Early Embryogenesis-Specific Expression of the Rice Transposon Ping Enhances Amplification of the MITE mPing

Miniature inverted-repeat transposable elements (MITEs) are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu), mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP), and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP). Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.


Introduction
Transposable elements (TEs) are DNA sequences that are capable of jumping from one genomic locus to another and make up a large fraction of eukaryotic genomes. More than 80% of the maize (Zea mays) and barley (Hordeum vulgare) genomes are composed of TEs [1], [2], and they constitute 35% and 14% of the genomes of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), respectively [3], [4]. TEs are harmful to the host because their mobilities perturb genome stability, whereas they play greatly generative roles in genome evolution such as alternation of gene structure, change of expression pattern, and rearrangement of chromosome structure [5], [6].
TEs are classified into two groups according to their transposition mechanisms: class I elements (retrotransposons) that transpose through a copy-and-paste mechanism via an RNA intermediate, and class II elements (transposons) that transpose through a cut-and-paste mechanism without undergoing an RNA intermediate. Class I elements easily attain tens of thousands of copies, whereas the majority of class II elements cannot amplify themselves to 50 copies at most. Unlike other class II elements, miniature inverted-repeat transposable elements (MITEs) have the capacity to amplify themselves to high copy numbers (hundreds or thousands) [7]- [9]. In the rice genome, MITEs are numerically predominant TEs [10], constituting 8.6% of the genome [11]. Because MITEs are too short (,600 bp) to encode any protein, their transpositions must depend on the proteins encoded by the autonomous elements. Well-studied MITEs are classified into the Stowaway and Tourist families, which belong to the Tc1/mariner and PIF/Harbinger superfamilies, respectively. Because MITEs are mainly deployed in gene-rich regions [10], [12] and affect adjacent gene expression [13], they are considered to play an important role in genome evolution. However, little is known about how MITEs attain high copy numbers.
Miniature Ping (mPing) is the first active MITE identified in the rice genome [14]- [16]. Although MITEs are deployed in the genome at a high copy number, the copy number of mPing exceptionally remains at a low level in most rice cultivars: indica and tropical japonica cultivars have fewer than 10 copies, and temperate japonica cultivars including Nipponbare have approximately 50 copies [14]. The transposition of mPing is suppressed in most rice cultivars, but, like other TEs, mPing is activated by exposure to various stress conditions such as gamma-ray irradiation [16], hydrostatic pressurization [17], cell culture [14], anther culture [15], and inhibition of topoisomerase II [18]. Introgression of distantly related genomes also causes mPing transposition [19], [20]. However, mPing is actively transposing without such stresses in the temperate japonica rice strain EG4 (cultivar Gimbozu) under natural growth conditions, and its copy number is approximately 1000 copies [21]. This indicates that mPing has overcome the silencing mechanism or established a novel strategy for its amplification in the EG4 genome. In this sense, mPing in EG4 is an appropriate material to study the amplification of MITEs in plant genomes.
The autonomous element Ping and its distantly related element Pong, which both belong to the PIF/Harbinger superfamily, provide two proteins required for mPing transposition. Both Ping and Pong have two open reading frames (ORFs), ORF1 and ORF2 [22], [23]. The former encodes a Myb-like DNA-binding protein, and the latter encodes a transposase lacking DNA binding domain. Transposase of most class II elements contains a conserved catalytic domain (DDE motif) and a DNA-binding domain [23], [24], whereas these domains are encoded separately by two ORFs in both Ping and Pong [22], [23]. The study of other members of the PIF/Harbinger superfamily suggested that the Myb-like DNAbinding protein directly binds to the subterminal regions of the transposon in order to recruit the transposase [25]. Both Myb-like protein and transposase of either Ping or Pong or both elements are necessary for mPing transposition [22], [23].
In this study, we demonstrate that mPing is actively transposing in the embryo of EG4 during the period from the regionalization of shoot apical meristem (SAM) and radicle to the formation of the first leaf primordium (3 to 5 days after pollination, DAP) with the aid of developmental stage-specific expression of Ping. Our results provide important evidence for the amplification mechanism not only of mPing but also of other MITEs.

Transpositions of mPing during gametogenesis
Plants have acquired the silencing mechanism of TEs in germ cells. In Arabidopsis, for example, TEs are activated specifically in the vegetative nucleus of the pollen, and siRNAs from the activated TEs accumulate in the sperm cells [26]. On the basis of these results, Slotkin and colleagues proposed that siRNAs derived from TEs activated in the vegetative nucleus silence TEs in the sperm cells [26]. We conceived that mPing might overcome such a silencing mechanism in EG4. To confirm this hypothesis, we developed two F 1 populations from reciprocal crosses between the mPing-active strain EG4 and the mPing-inactive cultivar Nipponbare, and investigated the transposition activity of mPing by transposon display (TD) analysis. Success of reciprocal crosses was confirmed by PCR analysis using locus-specific primers ( Figure  S1A). One of the results of TD analysis using two selective bases is shown in Figure 1A; all 16 possible primer combinations were analyzed. The banding patterns of F 1 plants were almost the same as those of EG4. The bands that appeared in all F 1 plants but not in the parental EG4 plant were derived from another parental Nipponbare plant ( Figure S1B). Furthermore, the bands that appeared in only one of eight F 1 plants but not in the parental EG4 plant are herein referred as de novo insertions. These bands were confirmed not to be PCR artifacts by sequence and locusspecific PCR analysis (Table S1 and Figure S2). We detected 15.5 de novo insertions per plant in the selfed progenies of EG4, whereas Nipponbare yielded no de novo insertions in the selfed progenies ( Figure 1B). This confirmed that mPing is active in EG4 under natural growth conditions but inactive in Nipponbare. If mPing was specifically activated in the pollen of EG4, we could obtain de novo insertions only in the F 1 plants from the Nipponbare/EG4 cross. However, we obtained de novo insertions in both Nipponbare/EG4 and EG4/Nipponbare populations ( Figure 1B). Moreover, there was no significant difference in the number of de novo insertions per plant between the two F 1 populations. This indicates that the activating factor(s) for the mPing transposition is present in both male and female gametes of EG4.

Transpositions of mPing during ontogeny of EG4 plants
We performed TD analysis of mPing using genomic DNA samples extracted from endosperm, radicle, and leaf blades of eight progenies (S 1 ) derived from a single parental EG4 plant (S 0 ), and investigated the mPing transposition during ontogeny of rice plants ( Figure 2A). One of the results of TD analysis using two selective bases is shown in Figure S3; all 16 possible primer combinations were analyzed. We examined de novo insertions in the same way as described above. Consequently, a total of 228 de novo insertions were detected. These insertions were divided into five groups ( Figure 2B): (1) endosperm-specific insertions that appeared only in the endosperm sample, (2) radicle-specific insertions that appeared only in the radicle sample, (3) leaf-specific insertions that appeared only in one sample from the 1st to 3rd leaf blades, but not in the 4th and 5th leaf blades, (4) shoot-specific insertions that appeared in at least one sample of 1st, 2nd, and 3rd leaf blades, and in at least one sample of 4th and 5th leaf blades, and (5) radicle/shoot-specific insertions that appeared in both radicle and leaf blade samples. These de novo insertions were confirmed by sequence and locus-specific PCR analysis (Table S2 and Figure  S4). Numbers of each insertion obtained in this study are summarized in Figure 2C. Plant development is divided roughly into three successive phases: embryogenesis, vegetative phase, and reproductive phase. If mPing transposed in the SAM of the S 0 plant during vegetative and/or reproductive phases, the de novo insertions would segregate according to Mendel's law among the S 1 progenies. We obtained no band that appeared in at least two S 1 progenies and was not seen in the S 0 plant. This indicates that the transmissible insertion of mPing to the next generation seldom (or never) arises during the vegetative and reproductive phases.

Author Summary
Transposable elements are major components of eukaryotic genomes, comprising a large portion of the genome in some species. Miniature inverted-repeat transposable elements (MITEs), which belong to the class II DNA transposable elements, are abundant in gene-rich regions, and their copy numbers are very high; therefore, they have been considered to contribute to genome evolution. Because MITEs are short and have no coding capacity, they cannot transpose their positions without the aid of transposase, provided in trans by their autonomous element(s). It has been unknown how MITEs amplify themselves to high copy numbers in the genome. Our results demonstrate that the rice active MITE mPing is mobilized in the embryo by the developmental stagespecific up-regulation of an autonomous element, Ping, and thereby successfully amplifies itself to a high copy number in the genome. The short-term expression of Ping is thought to be a strategy of the mPing family for amplifying mPing by escaping the silencing mechanism of the host genome.
Flowering plants have evolved a unique reproductive process called double fertilization. In this process, either of two sperm cells in pollen fuses with either an egg cell or a central cell in the ovule, and then the egg cell fertilized with the sperm cell initiates embryogenesis [27]. In rice, the SAM and radicle are regionalized in the embryo 3 DAP, and three leaves and the radicle are already present in the mature embryo [28]. We detected only three radicle/shoot-specific insertions ( Figure 2C), indicating that mPing scarcely transposes during the period from the onset of gametogenesis to the early stage (until 3 DAP) of embryogenesis. Among the 228 de novo insertions, 116 and 17 were shoot-specific and leafspecific insertions, respectively ( Figure 2C). This indicates that mPing actively transposes in the embryo during the period from the regionalization of SAM and radicle (at 3 DAP) to the formation of the 3rd leaf primordia (at 8 DAP). Of the 133 shoot-and leafspecific insertions, 108 were of the 1st leaf blade ( Figure 2D). Since the 1st leaf primordium is formed at 5 DAP, the most active phase of the mPing transposition was considered to be from 3 to 5 DAP. We detected a large number of radicle-specific insertions as well as shoot-specific insertions, and the sum of these insertions accounted for 90% of all insertions detected in this study ( Figure 2C). Taken together, we concluded that mPing in EG4 most actively transposes in the 3 to 5 DAP embryo.
Endosperm is a triploid tissue that is produced by fusing a central cell containing two polar nuclei with one of two sperm cells in no particular order. The endosperm formation occurs in parallel with embryogenesis. The endosperm-specific insertions result from the mPing transposition occurred in either gametogenesis or endosperm formation. We observed only two endospermspecific insertions ( Figure 2C), supporting that mPing scarcely transposes during the period from the onset of gametogenesis to the early stage of embryogenesis. The relationship between the banding patterns obtained in TD analysis and the timing of mPing transposition is summarized in Figure S5.

Inheritance of de novo mPing insertions to the next generation
In order for mPing to amplify, the de novo insertions must be transmitted to the next generation. We performed TD analysis using 12 progenies (S 2 ) derived from the main culm and the primary tiller of a single selfed parent (S 1 ) to investigate whether the de novo insertions detected in ontogenical analysis are inheritable ( Figure S6). Both radicle-specific and leaf-specific insertions in the S 1 plants were not detected in the S 2 progenies (0 of 15, 0 of 2, respectively). In contrast, 85% (11 of 13) of the shootspecific insertions that were detected in the S 1 plants also appeared in the S 2 progenies. This value (85%) is consistent with the estimated number of inheritable de novo insertions in our previous report [21]. Thus most of the de novo insertions that arose in the 3 to 5 DAP embryo were successfully inherited to the next generation.

Excisions of mPing during ontogeny of EG4 plants
We have already determined the sites of all mPing insertions (1163 in total) in the EG4 genome [13], and have investigated mPing excisions in a small EG4 population using locus-specific  Eight progenies (S 1 ) derived from a single parental EG4 plant (S 0 ) were grown in a greenhouse. Endosperm, radicle, and leaf blades (1st to 5th) of each S 1 plant were sampled for DNA extraction. S 2 seeds were harvested from the main culm and the primary tiller of each S 1 plant to investigate the inheritance of de novo mPing insertions. The 2nd leaf blade of S 0 and S 2 plants was also sampled. Detailed information is provided in the 'Materials and Methods'. (B) mPing insertions were detected by transposon display. Representative images of shoot-, leaf-, endosperm-, radicle-, and radicle/shoot-specific insertions are shown. White arrowheads indicate the bands representing the de novo mPing insertion. E: endosperm, R: radicle, L1-L5: 1st to 5th leaf blades. (C) The number of organ-specific de novo insertions in EG4. All 16 possible primer combinations were analyzed. En: endosperm-specific insertion, RS: radicle/shoot-specific insertion, R: radicle-specific insertion, Shoot: shoot-specific insertion, Leaf: leaf-specific insertion. (D) Percentage of leaf positions where the first de novo mPing insertion was found. Shoot: shoot-specific insertion, Leaf: leafspecific insertion. doi:10.1371/journal.pgen.1004396.g002 primer pairs [29], [30]. Here we examined the timing of the mPing excision with locus-specific PCR using the genomic DNA samples that were used for the ontogenical analysis of the de novo insertion. We randomly chose 48 markers for this study (Table S3). We divided the mPing excisions into five types with the same criteria as those used for the de novo insertions: endosperm-, radicle-, leaf-, shoot-, and radicle/shoot-specific excisions ( Figure S7). There were no endosperm-specific and radicle/shoot-specific excisions, indicating that no mPing transposition occurs during the period from the onset of gametogenesis to the early stage of embryogenesis. We detected seven radicle-specific, six leaf-specific, and three shoot-specific excisions. All shoot-specific excisions were detected from the 1st leaf blade sample. These results indicate that, like the de novo insertion, the mPing excision also occurs during the period from the regionalization of the SAM and radicle to the formation of the first leaf primordium, although we cannot completely rule out the possibility that these excisions occur also in somatic cells of mature tissues. Thus, in addition to the experimental results of the de novo insertion, we concluded that mPing of EG4 was most actively transposing in the 3 to 5 DAP embryo.

Expression pattern of Ping in EG4
Both Ping and Pong provide a Myb-like protein and a transposase, which are encoded by their ORF1 and ORF2, respectively ( Figure 3A), and have been considered as autonomous elements responsible for the mPing transposition. We investigated the expression of Ping-ORF1, Ping-ORF2, Pong-ORF1, and Pong-ORF2 during embryogenesis to evaluate which autonomous element plays a predominant role in driving the mPing transposition in EG4. Reverse transcription-PCR analysis revealed that Ping-ORF1 and Ping-ORF2 constitutively expressed in the ovary during embryogenesis ( Figure 3B). On the other hand, no transcriptions of Pong-ORF1 and Pong-ORF2 ( Figure 3B) were observed. This strongly suggests that Ping predominantly controls the mPing transposition in EG4.
We performed real-time quantitative PCR (qPCR) analysis to compare the expression level of Ping-ORF1 and -ORF2 between EG4 and Nipponbare during embryogenesis. In all developmental stages from 1 to 6 DAP, the expression levels of both Ping-ORF1 and -ORF2 were higher in EG4 than in Nipponbare ( Figure 3C, D). Since EG4 harbors seven copies of Ping, whereas Nipponbare has only one copy (Table S4), the difference in the expression levels between EG4 and Nipponbare is considered to be attributable to the different copy number of Ping. However, we found that Ping of EG4 showed different expression patterns from that of Nipponbare. In Nipponbare, the expression level of Ping-ORF1 and -ORF2 gradually declined until 3 DAP, and restored to the basal level at 6 DAP. In contrast, in EG4, the expression levels of both Ping-ORF1 and -ORF2 rapidly increased, with a peak at 3 DAP ( Figure 3C, D). The ratio of relative expression level (EG4/ Nipponbare) clearly demonstrated that Ping might be up-regulated in a developmental stage-specific manner in the ovary of EG4 ( Figure 3E). Since mPing transposed during the period from 3 to 5 DAP, the rapid increase in Ping expression most likely drive the mPing transposition.

Accumulation of Ping transcripts in the embryo triggers mPing transposition
We investigated the spatial expression pattern of Ping by in situ hybridization using Ping-specific probes. The probe positions were indicated in Figure 3A. The Ping transcripts were detected in all tissues, viz. embryo, endosperm, and ovary wall, in both EG4 and Nipponbare ( Figure 4A-C, S8). Among the tissues, the 3 DAP embryo of EG4 yielded an exceptionally strong signal, indicating a high accumulation of Ping transcripts ( Figure 4A), whereas the 5 DAP embryo showed a much lower accumulation of Ping transcripts in EG4 ( Figure 4D-F). Such a drastic change in accumulation quantity of Ping transcripts with the advance of embryogenesis was consistent with the change in the expression quantity of Ping with the advance of embryogenesis, which was investigated by real-time qPCR (Figure 3C-E). These results suggest that the tissue-and developmental stage-specific accumulation of the Ping transcripts triggers mPing transposition at this stage in EG4. To confirm this hypothesis, we evaluated the spatial expression pattern of Ping in the SAM during the vegetative phase. As described above, mPing hardly transposes in the SAM during this phase. The Ping transcripts were detected in all tissues including the SAM, and, as expected, there was no obvious difference in the signal intensity between EG4 and Nipponbare ( Figure 4G-I). Thus the Ping transcripts proved to accumulate developmental stage-specifically only in the tissue where mPing actively transposes. We therefore concluded that the high accumulation of Ping transcripts triggers the transposition of mPing in the 3 DAP embryo of EG4.

SNP in an intronic region of Ping-ORF1
EG4 has seven Ping elements (Ping-1 to -7), whereas Nipponbare has only one (Ping-N) (Table S4). When we sequenced and compared all Ping elements, a single nucleotide polymorphism (SNP) in the first intronic region of Ping-ORF1 was detected between EG4 and Nipponbare ( Figure 5A). Ping-N has a 'T' nucleotide on the SNP region, whereas all Ping elements in EG4 have a 'C' nucleotide. We named the former 'T-type Ping' and the latter 'C-type Ping'.
In addition to EG4, several Aikoku and Gimbozu landraces (hereafter AG strains) are known to exhibit high mPing activity [21]. We investigated the SNP-type of Ping and the copy number of Ping and mPing in 93 AG strains, and evaluated the effect of Ctype Ping on the mPing activity. These 93 AG strains were divided into three groups according to the SNP-type of the Ping allele (Table S4): strains harboring C-type Ping; strains harboring T-type Ping; and strains harboring no Ping. The strains with C-type Ping had more mPing copies than those with T-type Ping or no Ping ( Figure 5B, Steel-Dwass test, p,0.01). This implies that the Ctype Ping could drive the mPing transposition. We further investigated the expression patterns of Ping-ORF1 and -ORF2 in two mPing-active strains (A119 and A123) and two mPing-inactive strains (A105 and G190) during embryogenesis (from 1 to 6 DAP). A119 and A123 have six and ten copies of C-type Ping, respectively, and both A105 and G190 have one copy of T-type Ping (Table S4). Expression analysis revealed that A105 and G190 kept low expression levels of Ping-ORF1 and -ORF2, whereas A119 and A123 showed high expression levels with a peak around 3 DAP ( Figure 5C-F). This indicates that, in EG4, A119, and A123, the developmental stage-specific expression of Ping is controlled by the same factor(s) described in the Discussion.

Discussion
Our final goal was to elucidate how MITEs attain their high copy numbers in the genome. To this end, we chose mPing, which is the only active MITE identified in rice, as a material and analyzed the timing of mPing transposition in the mPing-active strain EG4. Consequently, we successfully found one mechanism of the mPing amplification; mPing most actively transposes during the period from the regionalization of the SAM and radicle to the formation of the first leaf primordium (3 to 5 DAP) by the developmental stage-specific up-regulation of the autonomous element Ping.
The transpositions of TEs are categorized into germinal and somatic types according to the type of cells where the transposition takes place. LORE1a in Lotus japonicus is activated in plants regenerated from de-differentiated cells and transposes in male germ cells by the pollen grain-specific LORE1a transcription, resulting in the asymmetric transposition of LORE1a in the reciprocal crosses between the active and non-active lines [31]. Tag1 in Arabidopsis shows germinal transposition activity in both male and female germ cells. Consequently, the reciprocal crosses show symmetric transposition of Tag1 [32]. These results demonstrate that the transposition activity in reciprocal crosses reflects the tissue specificity of germinal transposition. In this study, reciprocal crosses between EG4 and Nipponbare showed the same mPing transposition activity, which may suggest that mPing in EG4 transposes in both male and female germ cells. However, we obtained only a few de novo endosperm-specific and radicle/shootspecific insertions in the ontogenical analysis, although we detected a number of de novo shoot-specific and radicle-specific insertions. We therefore concluded that most mPing transposes not in germ cells but in somatic cells after pollination. Somatic transposition that occurs at the late stage of plant development often produces spotted and striped segments in tissues, such as maize seed coat variegation caused by Mutator excision from the bz2 gene [33], [34] and rice leaf color variegation by nDart1excision from the OsClpP5 gene [35]. In animals, somatic transposition is seldom transmitted to the next generation because germ cells are set aside from somatic cells at the early stage of embryogenesis. On the other hand, in plants, germ cells are generated from somatic cells at the reproductive stage. In rice, gametes are generated in the SAM; therefore, somatic transposition that occurred in the SAM can be transmitted to the next generation via gametes. In this study, we revealed that most mPing elements transposed in somatic cells of the embryo during the period from 3 to 5 DAP. Being a class II TE that transposes by a cut-and-paste mechanism, mPing is expected to be eliminated from genomic DNA with a certain frequency. However, a previous report demonstrated that the mPing excision sites would be repaired by utilizing a copy of mPing from either the sister chromatid or from the homologous chromosome [29]. The mPing excision site cannot be repaired if mPing transposes in germ cells, which are haploid. It is therefore considered that the somatic transposition of mPing is an important factor for mPing amplification in the genome.
The autonomous elements Ping and Pong mediate mPing transposition in the rice genome. Many japonica cultivars, including EG4 and Nipponbare, have both Ping and Pong. This study demonstrated that Ping plays a predominant role in mPing transposition in EG4. However, a heterologous expression assay using Arabidopsis and yeast showed that Pong had a higher catalytic capacity for mPing transposition than Ping [22], [23]. Furthermore, mPing transposition was observed under stress conditions in several rice cultivars harboring only Pong [14], [17], [19]. In this study, however, we detected very low expression of Pong through the development of rice plants, indicating that Pong would be epigenetically silenced at the transcriptional level in EG4. In contrast, Ping constitutively expressed in all organs including the SAM and embryo. Nevertheless, mPing could be transposing most actively in the embryo during the period from 3 to 5 DAP. Since the stage-specific up-regulation of Ping was observed during the period of mPing transposition, we hypothesized that the expression level of Ping needed to exceed a certain threshold of mPing transposition.
All mPing-active strains (EG4, A119, and A123) showed higher expression of Ping with a peak around 3 DAP than the mPinginactive strains (Nipponbare, A105, and G190). Although further experiments are needed to elucidate the mechanism of developmental stage-specific up-regulation of Ping expression, we propose two hypotheses: (1) position-and dosage-effect, and (2) effect of SNP. The details of the hypotheses are as follows.

Position-and dosage-effect
Chromosomal position and copy number of TE often affect the transposition activity. The former is known as 'position effect' and the latter as 'dosage effect'. Eight independent Tam3 copies residing in the Antirrhinum majus genome show different transposition activities from each other [36]. In Arabidopsis, germinal reversion frequency of Tag1 increases in proportion to its copy number [32]. The mPing-inactive strains Nipponbare, A105, and G190 have only one Ping at the same locus, whereas the mPingactive strains EG4, A119, and A123 have respectively seven, six, and ten copies of Ping at different loci except for the Ping-1 locus. Furthermore, the expression pattern of Ping showed slight variation among the mPing-active strains harboring only C-type Ping. These results suggest that the developmental stage-specific up-regulation   (A105 and G190). The results are presented as means of three biological replicates. Bars indicate SE. The ratio of (E) Ping-ORF1 and (F) -ORF2 expression level of A105, A119, A123, and G190 to that of Nipponbare. The means in (Fig. 3C) and (Fig. 3D)  of Ping expression is probably regulated by the position-effect and/ or the dosage-effect.

Effect of SNP
Intronic SNPs are known to cause drastic effects on gene expression. In humans, an intronic SNP in SLC22A4 affects transcriptional efficiency in vitro, owing to an allelic difference in affinity to the transcriptional factor RUNX1 [37]. Furthermore, a SNP located in the intronic enhancer region of the thyroid hormone receptor b gene enhances pituitary cell-specific transcriptional activity [38]. In this study, we demonstrated that a SNP is present in the intronic region of Ping-ORF1, and Ping elements in the AG strains were categorized into either T-type or C-type Ping according to the SNP-type. Since all strains that showed a peak in the expression analysis had only C-type Ping, the intronic SNP might influence the developmental stage-specific up-regulation of Ping expression. T-type Ping was present in 14 AG strains as one copy, and its chromosomal location did not differ among strains. In contrast, the copy number of C-type Ping varied from one to ten, and their chromosomal locations, except for Ping-1, differed from each other. These results indicate that T-type Ping has lost its activity, whereas C-type Ping may be still active in the rice genome. Furthermore, we found that the copy number of mPing was significantly larger in strains harboring C-type Ping than in strains harboring T-type Ping. This strongly supports that Ctype SNPs in the intronic region of Ping contribute to the amplification of mPing, presumably by the developmental stagespecific up-regulation of Ping expression.
Since the transposition of TEs often damages the host genome, TEs with high transposition activity are targeted by the silencing mechanisms. Nevertheless, MITEs amplify to very high copy numbers not only in plant genomes but also in animal genomes. Very little is known about how MITEs attain their high copy numbers by escaping the silencing mechanism. The transposition of mPing is transiently induced by various stresses [14]- [18], indicating that the activity of mPing is suppressed by the silencing mechanisms in many cultivars. Thus, mPing must overcome the silencing mechanism in order to maintain the transposition activity under natural growth conditions. Our results revealed that mPing in EG4 was mobilized by the sufficient supply of Ping transcripts produced only during the period of mPing transposition. This stagespecific activation is thought to be a strategy of the mPing family to amplify mPing by escaping from the silencing mechanism of the host genome. Since no active MITEs other than mPing so far have been identified, it is very difficult to elucidate if the other MITEs also attain their high copy numbers in the same way as mPing amplifies. Given that the other active MITEs are identified, however, our study will help to understand their amplification mechanisms. Our previous study documented the generation of new regulatory networks by a subset of mPing insertions that render adjacent genes stress inducible [13]. In addition to mPing, other MITEs also contribute to gene and genome evolution via providing new promoter regulatory sequences, transcriptional termination elements, and new alternative exons [39], suggesting that the amplification of MITEs causes gene and genome evolution. Our results provide clues to further understand not only the amplification mechanism of MITEs but also the coevolution of MITEs and the host genome.

Materials and Methods
Plant materials and sampling EG4 (cultivar Gimbozu), Nipponbare, and 94 Aikoku/Gimbozu landraces were used in this study (Table S4). Aikoku/Gimbozu landraces were provided from the GenBank project of the National Institute of Agrobiological Science, Ibaraki, Japan. Reciprocal crosses between EG4 and Nipponbare were made in a green house. Before pollination, all anthers were removed from the flowers of maternal plants. The pollinated flowers were covered with protective bags to prevent outcrossing until harvest. After harvesting, success of crosses was checked with the molecular markers. For ontogenical analysis, eight progenies of EG4 (S 1 ) derived from a single parental plant (S 0 ) were grown in a greenhouse, and all S 2 seeds were harvested. For S 1 plants, each seed was cut into two halves, and the half including the embryo was germinated and the other was sampled. After germination, the radicle and the 1st, 2nd, 3rd, 4th, and 5th leaf blades were sampled. The second leaf was collected from S 0 and S 2 plants. For estimation of Ping and mPing copy numbers, eight bulked plants were sampled. For RNA extraction, ovaries before pollination and ovaries at 1, 2, 3, 4, 5, and 6 DAP were collected. All samples were immediately frozen in liquid nitrogen and stored at 280uC until use.

DNA extraction and transposon display
DNA extraction and transposon display was performed following a published protocol [30]. For DNA extraction from endosperm, we used GM quicker 2 (Nippon Gene).

Locus-specific PCR
Sequencing of mPing-flanking fragments excised from transposon display gels and primer design were performed following a published protocol [30]. The genomic locations of the mPing insertion sites were forecasted by a BLAST search in the Rice Annotation Project Database (RAP-DB; http://rapdb.dna.affrc. go.jp/) [40], [41] using mPing flanking sequences as queries. To prepare enough templates for PCR, whole genome amplification was performed using an illustra GenomiPhi V2 Kit (GE Healthcare). mPing excision was detected by PCR with mPingsequence characterized amplified region (SCAR) markers [29]. PCR was performed in 10-ml reaction volumes containing 10 ng of the template DNA, 5 ml of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 mM of each primer (Table S3). PCR conditions were as follows: 94uC for 3 min; 40 cycles of 98uC for 10 s, 57uC for 30 s, and 72uC for 45 s; and 72uC for 5 min. To detect the presence of Ping-N, -1, -2, -3, -4, -5, -6, and -7, eight Ping-SCAR markers were used. The genomic locations of the Ping insertion sites were referred from a previous report [42]. For detection of the Ping-1 allele, PCR was performed in 10-ml reaction volumes containing 10 ng of template DNA, 0.2 U of KOD FX Neo (Toyobo), 16PCR buffer for KOD FX Neo (Toyobo), and 0.2 mM of each primer (Table S5). PCR conditions were as follows: 94uC for 3 min; 35 cycles of 98uC for 10 s, 60uC for 30 s, and 68uC for 90 s; and 72uC for 1 min. For detection of Ping-N, -2, -3, -4, -5, -6, and -7 alleles, PCR was performed in 10-ml reaction volumes containing 10 ng of template DNA, 5 ml of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 mM of each primer (Table S5). PCR conditions were as follows: 94uC for 3 min; 35 cycles of 98uC for 10 s, 60uC for 30 s, and 72uC for 45 s; and 72uC for 1 min.

RNA isolation and expression analysis
Total RNA was isolated using TriPure isolation reagent (Roche) and digested using RNase-free DNase (TaKaRa). First strand cDNA was synthesized using a Transcriptor first strand cDNA synthesis kit (Roche). For reverse transcription PCR, PCR was performed in 10 ml reaction volumes containing cDNA generated from 4 ng total RNA, 0.2 U of KOD FX Neo (Toyobo), 16PCR buffer for KOD FX Neo (Toyobo), and 0.5 mM of each primer. PCR conditions were as follows: 94uC for 3 min; 35 or 45 cycles of 98uC for 10 s, 60uC for 10 s, and 68uC for 10 s. Relative quantification of Ping-ORF1 and Ping-ORF2 were calculated by the 2 2DDCT method [43] using Light cycler 1.5 (Roche). The UBQ5 gene was used as the calibrator gene. The thermal profile consisted of 10 min at 95uC; and 45 cycles of 4 s at 95uC, 10 s at 60uC, and 1 s at 72uC. Amplification data were collected at the end of each extension step. The primer pairs used in this study are listed in Table S6.

Paraffin sectioning and in situ hybridization
Plant samples were fixed with 4% (w/v) paraformaldehyde and 1% Triton X in 0.1M sodium phosphate buffer for 48 h at 4uC. They were then dehydrated in a graded ethanol series, substituted with 1-butanol, and embedded in Paraplast Plus. The samples were sectioned at 8-mm thickness using a rotary microtome. Fragments of Ping-ORF1 (1091 bp) and Ping-ORF2 (1368 bp) were cloned into pBlueScript SK+ (Stratagene) and sequenced. For digoxigenin-labeled antisense/sense RNA probe synthesis, in vitro transcription was performed using T7 RNA polymerase and T3 RNA polymerase. In situ hybridization and immunological detection with alkaline phosphatase were performed according to Kouchi and Hata [44].

SNP detection
PCR was performed in 10-ml reaction volumes containing 10 ng of template of DNA, 5 ml of GoTaq Green Master mix (Promega), 5% DMSO, and 0.25 mM of each primer. PCR conditions were as follows: 94uC for 3 min; 35 cycles of 98uC for 10 s, 60uC for 30 s, and 72uC for 30 s; and 72uC for 1 min. PCR primers used in this study are listed in Table S6. Because the original sequence contained an Afa I restriction site, one mutation was introduced into the reverse primer. The 5-ml PCR products were mixed with 5 ml restriction mixture containing 1 U Afa I (TaKaRa), 33 mM Tris-acetate (pH 7.9), 10 mM Mg-acetate, 0.5 mM Dithiothreitol, 66 mM K-acetate, and 0.01% (w/v) bovine serum albumin. After 16 h incubation at 37uC, DNA gel electrophoresis was performed. PCR products (502 bp) including +1261T SNP were not digested, whereas PCR products including +1261C SNP were digested into two fragments (352 bp and 150 bp).

Estimation of Ping and mPing copy number
To determine the copy number of Ping by Southern blot analysis, genomic DNA samples were digested with Eco RI restriction enzyme. These samples were loaded onto an agarose gel, separated by electrophoresis, blotted onto a nylon membrane, and probed with the Ping fragment. The mPing copy number was determined by real-time quantitative PCR as described previously [45] with little modification. Quantitative PCR was performed using the Light-Cycler 480 system (Roche). PCR was performed in 20 ml reaction volumes containing 5 ml genomic DNA (0.4 ng/ml), 16LightCycler 480 SYBR Green I Master mix (Roche), and 0.5 mM of each primer. Specificity of the amplified PCR product was assessed by performing a melting curve analysis on the LightCycler 480 system.  Figure S3 Ontogenical analysis of mPing transposition in EG4 by transposon display. Eight progenies (S 1 ) were derived from a single parental EG4 plant (S 0 ). The 2nd leaf blade of the S 0 plant and the endosperm, radicle, and leaf blades of each S 1 plant were sampled and subjected to transposon display. White, red, and green arrowheads indicate shoot-, radicle-, and leaf-specific insertions, respectively. The rice plant has alternate distichous leaves; therefore, we analyzed the insertion in both [n+1]th and [n+2]th leaves to confirm whether the insertion detected in [n]th leaf is leaf-specific or shoot-specific. But we did not investigate the specificity of the insertions detected in the 4th and 5th leaves using their upper leaves. For this reason, we did not categorize such insertions and marked with the gray arrowhead. E: endosperm; R: radicle; L1-L5: 1st to 5th leaf. For progeny 2-8, samples are applied in the same order as for progeny 1.  Figure S5 Schematic representation of the relationship between banding patterns obtained in transposon display and the timing of mPing transposition. If mPing transposes in the period indicated by the red bar, the schematic banding patterns indicated by the arrows will be obtained. E: endosperm, R: radicle, L1-L5: 1st to 5th leaf blade, DAP: days after pollination, SAM: shoot apical meristem. (TIF) Figure S6 Inheritance of de novo mPing insertions in EG4. S 2 plants derived from the main culm and the primary tiller of a single S 1 plant were assayed. The shoot-specific insertion in the S 1 plant (white arrowhead) was inherited by S 2 plants, whereas the radicle-specific insertion (red arrowhead) was not. E: endosperm; R: radicle; L1-L5: 1st to 5th leaf. (TIF) Figure S7 mPing excisions in EG4. mPing excisions were detected by locus-specific PCR using the genomic DNA samples that were used for the ontogenical analysis of the de novo insertion. We analyzed 48 loci. Black and white arrowheads show the bands indicating the presence and absence of mPing, respectively. Figures  indicate (A, B) shoot-specific excisions, (C, D) leaf-specific excisions, and (E) radicle-specific excision. G: EG4 (S 0 plant); N: Nipponbare; E: endosperm; R: radicle; L1-L5: 1st to 5th leaf. (TIF) Figure S8 Detection of Ping-ORF2 spatial expression patterns by in situ hybridization analysis. Longitudinal sections through the ovary 3 days after pollination of (A) EG4 and (B, C) Nipponbare were hybridized with (A, B) antisense or (C) sense RNA probes. (TIF)

Author Contributions
Conceived and designed the experiments: TTs. Performed the experiments: ST. Analyzed the data: ST. Contributed reagents/materials/ analysis tools: YO TTa. Wrote the paper: ST TTs.