A Versatile Transposon-Based Activation Tag Vector System for Functional Genomics in Cereals and Other Monocot Plants^,

Abstract

Transposon insertional mutagenesis is an effective alternative to T-DNA mutagenesis when transformation through tissue culture is inefficient as is the case for many crop species. When used as activation tags, transposons can be exploited to generate novel gain-of-function phenotypes without transformation and are of particular value in the study of polyploid plants where gene knockouts will not have phenotypes. We have developed an in cis-activation-tagging Ac-Ds transposon system in which a T-DNA vector carries a Dissociation (Ds) element containing 4× cauliflower mosaic virus enhancers along with the Activator (Ac) transposase gene. Stable Ds insertions were selected using green fluorescent protein and red fluorescent protein genes driven by promoters that are functional in maize (Zea mays) and rice (Oryza sativa). The system has been tested in rice, where 638 stable Ds insertions were selected from an initial set of 26 primary transformants. By analysis of 311 flanking sequences mapped to the rice genome, we could demonstrate the wide distribution of the elements over the rice chromosomes. Enhanced expression of rice genes adjacent to Ds insertions was detected in the insertion lines using semiquantitative reverse transcription-PCR method. The in cis-two-element vector system requires minimal number of primary transformants and eliminates the need for crossing, while the use of fluorescent markers instead of antibiotic or herbicide resistance increases the applicability to other plants and eliminates problems with escapes. Because Ac-Ds has been shown to transpose widely in the plant kingdom, the activation vector system developed in this study should be of utility more generally to other monocots.

Genetic mutants have always played a central role as tools for functional analysis of plant genes. Many plant genes have been isolated by the strategy of insertional mutagens. In the model plants of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), large-scale T-DNA and transposon insertion libraries and flanking sequence tag (FST) databases have been generated, which serve the plant biologists worldwide for both forward and reverse genetics studies (Parinov et al., 1999; Tissier et al., 1999; Jeon et al., 2000; Ito et al., 2002; Kuromori et al., 2004; Ito et al., 2005). Transposon insertional mutagenesis is an effective alternative to T-DNA mutagenesis when transformation through tissue culture is inefficient, as is the case for many crop species. The strategy for transposon mutagenesis requires just a limited number of primary transformants, with insertions being generated through propagation.

On the other hand, although the classic insertional mutagenesis strategies play an important role in plant functional genomics studies, a limitation of such methods is the difficulty of identifying genes that are redundant in plant genomes and whose knockouts do not induce phenotypes. Activation tagging is an effective approach for overcoming this limitation. Activation tagging involves introduction of a T-DNA-containing regulatory sequence such as the enhancer of the cauliflower mosaic virus (CaMV) 35S promoter randomly into a plant genome to enhance expression of nearby genes, which potentially resulted in a gain-of-function phenotype (Kardailsky et al., 1999; Borevitz et al., 2000; Weigel et al., 2000; Jeong et al., 2002; Mathews et al., 2003; Jeong et al., 2006; Mori et al., 2007; Hsing et al., 2007).

As for the functional genomics of cereals and monocot plants, maize (Zea mays) transposable elements are useful tools for generating large collection of gene knockouts, because high-throughput T-DNA transformation is not a viable approach for those plant species. A number of maize genes have been isolated from transposon-tagged mutants. Several studies have shown that the maize Ac-Ds transposable elements function in barley (Hordeum vulgare), and mapped Ds launching pads were developed for the study of functional genomics (Koprek et al., 2000; Cooper et al., 2004; Singh et al., 2006; Zhao et al., 2006). Ayliffe et al. (2007) reported a novel Ac-Ds system in which a modified Ds element (UbiDs) carrying two maize ubiquitin 1 promoters (Christensen and Quail, 1996) was utilized for read-through transcription of adjacent flanking sequences.

We have been developing vectors using maize transposable elements for plant functional genomics. In rice, we previously developed an Ac-Ds insertional mutagenesis system in which a GFP fluorescence gene functioned as negative selection marker against the immobilized Ac and a Basta resistance marker worked for selection of transposed Ds elements (Kolesnik et al., 2004). In the development of an En/Spm-tagging system in rice, our transposition selection scheme was improved based on GFP and red fluorescent protein (RFP) double fluorescence markers (Kumar et al., 2005). In this study, we developed an activation tagging Ac-Ds in cis-system in which the advantage of GFP and RFP double fluorescence markers was utilized. The system was shown to be effective for distributing a large number of activation-tagging Ds elements in the rice genome. As the Ac-Ds has been shown to transpose widely in the plant kingdom, we anticipate the applicability of our activation vector system for studies in monocot plants, including other cereals.

RESULTS

Vector Construction and Production of Starter Lines

The T-DNA vector pSQ5 carrying a nonautonomous Ds element along with an immobilized Ac element in cis is shown in Figure 1. The immobilized Ac contains the Ac transposase gene under the control of the CaMV 35S promoter (Kolesnik et al., 2004). The Ds element has the 1,785-bp 5′ terminus and 222-bp 3′ terminus of the wild-type Ac element (Sundaresan et al., 1995). A tetramer of the transcriptional enhancer of CaMV 35S promoter and the Discosoma sp. RFP (DsRed) gene were cloned between the 5′ and 3′ Ds termini. The DsRed (or RFP) gene, encoding a RFP (Clontech; Baird et al., 2000), is driven by the maize ubiquitin 1 promoter (Christensen and Quail, 1996). The vector carries a hygromycin phosphotransferase gene for plant transformation selection. A synthetic GFP (sGFP) gene was cloned next to the immobilized Ac transposase source in the T-DNA. The GFP and RFP fluorescence markers make it possible to visually track the Ds element and the immobilized Ac element, respectively, in transgenic progeny.

Figure 1.

T-DNA of the activation-tagging Ac-Ds vector pSQ5. RB and LB, Right and left borders of the T-DNA; Ubi, maize ubiquitin 1 promoter; GFP, GFP gene as a negative selection marker; RFP, RFP (or DsRed) gene as selection marker for the Ds element; 4xEn, a tetramer of CaMV 35S enhancers; HPH, hygromycin phosphotransferase gene as plant transformation selection marker. The T-DNA is in the backbone of pCAMBIA-1300.

The pSQ5 Ac-Ds vector was introduced into rice sp. ‘Nipponbare’ via Agrobacterium tumefaciens-mediated rice transformation. Eighty fertile transformants that were double fluorescent (GFP⁺ and RFP⁺) were produced. In the T₂ generation, 50 seeds of each transformant were germinated and assayed for GFP and RFP. For GFP segregation, T₂ plants of 58 transformants showed a 3:1 ratio and therefore carried a single T-DNA locus in the rice genome. Eight transformants had multiple T-DNA integration loci based on the GFP segregation ratios (>3:1) of T₂ plants. The T₂ generation of 14 transformants did not have any plants showing GFP or RFP, possibly because of Ds transposition into the GFP gene or due to transgene silencing (see “Discussion”).

High Frequency of Germinal Transposition in T₁ and T₂ Transgenic Plants

Because the activation-tagging Ds element and the T-DNA-based Ac transposase can be tracked by RFP and GFP, respectively, we determined the GFP and RFP phenotype of each T₂ plant from 53 single-T-DNA-locus populations (Table I). An average of 48 T₂ plants in each population were assayed for GFP and RFP fluorescence. The phenotype of individual plants was determined as shown in Figure 2A. Four different phenotypes were observed in these T₂ populations. Because each T₂ population was generated from a single primary transformant (T₁) and in the T₁ generation the Ds element carrying the RFP gene can transpose, the RFP gene and the T-DNA-anchored GFP gene may have different chromosomal locations and may segregate in the T₂ plants (Fig. 2B). Therefore, it is likely that the GFP⁻RFP⁺ or GFP⁺RFP⁻ plants in the T₂ generation were derived from transposition events in the T₁ generation. In GFP and RFP assays of T₂ plants of 53 transformants (Table I), GFP⁻RFP⁺ plants were identified from T₂ populations of 23 transformants (nos. 19–41). These GFP⁻RFP⁺ plants indicated that 43.4% (23/53) of the T₁ transformants carried transposed Ds elements that were germinally transmitted to the T₂ generation. Among the 53 T₂ populations, 12 populations (nos. 42–53) had GFP⁺RFP⁻ plants but no GFP⁻RFP⁺ plants, which suggested that a fraction of excised Ds elements did not reinsert in the rice genome (see “Discussion”).

Table I.

GFP and RFP assays of 53 T₂ families

No.	Transformant	No. of T2 Plants	GFP+RFP+	GFP−RFP−	GFP+RFP−	GFP−RFP+
1	A3	50	38	12	0	0
2	A5	48	34	14	0	0
3	A14	50	39	11	0	0
4	A15	47	36	11	0	0
5	A28	57	42	15	0	0
6	A30	50	35	15	0	0
7	A39	50	33	17	0	0
8	A51	50	37	13	0	0
9	A52	49	34	15	0	0
10	A72	45	25	20	0	0
11	B4	49	36	13	0	0
12	B16	48	34	14	0	0
13	B30	49	33	16	0	0
14	B46	42	28	14	0	0
15	B49	45	28	17	0	0
16	B50	45	37	8	0	0
17	B52	50	38	12	0	0
18	B53	43	31	12	0	0
19	A35	50	36	11	2	1
20	A56	50	32	12	5	1
21	A57	47	29	11	6	1
22	A66	48	26	3	9	10
23	A68	50	24	9	13	4
24	A70	50	30	2	9	9
25	B9	50	26	11	10	3
26	B15	47	32	3	8	4
27	B17	48	28	9	4	7
28	B18	45	24	8	7	6
29	B23	49	29	12	6	2
30	B28	45	28	13	2	2
31	B31	49	36	12	0	1
32	B45	50	27	10	12	1
33	B47	47	29	7	3	8
34	B54	48	21	5	13	9
35	B62	47	30	10	6	1
36	B65	46	30	5	4	7
37	A13	31	23	1	1	6
38	A36	51	40	3	0	8
39	A37	78	56	9	0	13
40	A50	50	14	15	20	1
41	B36	49	38	5	0	6
42	A8	41	30	10	1	0
43	A34	48	37	10	1	0
44	A74	50	27	13	10	0
45	A83	45	23	11	11	0
46	B10	49	39	9	1	0
47	B12	49	27	15	7	0
48	B13	49	32	14	3	0
49	B24	47	34	12	1	0
50	B41	49	36	9	4	0
51	B48	19	13	5	1	0
52	B69	49	26	13	10	0
53	A55	49	10	9	30	0

Figure 2.

GFP and RFP fluorescence assay of young seedlings (T₂) derived from pSQ5 transgenic rice plants. A, GFP and RFP assays and the scheme to determine GFP and RFP phenotype of each T₂ plant. B, A diagram showing a transposition event in the primary transformant (T₁) and its germinal transmission to T₂ population.

To detect transposition in T₂ generation, we investigated T₃ populations of 37 transformants. For the 37 transformants, 18 transformants (Table I; nos.1–18) did not show transposition in T₂ generation, 14 transformants (Table I; nos. 19–21, 30–32, 35, 42–43, 46, 48–51) showed transposition, and T₂ families of five transformants (nos. 54–58) were not analyzed. The T₃ families of the 37 transformants were obtained by selfing heterozygous T₂ plants. As GFP heterozygous T₂ seedlings gave a lower level of GFP fluorescence than GFP homozygous seedlings (Kumar et al., 2005), we selected the heterozygous T₂ plants based on their lower GFP intensity. In this way we were able to reduce the T₂ homozygotes to 8.2% according to the results of GFP assays of T₃ generation (data not shown). In each T₃ family, 200 to 400 plants were assayed to identify GFP⁻RFP⁺ transposant plants.

For the numbers 1 to 18 transformants, at least one GFP⁻RFP⁺ plant was identified in T₃ families of 12 transformants (Table II; nos. 1–3, 6, 8–12, 14–15, 18). We were able to identify transposition events in the T₃ generation but not in their T₂ families, because just 42 to 50 plants from each T₂ family were analyzed (Table I; nos. 1–3, 6, 8–12, 14–15, 18). For the 14 transformants that showed transposition in the T₂ generation, at least one germinal transposition event was identified from T₃ families of 13 transformants (Table II; nos. 19–21, 30–32, 35, 42–43, 46, 48–51). In a total of 905 T₃ families derived from 37 primary transformants, germinal transposition events were identified in 295 (32.6%) families (Table II). These transposition events in the T₃ generation were derived from 30 (81.1%) of the transformants examined. Higher germinal transposition frequencies (20%–83.3%) were detected in the T₃ families of 19 single-T-DNA-locus transformants (Table II), which were among one-third (32.7%) of all 58 single-T-DNA-locus transformants.

Table II.

GFP and RFP assays of 905 T₃ families

No.	Transformant	T3 Families Tested	T3 Families with Germinal Transpositiona
1	A3	18	4 (22.2%)
2	A5	20	1 (5%)
3	A14	20	12 (60%)
4	A15	30	0
5	A28	10	0
6	A30	10	3 (30%)
7	A39	20	0
8	A51	20	3 (15%)
9	A52	20	1 (5%)
10	A72	6	5 (83.3%)
11	B4	30	10 (33.3%)
12	B16	10	2 (20%)
13	B30	20	0
14	B46	10	5 (50%)
15	B49	9	4 (44.4%)
16	B50	10	0
17	B52	10	0
18	B53	20	3 (15%)
19	A35	55	27 (49.1%)
20	A56	74	49 (66.2%)
21	A57	99	36 (36.4%)
30	B28	30	5 (16.7%)
31	B31	30	13 (43.3%)
32	B45	72	42 (58.3%)
35	B62	10	5 (50%)
42	A8	34	23 (67.6%)
43	A34	20	2 (10%)
46	B10	20	3 (15%)
48	B13	20	3 (15%)
49	B24	20	0
50	B41	24	13 (54.1%)
51	B48	10	3 (30%)
54	A29	21	1 (4.7%)
55	A62	10	5 (50%)
56	A73	30	10 (33.3%)
57	B3	23	1 (4.5%)
58	B76	10	1 (10%)
Total		905	295 (32.6%)

^aGerminal transposition was based on identification of ≥1 of GFP⁻RFP⁺ plant. In parentheses is percent of T₃ families containing germinal transposition.

Generation of Activation-Tagging Ds Lines of Rice and Isolation of Ds FSTs

We grew a total of 3,057 T₂ plants that were derived from 37 primary transformants. An average of 300 seeds (T₃) were produced from each T₂ plant. The GFP negative selection marker and the RFP positive selection marker were utilized to screen T₃ families to obtain putative stable transposants (GFP⁻RFP⁺ plants). Using the scheme as shown in Figure 3, we initially screened 905 T₃ families that were derived from the 37 transformants (Table II). In the next screening step, we did not continue with the T₃ families derived from transformants for which transposition frequencies in the T₃ generation were less than 10%. Instead, we focused on the 1,181 T₃ families of the 26 transformants that generated transposition frequencies in the T₃ of 10% to 83.3% (Table II). Among the 1,181 T₃ families, 343 families were found to have at least one GFP⁻RFP⁺ plant per family. Taken together, a total of 2,086 T₃ families were screened, and GFP⁻RFP⁺ transposants were selected from 638 (30.6%) of those families.

Figure 3.

Selection of stable Ds insertion lines and generation of Ds FSTs.

The putative stable transposants were subjected to adaptor-ligation PCR to determine Ds flanking sequence (Fig. 3). We analyzed 559 transposants that were derived from 26 primary transformants. A total of 463 sequences were obtained after adaptor-ligation PCR, sequencing, and filtration against the T-DNA sequence (to eliminate the Ds donor locus). In a Blast search against the Salk Institute Rice Functional Genomics database (http://signal.salk.edu/cgi-bin/RiceGE), 311 (67.2%) sequences were mapped on the rice chromosomes (Table III). The 152 remaining sequences were 73 (15.8%) redundant FSTs from siblings carrying the same insertions and 79 (17.1%) unmapped sequences due to insertions in rice repeat sequence regions. Our explanation for the redundant FSTs is that a small number of T₂ siblings carried the same Ds insertion due to transpositions in the T₁ parent (Table I), which was propagated by T₂ siblings to the T₃ generation (see “Discussion”). In further analysis of the FSTs, The Institute for Genomic Research rice genome database (http://www.tigr.org/tdb/e2k1/osa1/pseudomolecules/info.shtml) was searched for gene homology between the FSTs and rice cDNA sequences. There were 186 mapped FSTs with hits to rice cDNA sequences. Such FSTs were 40.2% (186/463) among the FSTs obtained and represented Ds insertions in the genic regions.

Table III.

Interchromosomal distribution of ds insertions derived from 26 transformants

Transformant	Rice Chromosomes												Total
	1	2	3	4	5	6	7	8	9	10	11	12
A62	9	7	9	4	5	3	3	4	4	3	3	7	61
A73	4	2	6	3	5	3	3	2	1	3	8	2	42
A14	8	3	5		6	3	2	1	1	3	2	2	36
A57	6	1	3	1	2	3	2	1	4	2	3	1	29
A35	4	2	1		2	2	3	1	1		1	3	20
B62	3		3	3			1	1	1	3	2	2	19
B28	5	1	2	2	3		1	3		1			18
A8	1	2	3	1	6		1	1	1	1			17
B4	1	3	4		2		1		3			1	15
A56		3	1	1	1	1		1					8
B31	2	1	2	1		1		1					8
B41	1	1		2	1			1	1				7
B49	1		1			1		1	1				5
B45			2			1						1	4
A72	1		1					1					3
B13		1				1	1						3
B53	1			1		1							3
A3			1	1									2
A34	1						1						2
B16	1						1						2
B46					1	1							2
A51					1								1
A52												1	1
A29										1			1
A30										1			1
B10				1									1
Total	49	27	44	21	35	21	20	19	18	18	19	20	311
%	15.8	8.7	14.1	6.8	11.3	6.8	6.4	6.1	5.8	5.8	6.1	6.4

We analyzed the distribution of Ds insertions among the rice chromosomes. For each single-T-DNA-locus transformant, in the T₃ generation, the Ds element was found to be distributed among different rice chromosomes (Table III). When all the 26 transformants are taken in account, the largest chromosomes (1 and 3) carry the largest numbers (49 and 44, respectively) of Ds insertions, and the smallest chromosomes (9 and 10) carry the smallest numbers of Ds insertions (18 and 18). Based on the size of each chromosome, the Ds insertions appear to be evenly distributed in the rice genome.

Enhanced Expression of Rice Genes Adjacent to the Activation-Tagging Ds

To examine whether expression of the Ds-tagged rice genes was altered, reverse transcriptase (RT)-PCR was performed on transposant lines with Ds insertion adjacent to the rice genes. We randomly selected 24 transposant lines and examined gene transcript levels using the semiquantitative RT-PCR method. Initially, we analyzed the closest rice gene for each Ds insertion, which was at a distance of 1 to 7 kb upstream or downstream of the Ds element. RNA was extracted from the leaves or roots of 60-d-old rice plants. RT-PCR was performed to compare gene expression levels in the 24 candidate lines to those in the wild-type rice ‘Nipponbare’. Two pairs of primers specific to each gene were tested and each RT-PCR experiment was repeated at least three times under the same condition. As shown in Figure 4A, gene expression was enhanced in the line ADS247 whose Ds insertion was 1,609 bp upstream of Os03g15050 as compared to ‘Nipponbare’. In ADS248, whose Ds insertion was 1,880 bp upstream of a plasma membrane-type ATPase gene (Os11g29490), the transcript level was significantly higher than in ‘Nipponbare’, as suggested by the RT-PCR results (Fig. 4B).

Figure 4.

Semiquantitative RT-PCR analysis of five Ds insertion lines. Left, Schematic representation of the Ds insertion and the CaMV 35S enhancers (4xEn) relative to the nearby genes. The map of each chromosomal region was based on the Rice GE genome database RiceGE Functional Genomics Database (http://signal.salk.edu/cgi-bin/RiceGE). Genes that were examined in semiquantitative RT-PCR are marked with dotted-line boxes. Right, RT-PCR analysis of activation-tagged genes. Rice Act1 transcript was amplified as control. WT, Wild-type rice ‘Nipponbare’. RT-PCR using leaf or root RNA was indicated. A, Line ADS247 was tagged by the activation-tagging Ds element 1,609 bp upstream of Os03g15050, which encodes the phosphenolpyruvate carboxykinase. B, Line ADS248 was tagged by a Ds 1,880 bp upstream of Os11g29490, which encodes a plasma membrane-type ATPase. C, Line ADS305 was tagged by a Ds 12.2 kb upstream of Os09g30439, which encodes a heat shock protein. D, Line ADS427 was tagged by a Ds 1,349 bp upstream of Os04g42840, which encodes a HEAT repeat family protein. E, Line ADS210 was tagged by a Ds 11.8 kb upstream of Os10g41410, which encodes the nucleoside diphosphate kinase 1. For each gene, the RT-PCR result was confirmed using two different pairs of specific primers and the result from using one pair of primers was shown. Each PCR experiment was repeated at least three times to confirm the result.

However, the other 22 transposant lines showed the same results as ‘Nipponbare’ in semiquantitative RT-PCR when just one gene was analyzed. We further tested eight of such transposant lines by amplifying other genes in the vicinity of the Ds insertion. Three overexpressed Ds lines were identified through RT-PCR examination of the additional genes (Fig. 4, C–E). In ADS305, whose Ds insertion was 12.2 kb upstream of the gene Os09g30439, the transcript level was slightly higher than in ‘Nipponbare’ as indicated in the results of both leaf and root tissues (Fig. 4C). In ADS427, where the Ds was inserted 1,349 bp upstream of Os04g42840, the transcript level was increased significantly based on the amount of the RT-PCR product in ADS427 and wild-type rice (Fig. 4D). In the RT-PCR analysis of ADS210, we examined expression levels of four genes that are 1.8 to 11.8 kb from the Ds insertion (Fig. 4E). Just for the gene Os10g41410, which is 11.8 kb upstream of the Ds element, transcript level was significantly enhanced in ADS210 as compared to ‘Nipponbare’.

It was observed that the CaMV enhancers in Ds in ADS210 activated the farthest gene Os10g41410 but did not affect expression of the other closer genes (Fig. 4E). In ADS305, as compared to Os09g30439, Os09g30458 is closer to the Ds insertion but did not get activated (Fig. 4C). On the other hand, in ADS427, the CaMV enhancers in Ds affected expression of only Os04g42840, which is closer to the Ds element than the gene Os04g42860 (Fig. 4D). Therefore, for the function of the CaMV enhancers, likelihood of gene activation was not directly related to the distance from the CaMV enhancers to the gene.

DISCUSSION

We have developed an in cis-activation-tagging Ac-Ds system in which a T-DNA vector carries a Ds element containing 4× CaMV enhancers along with the Ac transposase gene. In testing the vector in rice, a total of 638 stable Ds transposant plants were selected from seedlings (T₃) of 2,086 T₂ plants that were derived from 26 primary transformants. In analysis of 559 transposant lines, 311 of the Ds insertions were mapped on rice chromosomes.

Enrichment of unlinked transpositions is an important step for random insertional mutagenesis using the Ac-Ds transposable elements, as these elements have a feature of preferential transposition to closely linked sites. In the Ac-Ds vector systems we developed in Arabidopsis (Sundaresan et al., 1995) and rice (Kolesnik et al., 2004), a negative-positive selection scheme was successfully utilized for enrichment of unlinked transpositions. The Ac-Ds vectors were designed in such a way that an IaaH or GFP gene functioned as a counter selection (or negative selection) marker against the Ac T-DNA, the Ds-donor T-DNA, and linked Ds transpositions (Sundaresan et al., 1995; Kolesnik et al., 2004). In this study, the activation vector system utilizes an improved negative-positive scheme that is based on dual GFP and RFP fluorescence markers for high-throughput unlinked transposition selection (Kumar et al., 2005). Additionally, our vector system combines the merits of in cis-Ac-Ds elements that do not need genetic crossing and activation/knockout dual function in the Ds element.

The activation-tagging Ds element showed germinal transposition in the primary transformants (T₁). In analysis of 53 single-T-DNA-locus transformants, GFP⁻RFP⁺ plants were identified in the T₂ generation of 23 (43.4%) transformants. These GFP⁻RFP⁺ plants represented germinal transposition events that were transmitted from the T₁ generation. For 22.6% (12/53) of the transformants, a fraction of excised Ds elements did not reinsert in the rice genome because the T₂ populations had GFP⁺RFP⁻ plants but not the GFP⁻RFP⁺ plants. It should be noted that these results were obtained using just 42 to 50 T₂ plants per transformant. Because a rice plant produces 200 to 400 seeds, if more T₂ progeny were assayed for GFP and RFP fluorescence, the frequency of primary transformants generating germinal transposition events could be higher than 43.4%.

From analysis of the T₂ families, we also found the germinal transposition frequency varied depending on transformants. The T₃ families of one-third of the single-T-DNA-locus transformants showed high transposition frequencies (20%–83.3%). The result was similar to the result of Kolesnik et al. (2004) that various frequencies were observed in different cross combinations of Ds line and Ac line. Therefore, it was important to select the right starter transformants to establish tagging populations. First, when generating transgenic plants of the activation vector, we did GFP and RFP fluorescence assays of the hygromycin-resistant calluses and plantlets to select transformants with normal GFP and RFP function. Transgenic rice plants carrying multiple or rearranged T-DNA copies may result in transgene silencing (Sallaud et al., 2003). Second, single-T-DNA-locus transformants were identified based on GFP segregation in T₂ seedlings, and single-T-DNA-locus T₂ plants were selected for generating T₃ populations. In screen for putative stable Ds transposants, we initially performed a pilot screen of all T₃ families and then focused on families with high germinal transposition frequencies. These strategies together with the GFP and RFP visual method highly facilitated screening of stable transposants.

In the design of a transposon tagging system, it is also essential to prevent a large number of transposant siblings from entering the pipeline. In this study, an important step was to avoid using transformants whose T₂ generation exhibited several transposants (GFP⁻RFP⁺ and GFP⁺RFP⁻ plants). For each of the transformants, we performed GFP and RFP assays on a small number of T₂ plants to know about the frequency of transposants. In establishing most of the T₃ populations, we utilized transformants whose T₂ progeny contained no or a low to moderate rate of transposition events. Among the 463 Ds flanking sequences obtained, 73 (15.8%) sequences were redundant FSTs from siblings carrying the same Ds insertions. The number of sibling FSTs could be further reduced if the T₂ families for generating T₃ populations were more strictly selected.

In testing the activation tag vector system in rice, we obtained a total of 463 FSTs sequences, of which 311 (67.1%) FSTs were from mapped Ds insertions, 79 (17.1%) were from insertions in repeat sequences, and 73 (15.8%) were redundant sibling FSTs as discussed above. Among the 67.1% (311/463) mapped FSTs, 40.2% (186/463) had hits to rice cDNA sequences and represented Ds insertions in the genic regions. For the other 26.9% (125/463) FSTs that showed no homology to rice cDNA sequences, we were not able to make a consistently clear distinction between the FSTs from 5′ or 3′ regulatory sequences of rice genes and the FSTs from Ds insertions in the intergenic regions. These results confirmed the previous reports that the Ac-Ds elements preferentially transpose to genic regions (Enoki et al., 1999; Kolesnik et al., 2004; van Enckevort et al., 2005). For the strategy of activation tagging, Ds insertions in the intergenic regions are desirable as compared to the insertions in genic regions. However, for the transposant lines where the Ds element is inserted in a rice gene, the CaMV enhancers in Ds could still activate other neighboring genes. As indicated in the semiquantitative RT-PCR results, genes that were over 10 kb away from the Ds insertions were activated by the CaMV enhancers in Ds.

We analyzed interchromosomal distribution of the 311 Ds insertions that were mapped. It appeared that the insertions were evenly distributed in the rice genome and the insertion number of each chromosome was proportional to the chromosome size. This is different from the previous report (Kolesnik et al., 2004) in which Ds transposition showed a bias toward chromosome 1. The different results can be explained by the numbers of Ds starter lines that were used in generation of the Ds transposant collections. We used a total of 26 Ds starter lines, while Kolesnik et al. (2004) used just six starter lines. A single Ds launching pad might have preferential transposition on some chromosomes, but utilization of more starter lines might have reduced such bias. We did not analyze intrachromosomal distribution of Ds insertions due to the limited number of Ds insertions on each chromosome. The Ds element in our system may have preferential transposition regions like the hot spot of Ds insertion (Kolesnik et al., 2004) because a similar scheme of enrichment of unlinked transposition was used in this study.

We performed semiquantitative RT-PCR of rice genes in 24 transposant lines and observed enhanced expression of Ds-tagged genes in five lines. The RT-PCR results were confirmed in repeated experiments using the same condition. We never had a result that the RT-PCR product of a transposant line was less than that of wild-type rice. Therefore, the CaMV enhancers in the Ds element were capable of activating rice genes adjacent to the Ds insertion, which was similar to the result of T-DNA-based activation tagging in rice (Jeong et al., 2002, 2006). However, the frequency of activation-tagged lines was 20.8% (5/24) in this study and the frequency is lower than the frequency of 52.7% in the previous report using T-DNA in rice (Jeong et al., 2006). One reason might be that we analyzed just one gene in semiquantitative RT-PCR for most (16 among 24) of the candidate lines. For the other eight lines, two to four rice genes in each line that are close to the Ds insertion were examined in RT-PCR and enhanced expression of Ds-tagged genes was observed in three lines. Therefore, our rate of activation-tagged lines could be increased if more genes in the candidate lines were analyzed. For the function of the CaMV enhancers, it was reported that no good relationship was found between frequency of activation and distance from the CaMV enhancers to the gene and that no correlation was observed between degree of activation and distance (Jeong et al., 2006). We also observed the similar results in RT-PCR analysis of three activation-tagged lines.

Although large T-DNA insertion libraries have been generated in rice, an efficient T-DNA transformation system is available just for the japonica subspecies. For the indica rice subspecies that is more widely grown in rice farming areas around the world, T-DNA transformation is still difficult. Also, most of the T-DNA transformation methods involve a tissue culture step that generates high frequency of somaclonal variation, which disturbs the process of forward mutant screens. Therefore, transposon mutagenesis is an effective approach for plant functional genomics and can serve as an alternative to T-DNA when transformation through tissue culture is inefficient, as is the case for the indica rice species as well as many other crops. Transposable elements can be mobilized or immobilized on demand and the approach requires only a few primary transformants to generate a large collection of independent transposon insertions within the genome. For this reason, generation of insertions in heterologous plants mainly depends on propagation as in the case of transposon mutagenesis in maize.

The Ac-Ds elements have features like small size of cis-required sequence (approximately 600 bp of minimal Ds), large cargo insert capacity, and reasonable high transposition frequency, which have made the Ac-Ds elements particularly amenable for genetic studies. The maize Ac-Ds elements can efficiently transpose in many heterologous plant species. Also, the Ac-Ds showed trans-kingdom transposition in yeast (Saccharomyces cerevisiae) and animal species (Weil and Kunze, 2000; Emelyanov et al., 2006), which suggested that the Ac-Ds elements do not rely on host-specific factors for transposition and that host factors involved in their mobility mechanism are widely conserved. Activation-tagging strategies based on maize transposable elements have been developed in tobacco (Nicotiana tabacum; Suzuki et al., 2001) and Arabidopsis (Marsch-Martinez et al., 2002; Schneider et al., 2005). The activation-tagging Ac-Ds system developed in this study was aimed for rice and other monocot plants. This in cis-two-element system eliminates the need for crossing and utilizes RFP and GFP for tracking Ds element and counter selection against the Ac transposase, respectively, to stabilize transposed Ds elements. The use of fluorescent markers instead of antibiotic or herbicide resistance increases the applicability to other plants and eliminates problems with escapes. As the dual fluorescence markers were under control of the maize ubiquitin 1 promoter (Christensen and Quail, 1996) that is functional in maize, rice, barley, wheat (Triticum aestivum), and many grasses, the principles developed here are applicable to many other monocot plants. We find that the Ds element preferentially transposes into genic regions, which is similar to previous reports about the maize transposable elements (Enoki et al., 1999; Cowperthwaite et al., 2002; Kolesnik et al., 2004; van Enckevort et al., 2005). When utilized as the carrier of the CaMV enhancers, the Ds element offers the advantage of both activation-tagging and knockout mutations. These features make transposon-based activation tagging particularly useful for large genomes with many duplicated genes such as maize, as well as polyploid plant crops. The Ac-Ds-based activation vector system developed in this study is publicly available without intellectual property restrictions and should be applicable for the functional genomics of a range of plants and especially for that of cereals and monocot plants.

MATERIALS AND METHODS

Vector Construction

In construction of the activation-tagging Ac-Ds vector pSQ5, the Ubi-DsRed-Nos cassette was from pSK62 (Kumar et al., 2005), the 1,785-bp 5′Ds and 222-bp 3′Ds were from pWS32 (Sundaresan et al., 1995), and the 4× CaMV 35S enhancers were from the AcREH construct (Suzuki et al., 2001). First, pWS32 was digested with NcoI/EcoRI and ligated to an NcoI/HindIII/EcoRI linker to generate pSQ1. Second, the 4× CaMV 35S enhancers were released as a 1.4-kb HindIII fragment from the AcREH construct and cloned at the pSQ1 HindIII site to make pSQ2. At the third step, multi-DNA ligation was performed using (1) the 1.7-kb 3′Ds/4× enhancers fragment from SacI/EcoRI digested pSQ2; (2) the 1,785-bp 5′Ds from SacI/HindIII digested pWS32; (3) a 3.3-kb Ubi-DsRed-Nos cassette from HindIII/EcoRI digested pSK62. The multiligation products were digested with SacI and 6.8-kb product (i.e. the activation-tagging Ds element) was purified and cloned into a SacI site of pCAMBIA-1300. The activation-tagging Ds element was partially sequenced to confirm junctions of fragments of the multiligation. The Ds element was finally cloned at the SacI site of the pSSZ40 construct (Kolesnik et al., 2004) carrying the CaMV35S-Ac and Ubi-sGFP-Nos cassettes. The backbone of the pSQ5 vector was from pCAMBIA-1300 (GenBank accession no. AF234296). The vector pSQ5 is in the public domain and will be made available by the corresponding author upon request.

Plant Transformation

Mature seeds of rice sp. japonica ‘Nipponbare’ were cultured on NB medium (Li et al., 1993) for 3 to 4 weeks to induce embryogenic callus. The callus was cocultivated with Agrobacterium strain LBA4404 containing the pSQ5 Ac-Ds vector and selected on NB medium containing 50 μg L⁻¹ of hygromycin B (Roche Diagnostics) as described (Yin and Wang, 2000; Sallaud et al., 2003). GFP⁺RFP⁺ calluses were transferred to preregeneration medium (Yin and Wang, 2000) and cultured at 25°C and in dark for 10 to 15 d. The GFP⁺RFP⁺ calluses were further cultured on regeneration medium (Yin and Wang, 2000) under light for 2 to 3 weeks. GFP⁺RFP⁺ transformant plantlets were finally transferred to greenhouse.

GFP/RFP Fluorescence Assays

Transformed rice callus and plantlets were examined under a Zeiss SV11 fluorescence microscope (Zeiss) using appropriate filters of GFP and RFP fluorescence. Mature seeds of transgenic plants were germinated at 25°C or 30°C in dark for 4 to 6 d. The emerging seedlings were screened for GFP fluorescence using the Green Fluorescent Protein Macro Detector set (model GFP-MDS-20BB, BLS). RFP fluorescence visualization of seedlings was under the Zeiss SV11 fluorescence microscope.

Cloning of Ds Flanking Sequences Using Adaptor-Ligation PCR

Rice DNA of stable Ds transposants was isolated using the DNeasy 96 Plant kit (Qiagen). In adaptor-ligation PCR, about 100 ng of rice genomic DNA was added to digestion-ligation reaction mixture, which contained the HindIII and EcoRI adaptors, the enzymes of EcoRI, HindIII, and T4 DNA ligase (Alonso et al., 2003). PCR primers of the first-round PCR were Ds5-1A (5′-acggtcgggaaactagctctacc-3′) and AP1 (5′-gtaatacgactcactatagggc-3′; Alonso et al., 2003). Products of the first round PCR were diluted 10 times with water. The second round PCR was performed using primers Ds5-4 (5′-ctcgggttcgaaatcgatcgggat-3′) and AP2-C (5′-tggtcgacggcccgggctgc-3′). PCR products were sequenced using ABI Biosystems BigDye v3.1 and ABI 3730 × 1 DNA sequencers.

RT-PCR

Total RNA was extracted from leaves or roots of Ds insertion lines and wild-type rice using the Trizol Regent (Invitrogen, catalog no. 15596-018). One microgram of RNA was reverse transcribed in a total volume of 25 μL that contained 0.4 nmol of oligo(dT)₁₈ primer, 5 mm dNTPs, 20 units of SUPERase-in (Ambion, catalog no. AM2694), and 200 units of Moloney murine leukemia virus RT (Promega, catalog no. M1701) in the murine leukemia virus reaction buffer. PCR was performed in 25 μL of solution containing 1× TaKaRa Ex Taq polymerase buffer, 0.2 μm gene-specific primers, 2.5 mm dNTPs, 0.5 unit of TaKaRa Ex Taq polymerase, and 1 μL cDNA reaction product. The reaction condition included denaturing template DNA at 95°C for 5 min, 24 to 27 cycles of PCR (94°C, 30 s; 56°C, 40 s; 72°C, 50 s), and a final incubation step (72°C, 5 min).