10 Transposon Insertional Mutants: A Resource for ... - Springer Link

10 Transposon Insertional Mutants: A Resource for Rice Functional Genomics

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Qian-Hao Zhu , Moo Young Eun , Chang-deok Han , Chellian Santhosh 4 5 6 Kumar , Andy Pereira , Srinivasan Ramachandran , Venkatesan Sundare4 1 1 7 san , Andrew L. Eamens , Narayana M. Upadhyaya and Ray Wu 1

CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia; 2Rice Functional Genomics and Molecular Breeding Lab, Cell and Genetics Division, National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, Korea; 3 Division of Applied Life Science, BK21 Program, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Korea; 4Department of Plant Sciences, Life Sciences Addition 1002, University of California–Davis, Davis, CA 95616, USA; 5Virginia Bioinformatics Institute, Washington Street, MC 0477, Virginia Tech, Blacksburg, VA 24061, USA; 6Rice Functional Genomics Group, Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 117604, Singapore; 7Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA Reviewed by Tony Pryor and John M. Watson

10.1 Introduction............................................................................................224 10.2 Transposon Tagging Systems ................................................................225 10.2.1 Activity of Transposons in Rice .....................................................225 10.2.2 One-Element System versus Two-Element System .......................229 10.2.3 Design of Constructs ......................................................................232 10.2.4 Gene and Enhancer Traps...............................................................236 10.2.5 Transiently Expressed Transposase System ...................................238 10.2.6 A High-Throughput System to Index Transposants .......................238 10.2.7 Using Endogenous Transposons.....................................................240 10.2.8 Inducible Transposition ..................................................................243 10.3 Mutagenesis Strategies ..........................................................................245 10.3.1 Random or Non-targeted Mutagenesis ...........................................245 10.3.2 Localized or Targeted Mutagenesis................................................246 10.4 Transposon Insertional Mutant Populations...........................................247 10.4.1 CSIRO Plant Industry Population...................................................248 10.4.2 EU (Wageningen) Population.........................................................249 10.4.3 National University of Singapore Population.................................250

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10.4.4 Korea Population............................................................................251 10.4.5 UC Davis Population...................................................................... 254 10.5 Gene Discovery by Transposon Tagging............................................... 256 10.5.1 Forward and Reverse Genetics Strategies ...................................... 256 10.5.2 Other Approaches for Mutation Identification ............................... 259 10.5.3 Tagging Efficiency......................................................................... 260 10.5.4 Confirmation of Tagged Gene........................................................ 261 10.6 Future Prospects .................................................................................... 261 References ..................................................................................................... 262

10.1 Introduction With the completion of rice genome sequencing, the new challenge for the rice community is to unravel the biological functions of approximately 40,000 rice genes. To achieve this goal, a wide range of functional genomics tools, such as microarray, serial analysis of gene expression (SAGE), RNA interference (RNAi), insertional mutagenesis, and bioinformatics, have been established and employed. Insertional (T-DNA, transposon or retrotransposon) mutagenesis has proven to be one of the most efficient methodologies, because studies of mutants with detectable phenotypes have given us the greatest insight into the mechanisms underlying a wide range of biological processes in plants. Compared with T-DNA insertional mutagenesis, transposon insertional mutagenesis (or transposon tagging) has distinct advantages. Large-scale transposon mutagenized populations can be produced using a relatively small number of starter lines, as many independent insertions can be generated among the progeny of a single line. The tagged gene can be confirmed by revertants resulting from excision of the transposon. Transposons were first discovered by Barbara McClintock in the 1940s as the causative agent of variegated maize (Fedoroff 1989). Since then, transposons have been found to be ubiquitous genetic elements in both prokaryotes and eukaryotes. In rice, recent genome sequencing and annotation have shown that a large portion of the rice genome consists of transposable elements (Mao et al. 2000), and almost all of these endogenous transposable elements are inactive under normal conditions. However, transposons have played an important role in the evolution of the rice genome. According to the transposition mechanism and propagation mode of transposons, they are categorized into two groups: class I elements, also called retrotransposons, transpose via an RNA intermediate, and class II elements transpose via a DNA intermediate by a “cut and paste” mechanism (Saedler and Nevers 1985; Coen et al. 1989; Gorbunova and Levy 2000). Both class I and class II elements exist as autonomous and nonautonomous transposable elements. An autonomous transposon

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encodes its own transposase—a protein required for its transposition. A nonautonomous transposon does not encode its own transposase, but can be induced to transpose by the transposase expressed by an autonomous transposon elsewhere in the genome. This chapter focuses on the utilization of class II elements—Ac/Ds and Spm/dSpm (also called En/I ) in rice functional genomics. The study of transposon insertional mutagenesis and the utilization of transposons as mutagens were initially carried out in maize (Zea mays) and snapdragon (Antirrhinum majus), in which a high frequency of spontaneous mutations resulted from insertions of their endogenous transposons within genes. Transposons were first isolated from these two species in the 1980s (Fedoroff et al. 1983; Pereira et al. 1985; Sommer et al. 1985), and soon after genes were cloned via transposon tagging (Fedoroff et al. 1984; Martin et al. 1985). Engineered transposons were also found to retain their transposability in transgenic plants, including rice (Baker et al. 1986; van Sluys et al. 1987; Yoder et al. 1988; Frey et al. 1990; Izawa et al. 1991; Murai et al. 1991; Finnegan et al. 1993). Several genes were cloned via introduced transposons in Arabidopsis and petunia at the same time (Aarts et al. 1993; Bancroft et al. 1993; Chuck et al. 1993; Long et al. 1993). These efforts demonstrated the feasibility of transposon tagging in heterologous plant species. The utilization of the maize two-element Ac/Ds and Spm/dSpm transposons for gene tagging have been extensively investigated since the autonomous Ac element was proven to be active in transgenic rice plants (Izawa et al. 1991; Murai et al. 1991). During the last decade, sophisticated transposon tagging systems have been established to improve screening efficiencies, and a large number of transposon insertion lines have been generated. Several genes have been cloned by transposon tagging since the successful cloning of a gene (BFL1/FZP) that mediates the transition from spikelet to floret meristem (Komatsu et al. 2003a; Zhu et al. 2003). In this chapter, we discuss rice transposon tagging systems and methodologies, summarize the progresses made, and discuss the strategies for gene discovery using transposon mutagenized populations.

10.2 Transposon Tagging Systems 10.2.1 Activity of Transposons in Rice Retaining transposability of engineered transposons in transgenic rice is an obvious prerequisite for rice gene tagging systems based on transposon mutagenesis. To test the mobility of transposons in the rice genome, the autonomous Ac element was first introduced into the rice genome by

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electrophoration via a construct in which the Ac element is inserted between the Cauliflower mosaic virus (CaMV) 35S promoter and the hygromycin phosphotransferase (hph) gene. Transposition of the Ac element was R proven by recovering hygromycin-resistant (Hyg ) plants (Izawa et al. 1991; Murai et al. 1991). The nonautonomous Ds element was also shown to transpose in transgenic rice plants in the presence of Ac transposase (Shimamoto et al. 1993). This was also the first study to demonstrate that Ds could be transactivated and stably integrated into different chromosomes of the rice genome by the transiently expressed Ac transposase at the tissue culture stage. These results encouraged many groups to investigate further the transposition behavior of Ac/Ds and Spm/dSpm in transgenic rice plants to determine the feasibility of using these two-element systems as mutagens for large-scale gene tagging in rice (Chin et al. 1999; Enoki et al. 1999; Nakagawa et al. 2000; Greco et al. 2001a, 2003, 2004; Kohli et al. 2001; Upadhyaya et al. 2002, 2006; Eamens et al. 2004; Ito et al. 2004; Jin et al. 2004; Kim et al. 2004; Kolesnik et al. 2004; Kumar et al. 2005; Szeverenyi et al. 2005; van Enckevort et al. 2005). Enoki et al. (1999) analyzed the behavior of Ac in 559 rice plants derived from four independent transgenic progenitors through three successive generations by Southern blot hybridization analysis. The frequency of Ac transposition ranged from 8.3% to 40.9% in the four independent transgenic rice populations. This frequency was comparable to those reported in other heterologous systems except for Arabidopsis, in which the transposition frequency of Ac was shown to be very low (Schmidt and Willmitzer 1989). This study also demonstrated a preferential transposition of Ac into protein-coding sequences in rice, through the rescue and analysis of Ac flanking sequences. Two-thirds of the rescued flanking sequences were shown to be homologous to predicted rice gene sequences (Greco et al. 2001a, 2001b). The frequency (15% to 50%) of Ac transposition detected by Greco et al. (2001a, 2001b) was similar to that reported by Enoki et al. (1999). Preferential transposition of Ds into coding regions has also been recently reported, with one-third of Ds flanking sequences showing homology to either protein coding sequences or to expressed sequence tags (ESTs) in rice (Kolesnik et al. 2004). Greco et al. (2001a) also showed that the transposition frequency of Ac in rice was inversely proportional to the Ac dosage (or copy number). Transformant lines harboring multiple copies of Ac resulted in a single transpositional event, whereas transformants with a single copy of the Ac induced multiple early transpositional events. This inverse correlation between the number of Ac excision events and Ac copy number had been previously observed in maize, and the timing of Ac excision in maize kernel development could be delayed by increasing the Ac copy number (McClintock 1950, 1951), but this effect depended on the level of transposase as well as the dosage and composition of the transactivated


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element (Heinlein 1996). However, in dicots, there was a consistent increase in germinal transposition of Ac with increasing Ac copy numbers (Jones et al. 1989; Hehl and Baker 1990; Keller et al. 1993a). Very high levels of transposase expression, however, have been found to inhibit Ac transposition in maize, petunia, and tobacco, perhaps because of the aggregation of the transposase protein (Scofield et al. 1993; Heinlein et al. 1994). Once stably integrated Ds lines are generated, they can be crossed with Ac lines capable of producing active transposase. In the resulting F1 progeny, expression of the Ac transposase protein can induce active transposition of the Ds element (Shimamoto et al. 1993). It was shown by Southern blot analysis that transposition of Ds occurred in a high proportion of F2 plants (Izawa et al. 1997). Although transposition inhibition was observed in most Ds lines in later generations (Izawa et al. 1997), Ds inactivation may not be a general phenomenon. First, in the same study, Izawa et al. (1997) found one line with actively transposing Ds elements over several generations. Second, later studies have also shown that Ds transposition was active in subsequent generations. For example, the frequency of independent Ds transposition in the F2 generation was 3% to 20% in one study (Nakagawa et al. 2000) and the frequency of putative stable insertion lines was approximately 6% in the F2 and double transformant T1 (DtT1) generations, and 7% to 12% in the F3 and DtT2 generations in another study (Upadhyaya et al. 2002). Other studies have also shown activity of Ds even in F4 and F5 generations (Kolesnik et al. 2004; Szeverenyi et al. 2006). These results indicate that a high frequency of Ds transposition can be achieved throughout successive generations in rice using an Ac/Ds-based tagging system. However, it is important to note that the transposition frequency may vary greatly among different lines and crossing combinations (Izawa et al. 1997; Nakagawa et al. 2000; Greco et al. 2001a; Upadhyaya et al. 2002; Kolesnik et al. 2004). Many factors could have caused these differences. First, the integration position of the Ds element may affect the binding efficiency of the transposase owing to the conformation or configuration of chromatin itself. The conformation and structure of the chromatin may also influence the reinsertion activity. If the targets and/or donor sites are difficult to access, the frequency of reinsertions may be low (Nakagawa et al. 2000). Therefore, the initial insertion site of a Ds element may be very important in determining its transposition frequency. The available large number of Ds insertion lines and corresponding Ds flanking sequences are valuable tools for investigating the effect of the initial Ds insertion site on its subsequent transposition. Second, the transposition of Ds is likely to be affected by the length or composition of the Ds construct itself (Ito et al. 1999). Third, imprecise excision and integration, by which the

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termini of the transposon are deleted, may also result in transposon inactivation as the integrity of the terminal inverted repeats of the Ac and Ds are essential for their transposition (Ito et al. 2002). Fourth, inactivation of Ds could be promoted by either multiple Ds copies or by varying levels of transposase (Kolesnik et al. 2004). In cases in which inactivation of Ds was observed, multiple copies of Ds and/or the Ds elements coexisted with the Ac transposase (Izawa et al. 1997). Therefore it is important to select single-copy Ds transgenic plants as starter lines. Finally, the inhibition of Ds transposition may also result from epigenetic suppression. The epigenetic suppression could be relatively stable, resulting in inactivation of the Ds element, even in the presence of transposase (Wang and Kunze 1989; Kim et al. 2002). It has become clear that chromatin structure and methylation are the two main mechanisms involved in epigenetic regulation (Gendrel and Colot 2005). In the case of the maize Ac/Ds transposon, methylation of the terminal or subterminal regions of the element has been correlated with decreased mobility of these elements (Fedonoff and Chandler 1994). McClintock (1984) suggested that genomic stress could also trigger the activity of transposons. Reactivation of silent Ac in maize through tissue culture was found to be associated with alterations in the methylation pattern (Brettell and Dennis 1991). Reactivation of silent Ds in rice following tissue culture has also been reported (Kim et al. 2002). Therefore, the transposability of epigenetically inactivated transposons could be reversed via developmental reprogramming such as tissue culture. It has been shown that Ac and Ds preferentially transpose to genetically linked sites in maize (Dooner and Belachew 1989) and in heterologous plant species such as tobacco (Jones et al. 1990), Arabidopsis (Keller et al. 1993b; Raina et al. 2002), and barley (Koprek et al. 2000). In rice, the frequency of linked transposition has been shown to range from 35% to 80% (Nakagawa et al. 2000; Greco et al. 2001a; Upadhyaya et al. 2006). This is a disadvantage of using the Ac/Ds for genome-wide mutagenesis. In contrast, the Spm/dSpm transposon has been shown to produce a high frequency of unlinked transpositions in Arabidopsis (Aarts et al. 1995). The feasibility of using the Spm/dSpm system for large-scale mutagenesis in rice has been investigated by two groups. Greco et al. (2004) found low frequencies of transposition. However, Kumar et al. (2005), using a new fluorescence marker based screening system, observed high frequencies of stable dSpm insertion lines with a high proportion of insertions unlinked to the donor sites (launch pads). The conflicting results obtained in these two studies in rice could be due to the differences in the length of termini of dSpm employed (Kumar et al. 2005). The 5΄ and 3΄ terminal sequences of the dSpm element used by Greco et al. (2004) were 267 and 640 bp, respectively, whereas those of


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the element used by Kumar et al. (2005) were 1,014 and 1,193 bp, respectively. As in Arabidopsis, dSpm element is likely to transpose to unlinked sites in rice more frequently than Ds element, therefore dSpm element should be more efficient than Ds for genome-wide coverage (Kumar et al. 2005). The mutation frequency in progeny plants depends on the frequency of germinal transposition in the parental plant. Moreover, a high frequency of somatic transposition may create undesirable mutations due to secondary transpositions. In the Ac/Ds two-element system, transposition of Ds is achieved by expressing Ac-encoded transposase driven by a constitutive promoter such as CaMV 35S. To achieve maximal germinal transposition, an attempt has been made to control the expression of transposase by the use of a meiosis-specific promoter in rice (Morita et al. 2003). Although a much higher frequency of independent transposition was observed with the meiosis-specific promoter when compared to a constitutive promoter, the feasibility of using these promoters needs to be further investigated, as the overall transposition frequency reported so far is still very low. 10.2.2 One-Element System versus Two-Element System Both the one-element and two-element systems have been used in transposon mediated gene tagging in rice (Fig. 10.1). In the one-element system, an autonomous transposon (Ac or Spm) that encodes its own transposase, delivered through T-DNA, is used as a mutagen. The Ac element is usually inserted between a constitutively expressed promoter such as CaMV 35S and an excision marker. The excision marker is expressed upon excision of the Ac element, by which the transposition of Ac can be monitored (Izawa et al. 1991; Murai et al. 1991; Greco et al. 2001a). However, identification of Ac reinsertion in the rice genome relies solely on molecular analyses due to the lack of a selectable marker within the Ac element. In the two-element system, two independent transgenic lines are generated: one with an immobilized autonomous element (wings-clipped Ac or Spm) that provides the transposase and the other with a nonautonomous element (Ds or dSpm) that is capable of transposition only in the presence of a transposase gene. In the nonautonomous element, selectable markers such as antibiotic or herbicide resistance genes are incorporated to select progeny bearing transposed elements. To monitor the transposition event, the nonautonomous element can be inserted between a promoter and an excision marker gene so that excision results in expression of the excision marker. Transgenic plants bearing these two transposable elements are crossed to induce transposition of the nonautonomous element. Stable Ds (or dSpm) insertion

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lines can be easily selected based on the expression of selectable markers in the F2 or subsequent generations because the transposed nonautonomous element is likely to be unlinked to the autonomous element that encodes the transposase.

A

Ac

P

Ex

G

S

Ac

P

B

iAc

P

Ex

x

S

G

S

P

RM

Ex

Ds

G

S RM

iAc

P

S

Ex

P

RM P

Ex

S

S

Ds G

Ds

G

Fig. 10.1. One- and two-element transposon tagging systems (Ac/Ds as an example). (A) One-element system. Ac is inserted between a promoter and an excision marker, which will express on excision of Ac and is used to indicate the occurrence of transposition. Transposed Ac re-inserts somewhere in the rice genome. If the insertion happens to be in an expressed gene, the function of the gene is impaired. The genomic sequence of the mutated gene can be isolated using Ac as a molecular tag. (B) Two-element system. iAc (immobile Ac) and Ds are introduced into two different transgenic plants. To induce Ds transposition, transgenic plants harboring iAc and Ds are crossed to bring iAc and Ds together. In the F1 generation, Ds transposes from the T-DNA (launch pad) and reinserts elsewhere in the rice genome. The transposition and reinsertion of the Ds element are monitored by the expression of the excision marker and reinsertion marker. In F2 and subsequent generations, stable Ds insertion plants are selected by segregating iAc away. The presence or absence of the iAc element can be achieved by Ac-specific polymerase chain reaction (PCR) or Ac counterselection marker. Ac, Activator; Ds, Dissociation; Ex, excision marker; G, rice gene; P:, promoter; RM, reinsertion marker; S, selectable marker for transformation


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Numerous studies have shown that both the one- and two-element systems are suitable for gene tagging in rice (Chin et al. 1999; Enoki et al. 1999; Nakagawa et al. 2000; Greco et al. 2001a, 2003, 2004; Kohli et al. 2001; Upadhyaya et al. 2002, 2006; Eamens et al. 2004; Jin et al. 2004; Kim et al. 2004; Kolesnik et al. 2004; Kumar et al. 2005; Szeverenyi et al. 2005; van Enckevort et al. 2005). To simplify the discussion that follows, Ac and Ds will be used as examples of autonomous and nonautonomous elements, respectively, unless otherwise specified. In one-element tagging system, the insertion is unstable as the transposability of the autonomous transposon remains throughout the life of a plant resulting in unstable mutations. Moreover, versatile selectable markers cannot be integrated into the autonomous element. Screening of transposed Ac elements relies on labor-intensive and costly polymerase chain reaction (PCR) and/or Southern blot analysis, which decreases the overall screening efficiency. Therefore, most laboratories are developing transposon mutagenized populations using the two-element system. In this system, the Ac element provides transposase to induce the transposition of the Ds element. Usually, the Ac element is modified to disable its own transposition by removing its inverted terminal repeats (i.e., wingclipped). The Ds element is extensively modified to incorporate a multitude of marker genes such as excision and reinsertion markers as well as a trapping reporter gene that can be activated by insertion adjacent to cisacting elements (e.g., promoters and enhancers). In several recent studies, counterselective markers are also incorporated into the Ac construct to eliminate plants bearing the transposase gene to stabilize the germinally transmitted Ds insertion. By appropriate combinations of marker genes, the efficiency of screening stable Ds insertion plants can be significantly improved. In most two-element systems, the autonomous and the nonautonomous elements are constructed in two different vectors. They are brought together by crossing transgenic plants bearing either of these two elements. Alternatively, calli can be cotransformed with two vectors harboring either the autonomous or the nonautonomous element to regenerate double transformants. The two-element system can also be integrated in one T-DNA vector (Greco et al. 2003, 2004; Kumar et al. 2005). This system has been efficiently used in selection of unlinked transposition events (Kumar et al. 2005). However, if the transposed nonautonomous element remains linked to the launch pad from which the transposase is expressed, then the transposed nonautonomous element will be as unstable as that in the oneelement system.

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10.2.3 Design of Constructs In the one-element system, the three basic components of the construct are a selectable marker for producing primary transformants; an autonomous element (Ac or Spm), and an excision marker, that is, a gene into which the transposon is inserted and in which, on excision of the transposon, expression of the maker gene is restored. The most common selectable markers R R are antibiotic resistance genes such as nptII (Kan ), hph (Hyg ), or an herR bicide resistance gene such as bar (Basta ) (Table 10.1). The most frequently used selectable marker in rice is hph. In the two-element system, the T-DNA carrying the transposase needs only a selectable marker for transformation, but it can be modified to carry a negative selectable marker (NSM) to allow plants bearing the T-DNA to be counterselected or identified by screening (Fig. 10.2). This feature stabilizes the transposed nonautonomous transposon and the mutant phenotype. The basic components for the second T-DNA, which harbors the nonautonomous transposon, are a selectable marker for transformation and a reinsertion marker (RM) for tracing the transposition or reinsertion of the nonautonomous transposon. As discussed in the preceding text, this marker can be an antibiotic- or herbicide-resistance gene. The transactivation of the nonautonomous transposon can be detected with the reinsertion marker in combination with an excision marker (Fig. 10.2). Another feature of some constructs is a plasmid rescue system that consists of an ampicillin resistance gene (bla) and an E. coli bacterial plasmid origin of replication (pBR322 ori). This provides an alternative way to rescue transposon flanking sequences, although thermal asymmetric interlaced PCR (TAIL-PCR; Liu et al. 1995), inverse PCR (iPCR; Earp et al. 1990), and adapter ligation PCR (Siebert et al. 1995; Devic et al. 1997; Zhu et al. 2006a) are all more suitable approaches for large-scale flanking sequence tag (FST) rescue. Further improvements of the basic construct can be made to increase the frequency of clean single-copy T-DNA insertion lines, which will greatly benefit subsequent segregation analysis. For example, in a newly designed construct, pNU435, a maize ubiquitin promoter-driven, intron-interrupted barnase gene is used as a vector backbone (VB) counterselective gene. Transformed cell lines with VB-containing T-DNA inserts will be eliminated by the activity of the barnase gene. Moreover, in this construct a clean T-DNA insert will allow the ubiquitin promoter positioned near the left border (LB) to act as a dormant gene activator. A second copy of a promoter-less intron interrupted barnase-nosT cassette, placed within the T-DNA and next to the right border (RB) of this construct also has the potential to serve as a T-DNA direct repeat (RB-LB-RB-LB) counterselector. The rationale of this design is that a directly repeated T-DNA transgene will have a strong ubiquitin promoter upstream of the barnase gene


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adjacent to the RB and that the resulting cell lines will be eliminated by the activity of the barnase gene (Upadhyaya et al. 2006). Table 10.1. Selectable and screening marker genes used in production of transposon insertion lines Tagging system

TransExcision Launching position marker pad (reinsertion) indicator marker Na hph hph

Transposase counter selectable marker hph

Reference

Ac

Selectable marker for transformation hph

Ac

hph

Na

gfp

gfp/hph

hph/gfp

Ac/DsG

hph

bar

na

hph

na

Greco et al. 2001a; Kohli et al. 2001 Chin et al. 1999

Ac/Ds

hph nptII

hph hph

nptII SPT

nptII SPT

na na

Nakagawa et al. 2000

Ac/DsG Ac/DsE

hph

nptII

na

gfp/hph

hph

Upadhyaya et al. 2002

Ac/DsE

hph

bar

na

gfp/hph/ P450

gfp/P450

Greco et al. 2003

Ac/DsG

hph

hph

na

Na

gfp

Eamens et al. 2004

Ac/DsE

nptII

hph

ALS

ALS

na

Ito et al. 2004

Ac/DsG

hph

bar

na

gfp/hph

gfp

Kolesnik et al. 2004

Ac/DsG

P450

bar

na

P450

P450

Kim et al. 2004

Ac/DsG Ac/DsE

hph bar

nptII bar

bar hph

hph hph

gfp gfp

Upadhyaya et al. 2006

Spm/dSpm hph (En/I)

bar

na

gfp/hph/ P450

hph/P450

Greco et al. 2004

Spm/dSpm hph (En/I)

DsRed

na

gfp/hph

gfp/hph

Kumar et al. 2005

Enoki et al. 1999

Ac, Activator; ALS, acetolactate synthase gene; bar, Basta resistance gene; Ds, Dissociation; DsE, Ds enhancer trap; DsG, Ds gene trap; dSpm, defective suppressor-mutator; En, Enhancer; gfp, green fluorescent protein gene; hph, hygromycin phosphotransferase gene; I, Inhibitor; na, not applicable; nptII, neomycin phosphotransferase gene; Spm, suppressormutator; SPT, streptomycin phosphotransferase gene

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A

Transposase

RB

B

S

NSM

LB

RM

RB

C

Ex

P

RM

R

RB

D

E

RM

RB

P

R

RM

F

Bn

R

RB

PRS

NSM

R

RB

LB

S S

PRS

Ex

P

R

LB

PRS

Ex

LB

S

R

LB

Bn

RM

Transposase

NSM

S

LB

Fig. 10.2. Diagrams of typical constructs used in two-element transposon tagging system in rice. (A) T-DNA harboring transposase, which is encoded by an autonomous transposon (Ac or Spm). To increase the efficiency of screening stable insertion lines, a negative selection marker (NSM) is usually integrated in the


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Although the bar gene is not an ideal selectable marker at the callus stage, it is an excellent selectable marker at the plant stage and is the preferred transposition or reinsertion marker to select for the presence of the Ds transposon in rice (Chin et al. 1999; Kim et al. 2004; Kolesnik et al. 2004; Upadhyaya et al. 2006). This is due primarily to the fact that a single spray of Basta can eliminate Ds-null segregants and thus greatly increase the overall screening efficiency. A new positive selection marker DsRed (Discosoma sp. red fluorescence protein) has been used in an Spm/dSpm tagging system, which has been shown to be very efficient for the selection of transposants (Kumar et al. 2005). Currently, most transposon tagging systems used in rice do not have an excision marker to detect the excision of the Ds element from the donor site (Chin et al. 1999; Upadhyaya et al. 2002; Greco et al. 2003, 2004; Eamens et al. 2004; Kolesnik et al. 2004; Kumar et al. 2005). PCR-based analysis has to be performed to confirm the excision of the Ds element. Incorporation of an excision marker for the nonautonomous element will significantly increase the screening efficiency (Fig. 10.2; Upadhyaya et al. T-DNA for counterselection. (B) to (F) T-DNAs harboring nonautonomous transposon (inverted triangles). The basic components of these T-DNAs are selectable marker (S) for selection of primary transformants and reinsertion marker (RM) for tracing the reinsertion events, but more markers can be combined to facilitate selection of stable insertion lines. In construct B, transposition of the nonautonomous element is monitored via an excision marker (Ex), and the reinsertion marker also serves as a selectable marker of transformation (for details see Nakagawa et al. 2000). Construct C is the most frequently used vector, in which the selectable marker is different from the reinsertion marker, and another marker (NSM) is used to distinguish the transposition events linked or unlinked to the launch pad (LP) T-DNA. A reporter gene without or with only minimum promoter is fused at one end of the transposon to serve as a gene or enhancer trap reporter. Because there is no excision marker PCRs are required to confirm that the transposon has excised from the LP in the selected stable insertion lines. In some cases a plasmid rescue system (PRS) is incorporated within the transposon for isolation of the genomic DNA flanking the transposon. To increase screening and trapping efficiency, the nonautonomous transposon is inserted between a promoter (P) and an excision marker (Ex) that will express on excision of the transposon and both ends of the transposon are fused with reporter genes (construct D; for details see Upadhyaya et al. 2006). This construct is further enhanced by incorporation of two copies of barnase genes, one within the T-DNA next to the RB and another within the vector backbone outside the LB, for selection of lines with clean single-copy T-DNA insertions preferably in intergenic regions (construct E; for details see Upadhyaya et al. 2006). The autonomous and nonautonomous transposons can also be integrated in the same vector as shown in construct F. Same as construct C, confirmation of excision of the nonautonomous transposon relies on PCR (for details see Greco et al. 2003, 2004; Kumar et al. 2005)

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2006). For example, the constructs pNU393A1/B2 and pNU435 have a CaMV 35S promoter-driven intron-interrupted hph gene cassette as the excision marker (Upadhyaya et al. 2006). This choice was based on the experience that this cassette works well as a selectable marker in rice transformation using embryogenic calli as the target tissue (Upadhyaya et al. 2000). It has been shown that this excision marker is particularly advantageous for selecting callus lines with Ds excision using the transiently expressed transposase (TET) system (Upadhyaya et al. 2006). An excision marker, in combination with a Ds reinsertion marker, is particularly useful for distinguishing Ds insertions either linked or unlinked to the T-DNA launch pad. Another feature that is incorporated in most of the constructs used in producing transposon mutagenized populations in rice is the gene or enhancer trap system (see next section for details). 10.2.4 Gene and Enhancer Traps Insertional mutagenesis by transposon tagging is useful when disruption of a gene leads to an obvious phenotype. But in eukaryotes, disruption of a gene frequently does not result in a visible phenotype because of functional redundancy between gene family members (Sundaresan 1996; Ramachandran and Sundaresan 2001). To overcome this difficulty, gene and enhancer trap systems have been developed for use in transposon tagging systems in plants (Sundaresan et al. 1995; Springer 2000). An enhancer trap harbors a minimal promoter fused to the open reading frame of a reporter gene. On insertion at or near a host gene, the minimal promoter may be cis-activated by enhancer elements in the host gene leading to expression of the reporter gene. A promoter trap contains a promoter-less reporter gene that is expressed when the transposon inserts downstream of an active endogenous promoter. A gene trap contains an intron with multiple splice acceptor sites fused to the coding region of the reporter gene. A fusion protein of the reporter gene with the N-terminal portion of a host gene will be produced when the element is inserted into either an exon or an intron of the host gene in the same transcriptional orientation (Fig. 10.3). Both enhancer and gene trap systems have been used in rice by incorporation of these features into Ac/Ds or Spm/dSpm transposon tagging systems (Chin et al. 1999; Upadhyaya et al. 2002, 2006; Greco et al. 2003, 2004; Ito et al. 2004; Kim et al. 2004; Kolesnik et al. 2004). The most frequently used reporter genes are uidA (gusA) and green fluorescent protein (gfp). A clear advantage of these trap systems is that the expression pattern of the tagged gene can be studied in detail by analyzing the GUS or GFP expression pattern during plant development. Such detailed knowledge of the


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TE SA

A

R

E Gene

B

E

Gene

TATA

R TE

TE TATA

C

R

E Gene

Fig. 10.3. Gene and enhancer trap systems. (A) Gene trap system; the transposable element (TE) has a promoter-less reporter gene (R), which contains splice acceptor (SA), at its 5΄ or 3΄ end. The reporter gene is expressed when the TE inserts into an intron, due to the creation of a fusion transcript (and therefore a fusion protein) by the interaction of splice donor of the gene and the SA of the reporter gene. (B and C) Enhancer trap system. The minimal promoter (TATA) of the reporter gene (R) is activated by a chromosomal enhancer (E), which can be in the same or complementary orientation as the TE, resulting in the expression of the reporter gene

expression pattern can be very helpful in subsequent phenotypic analysis of homozygous insertion mutants. The disadvantage of such unidirectional trapping systems is that there is neither selection against insertions outside genes, nor against insertions in which the reporter gene is in the opposite orientation relative to transcription of a tagged gene (Maes et al. 1999). This drawback has partly been addressed in a bidirectional trap system developed by Eamens et al. (2004). In the first series of bidirectional gene trap constructs (pEU334a/b), immediately inside the RB and LB borders are the Ds5΄ and Ds3΄ sequences, respectively. A promoter-less gfp gene (sgfpS65T), fused to the fourth intron of the Arabidopsis G protein gene (GPA1), containing splice acceptor sites in all three reading frames and a nopaline synthase terminator (nosT), were placed in 5΄−3΄ orientation as the RB or Ds5΄ gene trap. A promoter-less gus gene (uidA), fused to a GPA1 intron and nosT, was included as the LB or Ds3΄ gene trap. A CaMV

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35S promoter-driven, intron-interrupted hph chimeric gene was incorporated in the same orientation as the GUS-based gene trap to act as either (1) a selectable marker following the initial Agrobacterium-mediated transformation event or (2) a subsequent Ds reinsertion marker following Ds transposition from the T-DNA launch pad. The more recent construct pNU435 contains not only this proven bidirectional gene trap Ds cassette (Ds3'-GPA1-SA-uidA-nosT and Ds5'-GPA1-SA-eyfp-nosT), but also harbors two barnase genes located inside the RB and outside of the LB to counterselect against directly repeated T-DNA or VB integrations, respectively (Upadhyaya et al. 2006). 10.2.5 Transiently Expressed Transposase System Transient expression of introduced foreign DNA in target plant cells, which occurs before any stable integration through illegitimate recombination or its breakdown by the plant surveillance system, is a well known phenomenon. A burst of transient expression of the genes carried by the introduced T-DNA can be visualized by reporter gene expression within 48 to 72 hours of cocultivation (N. M. Upadhyaya et al., unpublished data). A transient assay is usually used to assess the transactivation of the Ac transposase-mediated excision of the Ds element prior to its stable integration into the plant genome. This type of transient assay has been performed in barley, rice and wheat using a Ds-interrupted uidA reporter gene (McElroy et al. 1997; Solis et al. 1999; Takumi et al. 1999). By cotransformation with an Ac construct and a Ds-interrupted uidA construct, the Ac-mediated transactivation of the Ds element can be measured by the expression of the uidA gene. Recently, Upadhyaya et al. (2006) have developed a system where a transiently expressed transposase (TET) is used to produce stable Ds insertion lines (i.e., without an integrated Ac element) in rice. The main advantage of the TET system is that stable Ds insertion lines can be produced as primary transformants. In contrast, with the Ac/Ds crossing system, the first available screening population is F2. To overcome somaclonal variation induced by tissue culture, which has been a major drawback with T-DNA insertional mutagenesis (An et al. 2005), the tissue culture phase in the TET system is kept to the absolute minimum (Upadhyaya et al. 2006). 10.2.6 A High-Throughput System to Index Transposants In producing an indexed and saturated insertional-mutant library, the final step is to determine the chromosomal location of each Ds transposant (Fig. 10.4). Up to now, most investigators have mainly focused on producing random, saturation mutant libraries. The different laboratories together


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have produced more than 150,000 Ds transposants (Table 10.3). However, the chromosomal locations of only 12% of these transposants have been determined by flanking sequence analysis. For these analyses, most investigators use the TAIL-PCR method. Researchers in Cornell University have recently developed a novel long-PCR based high-throughput procedure to determine the chromosomal location of a large number of Ds transposants to construct an indexed, region-specific, insertional-mutant library (He et al. 2007). The procedure is based on the novel use of a longPCR based high-throughput system, coupled with an anchored population, which allows rapid and simultaneous determination of the chromosomal location of thousands of insertional mutants at the same time. The principle of this procedure is based on measuring the transposition distance between a Ds transposant and a specific genomic sequence of interest in rice. Since the long-PCR procedure can amplify a genomic sequence of greater than 10 kb, all the transposants that transpose within this region can be captured simultaneously. Measuring the size of the long-PCR products by comparing DNA size markers may have an error of up to 3%; thus a 10-kb fragment may be 10 ± 0.3 kb away from that of the specific Ds primer position. However, this degree of accuracy is sufficient to meet the requirements of this system. Ds plants x Ac-TPase plants F1 plants Self-pollination F2 families Basta and hygromycin selection BastaR and HygR families

BastaS and HygR families Self-pollination F3 families (BastaR and HygR)

DNA isolation & PCR to display transposition events EDS+ & Ds+ siblings/family Plant EDS+ & Ds+ siblings in soil Determine chromosomal location of each transposant by a high throughput long-PCR procedure

Fig. 10.4. A flow chart for generating a large-scale, indexed, Ds transposant population in rice. EDS, empty donor site

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This approach has been tested by attempting to determine the chromosomal location of transposants derived from three anchor lines (launch pads). The results from one of these anchor line are as follows. Out of a total of 249 transposants, 72 (29%) transposed to new positions on the same chromosome, and 20 (8%) of these were within a region of 400-kb flanking the anchor position. In principle, all transposants within this 400-kb region can be captured by using 40 pairs of PCR primers with one set of primers positioned every 10 kb. The recurrent primer sequence is based on a short sequence complementing a portion of the 5΄ Ds sequence, whereas the 40 variable sequences are based on the genomic sequence chosen from the DNA database. Only 249 transposants around this anchor line were collected, and thus many more are needed to truly saturate this 400-kb region. Since the transposition process is not random, approximately 5,000 transposants may be needed to saturate this region with the expectation that up to 400 transposants may be found. Based on previous experience, it takes approximately two person-months to obtain 500 flanking sequences via the TAIL-PCR procedure. Thus, 20 person-months would be needed to analyze 5,000 transposants. On the other hand, using the long-PCR procedure, it would only take four person-months to analyze 5,000 transposants. According to Muskett et al. (2003), genes of approximately 0.5 kb in size account for between 10% and 20% of all Arabidopsis genes. If rice has the same percentage of small genes, the number of transposants required would need to be increased from 5,000 to approximately 20,000 to saturate a 400-kb region and tag all genes, within this region. If one uses the TAIL-PCR procedure to determine the flanking sequences, the amount of work would be proportional to the number of transposants. Thus, 80 person-months would be needed to determine the flanking sequences of 20,000 transposants. On the other hand, by using the long-PCR procedure, only eight personmonths of work would be required to achieve the same goal. Thus, the long-PCR procedure would be ten times more efficient than the TAILPCR procedure (He et al. 2007). The reconstruction experiment has shown that at least 100 transposants can be pooled together for DNA isolation, and 100 pools can be employed simultaneously to analyze 10,000 transposants (He et al. 2007). In principle, the entire rice genome can be saturated using this method, with the participation of many scientists around the world, and each group working on several specific Ds anchor lines at a time. 10.2.7 Using Endogenous Transposons Transposable elements are a major component of the repetitive DNA that comprises more than 40% of the rice genome (Goff et al. 2002; Yu et al.


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2002). Four types of active endogenous transposable elements have been identified in rice. The LTR (long terminal repeat) retrotransposons Tos10, Tos17, and Tos19 were the first identified (Hirochika et al. 1996) and Tos17 has been used in large-scale mutagenesis in rice. Karma is a LINE (long interspersed nuclear element)-type retrotransposon showing continuous transposition in consecutive generations (Komatsu et al. 2003b). The presence of active MITE (miniature inverted repeat transposable element) sequences, such as miniature Ping (mPing), has also been revealed through the analysis of rice genomic sequences (Jiang et al. 2003; Kikuchi et al. 2003). All of these native rice transposable elements are dormant under normal conditions and become active during tissue culture (Hirochika et al. 1996; Jiang et al. 2003; Kikuchi et al. 2003; Komatsu et al. 2003b) or after treatment with inducible agents such as γ-irradiation (Nakazaki et al. 2003). Tos17 is 4,114-bp long. Its copy number in the rice genome is quite low compared with other endogenous retrotransposon classes. Nipponbare contains only two copies of Tos17 per haploid genome. After tissue culture-induced activation, Tos17 could be amplified to approximately 30 copies (Hirochika 2001). Three characteristics make Tos17 an ideal mutagen for saturation mutagenesis in rice. First, the copy number of Tos17 correlates with the duration of tissue culture, making it possible to control the number of Tos17 copies. Second, Tos17 tends to transpose to unlinked positions. Third, Tos17 prefers low-copy-number sequences and genes as integration targets (Yamazaki et al. 2001; Miyao et al. 2003). A population of approximately 50,000 Tos17 insertion lines containing approximately 500,000 mutated sites has been generated and is available for public use (Hirochika et al. 2004). The feasibility of using this Tos17 insertion population for screening of targeted mutants (i.e., forward genetics approach) has been demonstrated by cloning several important genes (Table 10.2). A reverse genetics strategy is perhaps more powerful because of the availability of three-dimensional DNA pools of Tos17 insertion mutants and more than 15,000 Tos17 FSTs have been categorized for searches of genes knocked out (http://tos.nias. affrc.go.jp/). As such the proportion of mutants, identified and characterized by reverse genetics strategy, is more than that by forward genetics approaches (Table 10.2). The mobility of mPing/Pong in rice has provided the possibility of using this type of transposon for gene tagging in a similar way to that used in Tos17 mutagenesis. However, their high copy number (dozens, or even more), and currently unclear transposition frequency makes them impracticable for large-scale mutagenesis in the foreseeable future. Recently, the fourth active endogenous transposable element—nDart (nonautonomous DNA-based active rice transposon)— a member of the hAT transposon superfamily has been identified by the analysis of spontaneous mutable alleles (Fujino et al. 2005; Tsugane et al. 2006). The nDart element has

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Table 10.2. Genes identified using Tos17 tagged mutants Gene name OsH15 OsABA1, OsTATC OsPHYA OsHOS59 OsMSP1

OsCesA4, 7 and 9 OsCHLH OsCPS1, OsKS1, OsKO2, OsKAO OsGAMYB OsPAIR1 and 2 OsUDT1 OsCAO1 OsTPC1 OsGS1;1 OsMADS1 OsMADS3

OsFON1

OsSSI OsCLC-1 and -2

Mutant phenotype Dwarf Precocious germination

Strategy Reverse Forward

Etiolated seedlings No phenotypic changes Excessive number of both male and female sporocytes, disordered anther wall layers and loss of the tapetum layer Brittle culm due to dramatically cellulose contents Albino Dwarf

Reverse Reverse Forward

Shortened internodes, defects in floral organ development pollen development Male and female sterility Male sterility Pale green leaves Reduced defensive response Severe retardation in growth rate and grain filling Complete conversion of lodicules, stamens, and carpels into lemmaand palea-like structures Transformation of stamens into lodicules and ectopic development of lodicules in the second whorl near the palea Semi-dwarf, less tillers and secondary rachis-braches, enlarged shoot apical meristem and altered floral organs Increased gelatinization temperature of endosperm starch, but no effect on the size and shape of seeds Inhibition of growth at all life stages

Reference Sato et al. 1999 Agrawal et al. 2001 Takano et al. 2001 Ito et al. 2002 Nonomura et al. 2003

Forward

Tanaka et al. 2003

Reverse Forward

Jung et al. 2003 Sakamoto et al. 2004

Reverse

Kaneko et al. 2004

Forward

Nonomura et al. 2004a, 2004b Jung et al. 2005 Lee et al. 2005 Kurusu et al. 2005 Tabuchi et al. 2005 Agrawal et al. 2005

Reverse Reverse Reverse Reverse Reverse Reverse

Yamaguchi et al. 2006

Reverse

Moon et al. 2006

Reverse

Fujita et al. 2006

Reverse

Nakamura et al. 2006

identical 19-bp terminal inverted repeats (TIRs), and generates 8 bp of target site duplication (TSD) on insertion. The transposition of the nDart element can be induced by crossing with a line containing aDart, the corresponding autonomous element. The nDart insertions can then be stabilized after segregation away the aDart element (Fujino et al. 2005; Tsugane et al. 2006). Therefore, the nDart/aDart forms an endogenous transposon


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mutagenesis system in rice and is a potential new tool for gene tagging in this species. Polymorphism analysis of several japonica and indica varieties has shown that nDart is amplified independently in the genomes of these two rice subspecies (Fujino et al. 2005), and that a high frequency of transposition of nDart is observed in lines, such as H-26, that carry the aDart elements (Tsugane et al. 2006). Sequence analyses have revealed that Nipponbare contains at least 18 nDart elements, 12 dormant iDart elements (inactive Dart) but no aDart element (Fujino et al. 2005; Tsugane et al. 2006). iDart elements are structurally similar to aDart but are epigenetically silenced because they can induce transposition of the nDart elements after treatment with 5-azaC (Tsugane et al. 2006). Unlike the MITE transposon mPing/Pong, which preferentially inserts into AT-rich regions (Jiang et al. 2003; Kikuchi et al. 2003), nDart elements seem to transpose randomly in the rice genome since no conserved TSDs are found. This is an advantage for using nDart/aDart in transposon mutagenesis in rice. Before nDart/aDart transposons can be employed as a functional genomics tool in rice, further investigations are required to determine the transposability (induced by aDart or chemical treatment) and target preferences of nDart. In addition, because of the high copy number of the nDart element, efficient approaches for progeny analysis also need to be developed. 10.2.8 Inducible Transposition The efficiency of the current transposon tagging systems used in rice depends on whether the transposons can be efficiently controlled and stabilized after their transposition. In the one-element system, the autonomous transposon (Ac or Spm) retains its potential to excise from the inserted gene, resulting in chimeric progeny plants. Although this disadvantage has been overcome by the two-element system, the transposition time of the nonautonomous transposon (Ds or dSpm) remains unregulated due to the constitutively expressed transposase. To overcome this disadvantage, a self-stabilizing Ac derivative (Ds303), which undergoes autonomous transposition from the T-DNA but is stabilized once integrated (unless activated again by a subsequently introduced transposase source), has been investigated in tomato (Schmitz and Theres 1994). An ideal strategy would be to control transposase expression by means of an inducible promoter. Transposon constructs in which the expression of the transposase is controlled by heat-shock or chemically inducible promoters have been developed and used in tobacco, tomato, rice, and Arabidopsis (Charng et al. 2000; Nishal et al. 2005; G-L Wang’s group). In the INAc (Inducible Ac) vector, the transposase is driven by the PR-1a promoter that is induced by

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salicylic acid, and this component, together with a selectable marker (hph), is inserted in the internal region of the Ds element that is, in turn, inserted between the 1΄ promoter and the 5΄ untranslated region of the luciferase (LUC) gene. In this construct, LUC is the excision marker and hph serves as both a transformation-selectable marker and a Ds transposition marker. Transposition of the Ds element is induced by the application of salicylic acid and is stabilized in the absence of salicylic acid (Charng et al. 2000). Spontaneous transposition of the Ds element is low in tobacco but much higher in tomato. The induced transposition frequency depends on the concentration of salicylic acid. This construct has also recently been used to produce transgenic rice plants. Inducible transposition has been demonstrated in a salicylic acid dose-dependent mode, but high spontaneous transposition occurred in some transgenic rice lines (Charng et al. 2007). The main drawback of the INAc construct is that the inducible transposase cannot be segregated away in the progeny because it is integrated as a part of the Ds element. The transposase source and the nonautonomous element should be separated, even for the inducible system, to avoid the undesired additional transpositions due to autonomously expressed transposase. A heatshock promoter fused to the Ac element has been shown to be able to induce the transposition of Ds in Arabidopsis (Balcells et al. 1994). More recently, this heat-shock promoter has been integrated into a gene trap system and successfully used in Arabidopsis to produce large numbers of Ds insertion lines (Nishal et al. 2005). In this system, the Ac transposase, whose expression is induced by heat-shock at the flowering stage, is engineered in the same vector as the Ds element, which has nptII as a reinsertion marker and SPT (streptomycin) as an excision marker. This system can be easily adopted in rice, but its feasibility in large-scale transposon mutagenesis in rice still needs to be further investigated because the optimal time for heat-shock treatment is during the reproductive stage (as shown in Arabidopsis), which may not be practicable in rice, particularly for large-scale treatment. Another inducible transposon tagging system being developed in rice is a dexamethasone (DEX) inducible activation-transposon-tagging system (G-L Wang’s Group). In this system, the Ac transposase and Ds transposition are controlled by the transcription activator GVG that is regulated by the application of DEX. An approach using the cre-lox site-specific recombination system, to delete the Ac transposase (thereby stabilizing the transposed Ds elements) once Ds transposition has been induced, is also being investigated in rice by this group.


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10.3 Mutagenesis Strategies 10.3.1 Random or Non-targeted Mutagenesis In this strategy, starter lines homozygous for the autonomous (usually immobilized) or nonautonomous transposon insertions are produced and selected for crossing to produce F1 progeny. The F1 progeny are heterozygous for both the autonomous and the nonautonomous element. In this generation, the nonautonomous element transposes to new locations from the T-DNA (launch pad) under the influence of the autonomous element. The F2 populations are then produced by selfing F1 plants and screened for the presence of the selectable marker (excision and transposition markers) or by PCR analyses to select stable insertion lines in which the nonautonomous element has excised from the launch pad and reinserted into the rice genome and the autonomous element has segregated away. This procedure can be used to generate a large number of plants with transposed nonautonomous elements. Assuming that the nonautonomous element transposes randomly and inactivates rice genes, genome-wide (i.e., 400 Mb) saturation mutagenesis of all rice genes (assuming as 60,000) would require a mutagenized population of 180,000 to 460,000 (Hirochika et al. 2004). The population required is affected by the number of transposon copies in the rice genome as well as the transposition frequency and integration patterns (linked or unlinked). Similar to previously observed results in other plant species, a high proportion of the transposed Ds elements insert at sites that are closely linked to the launch pad (Upadhyaya et al. 2002, 2006). Thus Ac/Ds may be an inefficient general mutagen, but could be highly efficient for regional mutagenesis. One way to facilitate global mutagenesis is to select unlinked transpositions using a launch pad indicator (e.g., the excision marker or other markers integrated in the Ds/T-DNA launch pad). Using GFP as a counterselective marker for the launch pads, unlinked transposition events are significantly enriched to reach more than 80% (Kolesnik et al. 2004). An alternative is to select for Ds starter lines with insertions evenly distributed throughout the rice genome and then to use these starter lines for localized saturation mutagenesis. Considering that 50% of transposed Ds elements insert within 1 Mb (approximately 4 cM in rice) of the genomic region flanking the donor site, 430 Ds starter lines that are evenly distributed throughout the rice genome at a 1-Mb interval and approximately 400,000 F2 plants (~930 F2 plants need to be produced from each Ds starter line) could be sufficient to saturate the whole genome. Other assumptions for this estimation are: the rice genome contains 50,000 genes; the size of the rice genome is 430 Mb; the frequency of independent transposition in the F1 generation is 50%; each gene within the 1-Mb genomic region flanking the Ds donor site has the same probability of

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being mutagenized. The outcome will be better if more Ds starter lines are used because fewer F2 plants are required for each cross combination, thus increasing the frequency of independent transpositions. The major difficulty with this approach is to establish the starter lines. Considering that a large number of Ds insertion lines have been generated in several laboratories, and a good number of Ds FSTs have been rescued and mapped, the first set of the Ds starter lines could be selected from currently available resources. The regions devoid of Ds insertions can then be mutagenized using these Ds starter lines for “transposon-walking” strategies. Toward this end, 74 singlecopy Ds/T-DNA launch pads that are relatively evenly distributed amongst the 12 rice chromosomes have been produced (Upadhyaya et al. 2006). The Spm/dSpm system can be used in a complementary way for saturation mutagenesis as it has been revealed that Spm/dSpm does not show preferential transposition in rice (Kumar et al. 2005). 10.3.2 Localized or Targeted Mutagenesis Although there is no distinct difference between localized and targeted mutagenesis, localized mutagenesis is more focused on saturation of a particular chromosomal region in a way similar to that discussed in the preceding text for localized saturation mutagenesis while the main aim of targeted mutagenesis is to identify specific genes. The utility of the Ac/Ds system for localized insertional mutagenesis in Arabidopsis has been demonstrated by several studies (Long et al. 1997; Dubois et al. 1998; Ito et al. 1999, 2002; Muskett et al. 2003) and has now been extended to rice (Upadhyaya et al. 2006). Targeted transposon mutagenesis was first developed in Drosophila as a means of isolating mutants associated with a cloned gene (Kaiser and Goodwin 1990). The first successful example of using this approach in plant gene identification was the isolation of the tomato fungal resistance gene Cf-9 using a Ds located 3 cM away (Jones et al. 1994). The Ac/Ds system has also been successfully employed in a targeted tagging strategy where the FAT ACID ELONGATION1 gene was targeted and cloned using Ac as a molecular tag (James et al. 1995). In rice, a large number of genes of interest have been mapped based on QTL analysis or other methodologies. With the availability of the whole rice genome sequence, these genes of interest could be isolated by map-based cloning approaches, but this is a time-consuming process. A straightforward strategy for cloning these target genes is to use targeted transposon mutagenesis. In this strategy, an insertion line with the transposon insertion genetically linked to the gene of interest is retrieved from the insertion mutant libraries and crossed with a line containing transposase to generate multiple mutant alleles based on the fact that most Ds transpositions occur


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in genetically linked positions. Large populations of F2 or subsequent generations are then screened for mutant phenotypes tightly linked to the transposon insertion. In Arabidopsis, six independent Ac insertion alleles of DETERMINATE INFERTILE1 were generated from the same donor T-DNA by targeted mutagenesis (Bhatt et al. 1996). Seedling vigor has been selected as a trait for targeted mutagenesis in rice by CSIRO researchers (N.M. Upadhyaya et al., unpublished data). To do this, lines with Ds/T-DNA launch pads in the vicinity of previously identified seedling vigor QTLs are supertransformed with Agrobacterium harboring an Ac construct to induce Ds transposition. The DtT1 generations are being screened for seedling vigor mutants.

10.4 Transposon Insertional Mutant Populations Several groups are developing large-scale transposon insertional mutagenesis populations in rice using the one-element Ac system (Enoki et al. 1999; Greco et al. 2001a), the two-element Ac/Ds (Chin et al. 1999; Upadhyaya et al. 2002, 2006; Greco et al. 2003; Ito et al. 2004; Kim et al. 2004; Kolensik et al. 2004; Szeverenyi et al. 2005; van Enckevort et al. 2005) or Spm/dSpm systems (Greco et al. 2004; Kumar et al. 2005). The available transposon insertion populations and the rescued rice genomic sequences flanking transposon insertions are listed in Table 10.3. Table 10.3. Available transposon mutant populations Country Variety (institution)

Tagging system

Population No. of size FSTs

Approach for FST rescue

Reference

Australia

Ac/Ds

17,000

1,000

TAIL-PCR, plasmid rescue, adapter ligation PCR

Upadhyaya et al. 2002, 2006

China

Zhonghua 11 Ac/Ds

>5,000

na

Na

Xue et al. 2003

European Union

Nipponbare, Ac Bengal and Ac/Ds Pusa Basmati Spm/dSpm

>10,000

>5,000

TAIL-PCR or van Enckevort Adapter ligation et al. 2005 PCR

Korea

Dongjin

Ac/Ds

98,000

11,386

TAIL-PCR

Chin et al. 1999; Kim et al. 2004

Singapore

Nipponbare

Ac/Ds

23,000

3,000

TAIL-PCR

Kolesnik et al. 2004

United States

Nipponbare

Spm/dSpm Ac/Ds

10,500

7,400

TAIL-PCR or Kumar et al. Adapter ligation 2005 PCR

Nipponbare

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10.4.1 CSIRO Plant Industry Population At CSIRO, the Ds insertion populations are produced by crossing iAc and Ds (enhancer or gene trap) transgenic lines generated by Agrobacteriummediated transformation. Alternatively, cotransformation of iAc and Ds vectors or supertransformation of calli derived from Ds launch pads with the iAc vector is also used to produce mutagenized populations. Stable Ds – + insertion lines (iAc Ds ) with transposed Ds from subsequent generations are screened by either PCR analyses or via selectable markers, depending on the constructs used. The initial constructs used (pSK100 and pSK200) have a nonfunctional nptII (a Ds reinsertion marker) and hence screening for the presence of Ds and the absence of Ac required Ds- and Ac-specific PCR analyses, making the screening process very laborious and time consuming (Upadhyaya et al. 2002). To increase the screening efficiency, both iAc and Ds constructs have been modified to incorporate selectable and/or visual markers. In the pNU393A1/B2 construct, hygromycin and Basta resistance genes are used as Ds excision and reinsertion markers, respectively. In the iAc construct, pNU400 the GFP gene (sgfpS65T) is used as visual marker. Identification of plants with stable Ds insertions in the resulting screening population relies completely on these selectable and visual markers and PCR analyses are performed only for definite confirmation. Further improvement has been made to the Ds construct (pNU435) by the incorporation of the counterselective gene, barnase, that is under the control of the strong ubiquitin promoter upstream of the RB. With a RB-LB-RB-LB direct T-DNA repeat integration, the barnase gene will be expressed to kill the transformed cells containing this type of T-DNA repeat. CSIRO researchers are now focusing on producing single-copy T-DNA insertion lines or Ds launch pads that are evenly distributed in the rice genome using this Ds construct for localized mutagenesis and traittargeted mutagenesis via the TET system (Fig. 10.5; Upadhyaya et al. 2006). To date, approximately 1,000 Ds launch pads (LPs) have been produced, approximately 350 of these are single-copy Ds/T-DNA LP lines and approximately 100 of these single copy lines have had their FSTs mapped (Upadhyaya et al. 2002, 2006; see http://www.pi.csiro.au/fgrttpub for updates). Approximately 17,000 stable Ds insertion lines have been generated by crossing, co- or supertransformation and the majority of these are gene trap lines. Ds flanking sequences of these lines are being rescued by TAIL-PCR, adapter ligation PCR or plasmid rescue and approximately 700 FSTs have been deposited in public databases. Phenotyping has been performed for approximately 1,500 stable Ds insertion lines under normal glasshouse conditions and approximately 30% of lines show visible mutant phenotypes including late germination, defective shoot apex formation, low seedling vigor, seedling lethality, dwarfism, variegated or twisted


DtT0/F1

Single copy Ds (with hpt as Ds excision marker and bar as Ds reinsertion marker) callus lines from primary transformants or heterozygous T1 seeds (BastaR), co-transformtion with iAc binary vector (with gfp as visual marker)

Regeneration DtT0 GFP-, BastaR, HygR (stable insertion lines) Confirmation by PCR FST rescue, progeny analysis, phenotyping

DtT1/F2 GFP+/ GFPBastas

Eliminated

GFP+ BastaR

Repeat segregation analysis (DtT2/F3)

GFP+, BastaR, HygR

GFP-, HygS BastaR

Confirmation as SI lines unlinked to LP by PCR

Supertransformation (iAc Ds or Ds iAc)

GFP-, HygR BastaR

Confirmation as SI lines linked to LP by PCR

Transiently expressed transposase Ds excision (HygR) and reinsertion (BastaR) selection

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DsE/DsG X iAc

Fig. 10.5. Strategy for generating and screening stable Ds insertion lines by transiently expressed transposase (TET) system (shaded), crossing or double transformation (unshaded).

leaves, early or late flowering, partial or complete sterility, deformed spikelets, and small seeds. An analysis of 350 stable Ds insertion lines has shown that 15% and 70% of these lines expressed the GUS reporter gene in leaves and spikelets, respectively (Q.-H. Zhu et al., 2006b). Phenotyping is also being performed under field conditions. 10.4.2 EU (Wageningen) Population Both Ac one-element and Ac/Ds or Spm/dSpm two-element systems have been employed to develop transposon mutagenized populations. In the oneelement system, the Ac element is inserted between the CaMV 35S promoter and the gfp gene so that the expression of GFP is restored on excision of the Ac element (Greco et al. 2001a). In the Ac/Ds and Spm/dSpm twoelement systems, the immobilized Ac or Spm element driven by the CaMV 35S promoter is constructed in the same binary T-DNA vector as the Ds or dSpm element, in which bar is used to monitor and trace the mobilization of the nonautonomous element (i.e., Ds or dSpm) (Greco et al. 2003, 2004). The T-DNA construct also contains a negative selection maker (a cytochrome P450 gene, SU1, which converts the pro-herbicide 7042 into a cytotoxic form) for the transposase gene (i.e., Ac or Spm). Using this construct, transposition of the Ds or the dSpm element could occur directly after transformation in the transgenic calli or in the regenerating T0 plants. Theoretically stable transposants can be selected simply by application of Basta and R7042. The use of bar and SU1 genes as positive and negative selection markers seems to be highly efficient for screening in Arabidopsis (Tissier

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et al. 1999), but in rice only the bar gene has proven to work efficiently, while the effectiveness of SU1 is still remains to be determined. A core collection of 58 Ac/Ds T0 lines has been used to develop 1,421 T1 plants, from which more than 200,000 T2 seeds have been produced. Nearly 10,000 T2 plants have been analyzed in detail. In addition, more than 3,000 Ac lines that showed high frequency of Ac transposition have also been generated (van Enckevort et al. 2005). Transposon FSTs are isolated by TAIL-PCR or adapter ligation PCR. About half of the PCR products generated were of good quality as revealed by sequencing. After BLAST searching, it was found that 59% of the transposons inserted in annotated genes, while the remaining insertions were in intergenic regions. The mapping information of all these FSTs can be found in the database, OryGenesDB (http://orygenesdb.cines.fr/) and insertion lines are publicly available. 10.4.3 National University of Singapore Population Generation of Ds Insertion Lines

A two-element Ac/Ds gene trap system was used to generate a large collection (more than 20,000 lines) of stable, unlinked single-copy Ds transposants in rice (O. sativa ssp. japonica cv. Nipponbare). An immobilized Ac under the control of the CaMV 35S promoter was used to generate transposase R lines. The nonautonomous Ds element containing the bar gene (Basta ) as a transposition marker and a modified promoter-less uidA gene encoding β-glucuronidase as a reporter gene was transformed into rice to obtain Ds parental lines. The synthetic green fluorescence protein (sgfp, Chiu et al. 1996) and the enhanced yellow fluorescent protein (eyfp, Clontech, Mountain View, CA) genes both under the control of maize ubiquitin promoter were used as counterselection markers for Ac and Ds/T-DNA launch pads, respectively. Frequency and Timing of Transposition

Different cross combinations of homozygous Ac and Ds starter (parental) lines were used to establish the collection of Ds insertion lines. Altogether 4,413 F2 families were analyzed for transposants and the results showed an average germinal transposition frequency of 51%. Study of Ds transposition pattern in siblings of several F2 families revealed that 79% had at least two different insertions, suggesting late transposition during rice development, resulting in several independent single copy Ds lines within a family (Kolesnik et al. 2004). Further analysis on the timing of transposition during rice development (by analyzing possible footprints with reciprocal PCRs among siblings) showed that the independent events among siblings were due to primary transposition events. This analysis provided evidence that Ds transposed late after tiller formation (Szeverenyi et al. 2006).


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Stability of Parental and Transposed Lines

Several reports on Ac/Ds transposon mutagenesis showed that both starter lines and stable transposants become silenced in later generations, which cast doubts on the applicability of this approach for large-scale mutagenesis. Systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare) was carried out to show the stability of Ds through progressive generations. The high somatic and germinal transpositional frequencies observed in earlier generations were maintained as late as T4 and T5 generations indicating that the propagation of such parental lines did not induce transposon silencing. The stably transposed Ds was active even after the F5 generation as it could be remobilized (as shown by footprint analysis of several revertants). Apart from these, in more than a thousand stably transposed Ds lines, the bar gene expression was examined from F3 to F6 generations and notably substantial transgene silencing was not observed in the lines tested (Szevernyi et al. 2006). Chromosomal Distribution of Ds Insertions

The Ds flanking sequences of 2,057 putative transformants were obtained by TAIL-PCR and sequencing. Analysis of these sequences showed that 88% were unique. The remaining insertions were within the T-DNA with ~4% inserted in the resident negative selection marker, the gfp gene. Further analysis of the flanking sequences by BLAST search and annotation using Rice Genome Program’s Rice GAAS annotation program (http://ricegaas.dna.affrc.go.jp/rgadb/) revealed their distribution throughout the genome but with a bias (approximately twofold) toward chromosomes 4 and 7. Further, anchoring of more than 800 insertions to a YACbased EST map suggested preferential transposition of Ds into regions rich in expressed sequences (Kolesnik et al. 2004). 10.4.4 Korea Population An Ac/Ds-Mediated Gene Trap System

Ac and Ds were separately introduced into a japonica rice cultivar, Dongjin, via an Agrobacterium T-DNA vector. As Ac and Ds starter lines containing a single copy of Ac or Ds were selected and maintained. The Ac/Ds-based gene trap system consisted of three genetic components: Ac, gene trap Ds (DsG), and a counterselective marker. Ac cDNA was used as the transposase source that was under the control of a CaMV 35S promoter (Chin et al. 1999). The bar gene and uidA coding region were oriented so as to be transcribed from either end of Ds toward the middle of the element.

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The intron used in the DsG construct was the same as that used in Arabidopsis, i.e., the 4th intron of the Arabidopsis G-protein (GPA1) gene (Sundaresan et al. 1995). In rice, fusion of the uidA gene with a host gene was achieved by utilizing three out of four putative splicing donor sites at the 3΄ end of Ds and two out of three putative splice acceptor sites at the 5΄ end of uidA coding region (Chin et al. 1999) A modified bacterial cytochrome P450 gene was used as the counterselective marker in this system. Although cytochrome P450 was successfully used for negative selection (O'Keefe et al. 1994; Tissier et al. 1999), this group found that cytochrome P450/R4702 is not a reliable marker to screen a large population of rice. Germinal Transposition Rates in F2 Progeny and the Limitation of Genetic Crosses for a Large-Scale Mutagenesis

Single-copy Ac and Ds starter lines were crossed to assess the germinal transmission frequency of Ds. More than 10,000 F2 plants were individually analyzed via Southern blot analysis. The overall frequencies of independent germinal transposition in two F2 populations were 10% to 15% (Kim et al. 2004). With the repeated use of the same starter lines maintained by selfing, the frequency of germinal transposition of Ds in the F2 generation decreased. Therefore, the extent to which the use of genetic crossing contributes to the development of a highly saturated insertional mutant population depends largely on the availability of effective selectable markers for large-scale screening. High Proportion of Independent Ds Transposants in a Population of Regenerated Plants

To overcome the dependence on marker genes and the ongoing monitoring of Ac/Ds activity, plant regeneration was adapted as a Ds-mediated genetagging strategy. Ds transposition was analyzed by Southern blot analysis in more than 2,000 R1 plants derived from callus culture of seeds carrying both Ac and Ds. From 70% to 80% of regenerated plants carried new Ds insertions (Kim et al. 2002). Only 10% to 20% of the population carried Ac alone and/or was devoid of Ds (Basta sensitive). Monitoring of the transmission of Ds in R2 plants indicated that Ds elements of R1 plants were stably maintained in the subsequent generation. Also, most of the regenerated plants from any one callus culture carried different Ds insertions. The data showed that the majority of regenerated plants carried independently transposed elements. Therefore, rapid generation of a large Ds transposant population could be achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements obviating the need for any elaborate screening for transposed Ds.


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Chromosomal Distribution of Ds

For mass production of Ds FSTs, TAIL-PCR was primarily employed. The primer sets for amplification of the 5΄ or 3΄ end of Ds and optimal AD (arbitrary degenerate) primers sets were described by Kim et al. (2004). FSTs were mapped on rice pseudomolecules version 4 (http://www.tigr. org). The patterns of Ds distribution were very similar among several populations derived either by genetic crossing or tissue culture. Ds transposed to all chromosomes with preference near donor sites and some physically unlinked arms. Table 10.4 shows the chromosomal location of transposed Ds elements. The relatively high proportion of Ds elements in chromosomes 3 and 4 resulted from the locations of original donor sites in these chromosomes. Generation of Ds Population and FST Analysis

Owing to the nonrandom distribution of insertion loci, it is essential to create an evenly distributed population of original Ds elements throughout the rice genome for random mutagenesis. Using several Ac and Ds starter lines that were distributed on different chromosomes, a large-scale regeneration population has been developed. From 2001 to 2005, a total of 98,000 regeneration lines were developed. Because 70% to 80% of the population carried a transposed Ds, 73,000 lines are expected to carry independent Ds insertion events. To build up the database of FSTs, 11,386 Ds insertion sites were mapped on rice chromosomes. This material and FST data will be publicly accessible via http://genebank.rda.go.kr/dstag. Table 10.4. Chromosomal distribution of 11,386 Ds insertion sites Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 Un-mapped BAC Total mapped Ds

No. of Ds insertion sites 1,295 780 3,949 1,233 502 475 613 433 385 712 383 452 174 11,386

Proportion (%) 11.4 6.9 34.7 10.8 4.4 4.2 5.4 3.8 3.4 6.2 3.4 4.0 1.4 100

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10.4.5 UC Davis Population The UC Davis insertion lines are based on using the maize Spm/dSpm and Ac/Ds elements for large-scale genome-wide random insertional mutagenesis in japonica cv. Nipponbare. A complete description of this system utilizing the dSpm element has been published (Kumar et al. 2005). In this system insertion lines are generated using a single T-DNA vector carrying an immobilized Spm or Ac transposase gene as well as the corresponding nonautonomous transposon dSpm or Ds in cis. To track the presence of mobile dSpm or Ds elements in the plants, these elements are equipped with a positive selection marker, the DsRed gene that confers red fluorescence. The DsRed marker gene has been shown to work efficiently with no escapes recorded (i.e., all of the selected red fluorescent plants carried the Ds or dSpm element). The sgfp is used as the negative selection marker to select for unlinked transposition events and to select against the Spm or Ac transposase (Chiu et al. 1996) in the T-DNA. The use of a combination of fluorescent protein marker genes (sgfp and DsRed) as negative and positive selection markers enables quick and easy identification of insertion lines from germinated seedlings. The strategy for generating insertion lines using the aforementioned system is shown in Fig. 10.6A. Primary transformants (T1) carrying single cus/copy T-DNA, expressing gfp and DsRed genes are selected as starter lines. From these starter lines T2 heterozygous progeny are identified based on the GFP fluorescence levels. They are then propagated and allowed to self-pollinate to obtain T3 seeds. Finally the screening for the insertion lines + is carried out in the T3 seedlings (4 to 7 days old) by selecting GFP DsRed seedlings (Fig. 10.6B). The dSpm or Ds flanking sequences from the T3 insertion lines are recovered either by TAIL-PCR or by adapter ligation PCR. The flanking sequences are submitted to GenBank, and also maintained in a searchable FST Database (http://sundarlab.ucdavis.edu/rice/blast/blast.html). With the Spm/dSpm system, the frequency of presumptive unlinked transpositions of the dSpm element is about 45% to 50% (measured as the + percentage of T3 families with at least one GFP DsRed seedling), and that for the Ac/Ds system is approximately 40% to 45%. So far, this group has generated more than 3,500 dSpm insertion lines and sequenced FSTs from 1,800 lines. Using the Ac/Ds system 7,000 insertion lines have been generated and 5,600 FSTs have been sequenced. Analysis of dSpm and Ds flanking sequences revealed that both dSpm and Ds preferentially insert into genes or genic regions. The frequency of insertion within the T-DNA is less than 3% for the dSpm element, while for the Ds element it was about 12%. Further study also indicated that the transposition of dSpm element occurs relatively late in development hence multiple independent insertion lines can be recovered from a single T2 heterozygous parent.


A

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1 Single copy transformant (T1) Selfing ~400 T2 Seeds Screening for Heterozygous (GFP fluorescence levels) ~200 T2 Heterozygous plants Selfing 200 T3 families Screening for stable transposants (GFP- & DsRed+) ~80 Transposants (40% frequency)

B

Maintenance of dSpm or Ds insertion lines FST sequencing & Database

Fig. 10.6. (A) Strategy for generating dSpmTab or Ds insertions by in cis strategy. (B) Screening for stable transposants by GFP and DsRed fluorescence. White arrows indicate a putative stable transposant which is GFP – (left) and DsRed + (right) (See also color plate section).

The dSpm insertions appear to differ from Ds elements in genomic distribution and exhibit a greater fraction of unlinked transpositions when compared to Ds elements. The results suggest that Ds and dSpm elements may exhibit different preferences for insertion in the rice genome, and hence different genome coverage is likely to be achieved using these elements. The insertion mutant population carrying the dSpm elements can complement other existing mutagens such as Tos17, T-DNA, and Ac/Ds and fill gaps left by these elements in the rice genome. Further, as this approach uses fluorescent protein markers that can be potentially automated – + for the fluorescent sorting of GFP and DsRed seedlings, this seems to be an ideal system for high-throughput insertion line production. The seeds from rice dSpm and Ds insertion lines generated at UC Davis are publicly available (http://sundarlab.ucdavis.edu/rice/blast/blast.html).

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10.5 Gene Discovery by Transposon Tagging 10.5.1 Forward and Reverse Genetics Strategies In the previous sections, different gene tagging systems for generating a large population of transposon insertion mutants are discussed. Compared with other mutagenesis approaches, transposon mutagenesis has several advantages and disadvantages (Table 10.5). To identify tagged genes in transposon mutagenized populations, forward and reverse genetics strategies are currently being employing (Fig. 10.7). Forward genetics is a traditional strategy that has been used successfully for many years and is aimed at cloning genes that have been identified by a mutant phenotype or function. This approach is straightforward but relies on the identification of visible mutant phenotypes. In contrast, reverse genetics starts with the gene of interest and aims to determine the function of the gene by generating and analysing the phenotype(s) in the corresponding knockout mutant. The prerequisite of an efficient reverse genetics system is that it should be possible to determine the presence and absence of a knockout mutant of a gene of interest in the mutagenized population, which is particularly important as gene knockouts might not lead to an easily identifiable phenotype for the majority of genes. With the forward genetics strategy, new genes can be identified without prior knowledge of the identity of the gene or the gene product. In rice four genes have been identified by transposon mutagenesis using a forward genetics approach (Table 10.6). This strategy can also be used in trait-targeted mutagenesis as discussed in Section 10.3.2. To carry out trait-orientated screening, not only does a large mutagenized population need to be generated, but careful observation and analysis are required as the deleterious effects of a given mutation are often difficult to detect. Once a mutant is identified, plant genomic sequences flanking the transposon can be isolated by iPCR, TAIL-PCR, adapter ligation PCR or plasmid rescue. The subsequent gene cloning process is now relatively straightforward in rice owing to the availability of the entire genomic sequence. In the case of reverse genetics strategy, two approaches are employed to find knockout mutants by screening the mutagenized population. One can randomly amplify and sequence transposon insertion flanking sequences, or specifically screen for insertions in genes of interest. The PCR and plasmid rescue methods mentioned above are only efficient for isolation of transposon FSTs from single- or low-copy-number insertion lines. PCRrelated techniques such as transposon display (van den Broeck et al. 1998) and amplification of insertion-mutagenized sites (AIMS; Frey et al. 1998) have been successfully used to isolate the transposon FSTs in highcopy-number insertion lines. In rice, the most frequently used methods are

Loss-of-function (chemical induced: point mutation; physical induced: deletion mutation); Natural

Stable

Forward and reverse genetics

Map-based cloning, TILLING

N/A

Complementation Additional alleles

Impossible

Stability of mutation

Strategy of gene discovery

Method of gene cloning

Co-segregation analysis

Functional confirmation

Targeted or localized mutagenesis

Chemical and physical agent Very easy

Type of mutation

Generation of the mutagenized population

Mutagen

Impossible


Enhanced by selectable markers but deteriorated by somaclonal variation and complicated T-DNA integration

FST rescue


Stable

Loss-of-function or gain-offunction (with activation tagging, it makes possible to clone genes whose knockout mutant is lethal); Transgenic

Transformation and largescale tissue culture

T-DNA

Possible

Complementation Additional alleles Revertants

Enhanced by selectable markers but deteriorated by excision footprints of transposon

FST rescue


Stable but unstable for the mutations induced by the autonomous elements

Loss-of-function or gainof-function; Transgenic

Transformation but only need relatively small number of starter lines

Transposon

Table 10.5. Comparison of mutagenesis methodologies for gene discovery

Impossible


No selectable markers available and deteriorated by somaclonal variation and multiple copies of the retrotransposon

FST rescue


Stable but un-stable under tissue culture

Loss-of-function; Natural

Large-scale tissue culture

Retrotransposon

10 Transposon Insertional Mutants 257

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Forward genetics Transposon insertion population

FST rescue

Mutants discovered by phenotyping

Database search

Co-segregation analysis of transposon and the mutant phenotype using transposon as a probe Rescue transposon flanking sequence and confirm co-segregation relationship

Insertion in interested gene

Interested gene PCR screening transposon insertion population using gene-specific and insert-specific primers Find insertion line

Identify homozygous insertion plants and investigate phenotypic changes Co-segregation analysis of the insert and the mutant phenotype using gene specific probe

Gene cloning Confirmation by complementation and/or additional alleles

Fig. 10.7. Application of forward and reverse genetics strategies in gene identification using a transposon mutagenized population

TAIL-PCR and iPCR. To determine transposon insertion flanking sequence from single-copy lines, PCR-amplified products can be directly sequenced. In multiple-copy-number lines, the amplification products derived from different insertion sites are resolved on sequencing gels, isolated, reamplified and sequenced individually. Several systematically catalogued databases of transposon FSTs have been established for rice in different laboratories around the world (Upadhyaya et al. 2002; Greco et al. 2003; Kolesnik et al. 2004; Kumar et al. 2005; Szeverenyi et al. 2005; van Enckevort et al. 2005). These databases will significantly facilitate gene identification in rice. With the mapping information of the mutation, the genomic sequence around the mutation is retrieved and annotated to pinpoint the candidate genes that are likely to be affected in the mutant. The lines containing transposon insertions in these genes are then retrieved from the insertion mutant libraries. The knockout phenotypes can then be examined in the homozygotes (Maes et al. 1999). To screen transposon insertion mutants of a specific gene, PCR-based strategies can be used to identify mutants through amplification of a PCR product using gene- and insert-specific primers. Usually, insertion lines are identified using DNA pools containing many insertion lines. The sensitivity of the PCR technique, especially after hybridization of the PCR products with a genespecific probe, allows the easy detection of a single gene hit within a pool of hundreds or thousands of individuals. Screenings of DNA pools are generally organized in a three-dimensional array, to allow easy identification of the tagged individuals.


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Table 10.6. Genes discovered by forward genetics approach Gene name BFL1a

Tagged by

Mutant phenotype

Putative function

Reference

Ds

An AP2 domain transcription factor mediate the transition from spikelet to floret meristem

Zhu et al. 2003

FZPa

Ac

The formation of florets is replaced by sequential rounds of branching as several rudimentary glumes are formed in each ectopic branch and axillary meristems are formed in the axils of rudimentary glumes. The panicle is seedless. As above

As above

AID1

Ds

Komatsu et al. 2003a Zhu et al. 2004

OsKS1

Ds

OsNOP

Ds

Anther indehiscence and partial to complete spikelet sterility Sever dwarfism, dark green leaf and failure to initiate reproductive growth Pollenless and male sterility

A single MYB domain gene functions at late stages of anther development Encoding entkaurene synthase catalyzing the second step of the gibberellin biosynthesis Containing C2GRAM domain and functioning during late stage of pollen development and its germination by cross-linking both calcium and phosphoinositide signaling pathways.

MargisPinheiro et al. 2005 Jiang et al. 2005

a

bfl1 and fzp are alleles

10.5.2 Other Approaches for Mutation Identification The limitation of the aforementioned approaches of screening DNA pools is that only one or a small number of genes can be screened for at once. To enhance the utility of transposon insertional libraries approaches that allow DNA pools representing many lines to be screened for insertions in many genes at once are desirable. To this end, Mahalingam and Fedoroff (2001) have developed a microarray-based method to screen DNA pools from multiple transposon lines for simultaneous detection of insertions in

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different genes. In this approach, transposon FSTs are amplified preferentially by TAIL-PCR and hybridized to a cDNA microarray; FSTs that overlap genes represented on the microarray will hybridize with their respective cDNAs, thereby identifying genes containing insertion mutations in or near these genes (Mahalingam and Fedoroff 2001). It has been shown that microarray hybridization of TAIL-PCR amplified FSTs can detect individual Arabidopsis Ds insertion lines from a DNA pool comprised of as many as 100 lines. But this approach is likely to favor the identification of insertions in or very close to genes because the Ds insertions tend to cluster around the translational start site (Parinov et al. 1999). Moreover, TAIL-PCR products tend to be short. A tagged transcriptome display (TTD) strategy has been developed in rice to detect the transposon insertions located in transcribed sequences (Kohli et al. 2001). In this approach, a CpG methylation-sensitive enzyme such as SalI is used to preferentially cut rice genomic DNA in transcriptionally active chromosomal regions. The transposon (Ac) FSTs are then amplified by adapter ligation PCR, blotted onto a membrane and hybridized with labeled leaf cDNA to reveal insertions in transcribed genes specifically expressed in leaf. This strategy can be used not only to detect transposon insertions in specific tissues, but also in genes that are transcribed in response to particular biotic and abiotic stresses (Kohli et al. 2001). For maximum efficiency, more than one methylation-sensitive enzyme should be used for a given line to maximize the recovery of all potential gene-tagged transposon insertions. 10.5.3 Tagging Efficiency Transposon tagging has been proven to be a powerful tool for functional genomics in plants. To increase tagging efficiency, insertions within exons are preferred as transposons may be spliced out when they insert within an intron. Most studies have shown that Ac/Ds transposes into gene coding regions (Enoki et al. 1999; Greco et al. 2001a; Kolesnik et al. 2004), but it seems that exons and introns are equal targets for transposon insertion (Kolesnik et al. 2004; Q.-H. Zhu et al. 2006b). Cases have been reported in which a phenotype is not linked to the Ds element. One possible explanation for this is that the Ds element transposes more than once in the F1 (DtT0), or in subsequent generations in the presence of the Ac transposase, leaving footprints in a coding sequence and thereby altering the reading frame to result in a mutated gene product. This is most likely the case of the csl1 (compact shoot and leafy head 1) mutant, in which all primary branches of the panicle are converted to vegetative plantlets (Q.-H. Zhu et al. 2006b). Other background mutations that induce genetic and epigenetic changes may be induced by tissue culture. Particular


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attention therefore needs to be paid when performing double transformation as background mutations may be introduced by both tissue culture and secondary transposition during cocultivation of Ac and Ds vectors. It has also been reported that transposons can create large chromosome deletions on mobilization. In the case of Osnop mutant, a deletion of 65 kb of genomic DNA containing 14 genes together with 3.8 kb of the 5΄ Ds element itself was found at the Ds insertion site (Jiang et al. 2005). The exact mechanism of such a deletion is not clear, however, endogenous repetitive sequences of Ds interacting with the transformed Ds resulting in unequal homologous recombination events might be the causal factor (Page et al. 2004). As described in the preceding text, gene and enhancer trap systems allow the identification of genes and regulatory elements that are not amenable to classical genetic analysis. Hence, novel genes are likely to be identified in such trapped lines. This approach has been very successful in Arabidopsis (Springer 2000), but in rice no gene or enhancer has so far been found using these trap systems in the transposon mutagenesis populations. 10.5.4 Confirmation of Tagged Gene After establishment of the cosegregation relationship between the mutant phenotype and a transposon insertion, the simplest and most straightforward way to confirm that the mutant phenotype is the result of a mutation due to transposon insertion, is to check whether there are other alleles that have been independently identified. With transposon-tagged mutations, it is also possible to generate more alleles or revertants by crossing the mutant with a transposase-expressing line. Both will provide additional evidence that the tagged gene is responsible for the mutant phenotype. Complementation with the wild-type copy of the tagged gene is another standard but labor-intensive procedure for confirmation. Another way to confirm the relationship between the mutant phenotype and a transposon insertion is to use RNAi to mimic the knockout phenotype.

10.6 Future Prospects Transposon-induced phenotypic changes can provide strong evidence for the biological function of a gene. Substantial populations with transposon insertions have been established in rice, but a great deal of further work is required to achieve saturation mutagenesis. Localized mutagenesis will play an important role toward the achievement of this goal. More importantly, it is now the time to shift our focus to serious and systematic phenotyping using forward or reverse genetics approaches. The challenge is to

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develop sophisticated screening systems for the identification of phenotypes of transposon-induced mutations. Conditional and/or customized phenotyping will also be required, since the essential function of a large number of genes may not be revealed under normal growth conditions. Transposon mutagenesis, together with other functional genomics tools, will ultimately help us understand the function of the more than 40,000 rice genes, and their interactive networks.

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