCR can be used to recover flanking plant sequences by using a random primer and primers based on T-DNA sequences (C). Gene walking relies on cleaving the genomic DNA outside of the T-DNA insert, adding linkers (open box) and carrying out asymmetric PCR using primers based on the linker sequence and the T-DNA (D). Location of primers used in PCR are indicated throughout by small arrows. PCR products indicated by lines.

Thermal asymmetric interlaced PCR (TAILPCR) uses three nested specific primers based on the T-DNA sequence and a shorter arbitrary degenerate primer (Lin et al., 1995; Figure 2). Variation in the temperatures of the PCR amplifications carried out on genomic DNA is used to manipulate the relative amplification of specific vs. nonspecific priming events so that ultimately the T-DNA and flanking plant DNA is predominantly amplified. This is a highly effective means of recovering flanking DNA with a better than 95% success rate reported. However, it does require a pre-screen for single T-DNA inserts. Genewalker (Clontech) was developed to enable walking upstream or downstream in a ge-

nomic DNA from a defined sequence, identified, for example, from a cDNA. Genomic DNA from the mutant is isolated and cleaved with a restriction enzyme to produce blunt ends (Siebert et al., 1995). Adapters containing an amine group blocking 3' extension of the 3' end of the ligated fragments are ligated to the fragments of genomic DNA. The adapter sequences allow binding of two nested primers for PCR, an outer and an inner primer. Nested primers are also developed from the T-DNA sequence. A first round of PCR is used to amplify from the outer primers of the adapter and the T-DNA. The reaction mix is diluted and used in a secondary round of PCR primed with the nested inner primers of the adapter

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and the T-DNA. From this reaction a PCR product can be isolated and sequenced to identify the T-DNA sequences and flanking plant genomic sequences PCR-based approaches for screening site of insertions are of sufficiently high throughput that it allows the screening of T-DNA transformed individuals with such relative ease that it can form the basis of reverse genetic screens.

V. REVERSE GENETIC SCREENING Reverse genetic screening depends on two factors: large populations of independently transformed individuals and the sequence of potential target genes for insertion. In Arabidopsis, reverse genetics using T-DNA is feasible because of the ability to generate large populations of transgenic individuals via in planta transformation and advances in sequencing of both genomic and cDNAs. Indeed, with the completion of the Arabidopsis genomic sequencing program, it should be feasible to isolate mutations in any desired gene in this plant. Key to reverse genetic screening is the isolation and pooling of DNAs from populations of tens of thousands of transformed plants in a way that allows subsequent identification of an individual carrying a T-DNA insert in a predetermined gene via PCR. Based on concepts developed for screening transposon insertions in other experimental systems such as Drosophila and maize, procedures have been developed for screening isolated genomic DNAs as pooled samples (McKinney et al., 1995; Krysan et al., 1996; Azpiroz-Leehan and Feldmann 1997; Winkler and Feldmann, 1998; Winkler et al., 1998; Krysan et al., 1999; Young et al., 2001). The strategies developed for reverse genetic screening in Arabidopsis very much depend on limiting the amount of labor required to build the populations of transgenic plants and to carry out the PCR reactions effectively. Protocols adopted have varied depending on the initial number of transgenic plants available for screening as well as the conditions of the PCR reactions adopted (McKinney et al., 1995; Krysan, et al., 1996; Young et al., 2001). The process is initiated by

the in planta transformation of Arabidopsis either by seedling inoculation or vacuum infiltration (Figure 3). Resulting plants, the T1 generation, are allowed to set seed and seeds of the T2 generation are collected. T2 seedlings are grown in the presence of an antibiotic to select for transgenics. These are then transferred to pots or trays and grown to maturity and T3 seed collected as a bulked pool. Where relatively small numbers of transformants are involved, seeds from each pool can be germinated and used for bulk preparations of DNA. The DNA can be aliquoted in arrays of rows and columns. The rows and columns in turn can be pooled to produce 20 pools (rows and columns) that can be then be screened by PCR and positive clones traced. T3 seeds can also be planted out to screen for any obvious phenotypic changes (Azpiroz-Leehan, 1997; Winkler and Feldmann, 1998). This type of approach has been effective with relatively small populations of plants (< 10,000) recovering TDNA insertions in two actin genes (McKinney et al., 1995) and 12 P450 genes (Winkler et al., 1998). Interestingly, in the case of insertion in the P450 genes the inserts were located throughout the genes tagged. Four occurred in 5' regions, four in exons, one in an intron, and one in the 3' untranslated region and the three remaining were found at differing positions 3' to the transcribed regions (Winkler et al., 1998). This points to the random nature of the inserts in the transcribed region of the genes. A similar scheme was used to search for T-DNA insertions in 63 gene family members whose products are thought to be involved in signal transduction and ion transport. seventeen inserts were identified, a success close to what might have been expected with the size of population of plants screened (Krysan et al., 1996). Similarly, screening a population of 14,200 differing T-DNA lines (notionally containing 20,000 inserts) and insert in the AKT1 potassium channel gene has been isolated (Hirsch et al., 2001). To increase the possibility of isolating inserts in predetermined genes, a population of 60,480 Arabidopsis plants transformed with knockout tags has been assembled for screening and a resource for the research community (www.biotech.wisc.edu/ Arabidopsis, Krysan et al., 1999; Sussman et al., 2000). Here the pooling strategy relies on bulking

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FIGURE 3. Pooling strategies of reverse genetics. (For details see text.)

the seeds of 9 T3 individuals (pools of nine) and aliquoting equal amounts of 25 of these pools to form pools of 225. Seeds from these pools are germinated and used to prepare DNA. The DNA of nine pools of 225 is bulked to form super pools, which are then subjected to PCR screening. PCR products and positive Southern hybridisation are used to identify samples harboring T-DNA inserts in a specific gene. This population has been used to isolate three mutant alleles of a phloem-specific plasma membrane sucrose transporter (Gottwald et al., 2000). The potential of this population has been demonstrated recently by the isolation of 10 of the 12 known isoforms of AHA gene family, multiple mutant alleles for 7 of the 12 AHA gene family members (Young et al., 2001). More recently, Sygenta Research and Technology have offered access to a T-DNA insert population of approximately 100,000 individual Arabidopsis plants. Flanking plant sequences have been obtained by TAIL PCR and sequences of at least 70,000 insertions lying within genes or their promoters are available for screening (www.nadii.com). Access to a publically funded program of T-DNA insert analy-

sis has also become available recently (http:// signal.salk.edu/tdna_FAQs.html).

A. Linking Gene Action to Gene to Phenotype In forward genetics the definitive means of linking a tagged gene sequence to putative gene function depends on the complementation of the mutant phenotype. This relies on the transformation of the mutant with wild-type genomic DNA or a cDNA linked to a constitutive promoter. In reverse genetics where it is feasible to have an allelic series of inserts in a particular gene with a linked phenotype, this may suffice.

CONCLUSIONS Initial attempts at using T-DNA as a tag in plants could well have been considered as inefficient, labor intensive and fortuitious means of plant mutant creation and gene isolation. This

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was particularly true when comparing T-DNA tagging with the advantages of transposon tagging (Walbot, 1992). It was, in part, due to the difficulties at the time in creating populations of T-DNA transformed plants of a suitable size to allow subsequent screening for obvious phenotypic changes based on a strategy of gene knockout. However, in the meantime tagging strategies and transformation protocols coupled with genomic sequencing have advanced so much that we can begin to consider saturation mutagenesis of plant genomes. On the one hand, in planta transformaton has developed to a point with Arabidopsis where transgenic populations of tens of thousands are available for screening (Bent, 2000; Sussman et al., 2001). On the other hand, plant protoplast transformation, for example, with Catharanthus, can produce populations of transformant cells of hundreds of thousands that can be screened in vitro following activation tagging (Van der Fits and Memelink, 2000). Thus, in the foreseeable future it is not difficult to imagine that in Arabidopsis, at least, collections of seed will be available where individual accessions contain a T-DNA insert, whether a knockout or an activation tag within, or near each Arabidopsis gene. Indeed, coupled with transposon tagging this is one of the goals of the ambitious 2010 Project (Somerville and Dangl, 2000, http:// signal.salk.edu/smission.html). This is all heady stuff, but what are the limitations of T-DNA tagging and are there other directions that tagging might take? Possibly the greatest current limitation with T-DNA tagging is our inability to target the insertion to a preordained site. Despite much effort and scattered reports of success no reliable, general purpose protocol has emerged to allow efficient gene targeting into the nuclear genome of a higher plant. This is probably a result of homologous recombination within the plant nucleus occurring at frequencies below the detection level of current technology (Paszkowski, 1994; Miao and Lam, 1995; Babichuk et al., 1997; Liljegren and Yanofsky, 1998). Overcoming this limitation has forced efforts recovering T-DNA inserts in specific genes by reverse genetics. Is it currently feasible to obtain a T-DNA knockout in a specific gene in Arabidopsis? Ma-

jor advances in this direction by Sussman and coworkers has led to the establishment of an Arabidopsis T-DNA knockout facility at the University of Wisconsin (Krysan et al., 1999; Sussman et al., 2001). At the time of this writing 60,480 transformed lines have reported as being pooled and available for screening in pools of nine parental lines. The possibility of an insert being found in a specific gene depends on its size, the size of the plant genome, and the distribution of T-DNA inserts in a population of plants. Assuming random site insertion, it has been calculated that when considering a 5 kb target gene 110,000 lines need to be screened to achieve a 99% probability of detecting an insert with the number rising to 550,000 when the gene is 1 kb (Krysan et al., 1999). Given that the average size of Arabidopsis gene is about 2 kb (The Arabidopsis Genome Consortium, 2000) a population of approximately 280,000 is required for a 99% chance of obtaining an insert decreasing to approximately 180,000 for a 95% probability. Hence, success in obtaining a knockout in a gene of Arabidopsis at present remains a matter of luck. However, with increases in transformed population sizes the probability of success is likely to steadily improve. In the meantime the power of this approach has been demonstrated by the report of the isolation of individuals with T-DNA inserts in 10 of the 12 members of the H+ ATPase gene family (ranging from 4.5 to 6.5 kb) being identified from the population (Young et al., 2001). With sufficiently large populations of T-DNA transformed plants, will it be feasible to recover T-DNA insertions in every Arabidopsis gene? Current evidence suggests that T-DNA can insert throughout the genome and in differing regions of particular gene family members. However, it is perhaps too early to judge whether insertions are truly randomly distributed throughout the genome. The majority of T-DNA insert sites sequenced to date arise from mutants displaying an obvious phenotype. This will preferentially reveal inserts in transcribed regions. Although not based on phenotype, reverse genetics will also predominantly recover inserts in transcribed regions as a result of PCR primer selection. The issue will probably only be resolved by the systematic sequencing of a large number of T-DNA flanking

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regions. Random insertions of T-DNAs containing promoterless marker genes suggest preferential insertion into potentially transcribed regions of the genome (Koncz et al., 1989; Lindsey and Topping, 1995). This in turn raises the question of whether all transcribed regions are potential targets of insertion per se or just those that are being transcribed at the time of transformation. It has been suggested that the latter is the case with subsets of genes being potential targets depending on the tissue and the conditions used in the transformation (Martirani et al., 1999). If confirmed, this would suggest that in attempting to saturate a genome with T-DNA inserts it would be of value to use differing transformation strategies to construct transgenic populations. While saturation of the Arabidopsis genome with T-DNA is still some distance in the future, interest has increased in applying T-DNA as a mutagen in other model plants to study processes that might not be experimentally accessible in Arabidopsis. One example of this is nitrogen fixation, and with this in mind reports have appeared describing the scaling up of T-DNA insertion experiments in Medicago (Frugoli and Harris, 2000). Such projects, along with experiments to carry out T-DNA tagging in rice (Jeon et al., 2000) are labor intensive and will require a great amount of effort to achieve full genome coverage unless efficiencies of transformation and screening are not increased. To be truly effective, such experiments require transformation protocols allowing scale up and high throughput as well as efficient transformation. As we have seen in planta transformation, in one form or another, it appears to be generally applicable in differing plants and might offer a partial solution to this problem. However, the question remains as to whether transformation events can be detected in plants where seed production is not as prolific as in the Brassicas. Increasing transformation frequency may help. While this is limited by several factors, such as vir gene induction, or binding of Agrobacterium to the host plant cell, a major limiting factor appears to be integration into the plant genome (Gelvin, 2000). This also appears to account for differences in transformation frequencies of differing plant species, cultivars, or ecotypes. It is likely that T-DNA integration is dependent on host enzymes involved in

DNA replication or transcription, and this notion has been supported recently by the finding that the histone H2A gene in Arabidopsis plays an important role, probably as a result of mediating chromatin decondensation (Mysore et al., 2000b). Interestingly, overexpression of the gene either in stable transformants, or in a transient fashion, increases transformation frequency. It will be interesting to learn whether expression of such genes might serve to enhance transformation frequencies in model species currently recalcitrant to transformation by Agrobacterium thus increasing the range of application of T-DNA tagging. Some of the limitations of T-DNA tagging based on gene knockout are overcome in activation tagging by virtue of the fact that the mutants are dominant and thus operational at the level of primary transformant plant or cell. Recently, the technique has come into the spotlight with the appearance of activation tagged libraries of Arabidopsis (Weigel et al., 2000). However, its potential in gene isolation from more complex genomes has been demonstrated with the isolation of transcriptional factors controlling defined biochemical pathways in Catharanthus (Van der Fits and Mermelink, 2000). There may be concerns that when compared with gene knockout gene function can only be indirectly inferred as a result of product overexpression. Nevertheless, it is the authors opinion that a strength of the approach lies in dissecting signaling or biochemical pathways. There have been two recent demonstrations of this. First, the finding that overexpression of a flavin monooxygenase-like enzyme produces auxin-like effects and that the enzyme might be the rate limiting step of tryptophan-dependent auxin biosynthesis (Zhao et al., 2001). Second, the identification of a myb regulator of phenylpropanoid biosynthesis by activation tagging (Borevitz et al., 2000). The later work, along with that described with Catharanthus (Van der Fits and Mermelink, 2000), is significant in that it demonstrates the potential of up-regulating the biosynthesis of commercially relevant natural products. Moreover, via reverse genetics and using chip technology in transcript profiling activation tagging may provide a means of assigning a function to each of the myriad of myb genes and other transcriptional regulators present in plants (Meissner et al., 1999; Riechmann et al., 2000). However, throughout the secret of success with activation tag-

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ging is in devising adequate selection, or screening, systems. T-DNA tagging has made great advances from initial small-scale experiments through the development of in planta transformation to the development of large-scale screening strategies based on reverse genetics. Indeed, with the finding that Agrobacterium can transform filamentous fungi (Bundock et al., 1998), its impact may spread beyond plants where T-DNA tagging may provide an alternative to mutational analysis based on REMI (Nunn-Coleman and Wang 1998). The goal of tagging every Arabidopsis gene to assign function is an exciting and an appropriate goal of the post-genomic era. However, a real challenge will be to develop the means and the strategies based on traditional Agrobacterium infection to apply T-DNA tagging to the dissection of aspects of plant development that are currently unapproachable in Arabidopsis.

ACKNOWLEDGMENTS Thanks to Andrea Massiah, Brian Thomas, and Ian Puddephat for helpful comments on the manuscript and Penny Greeve for the artwork. R.W. is funded by the BBSRC and the East Malling Trust for Horticultural Research. Thanks to Jeff Dangl for the gravestone caption.

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