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Plant Cell. May 2005; 17(5): 1559–1568.
PMCID: PMC1091774

A Two-Edged Role for the Transposable Element Kiddo in the rice ubiquitin2 PromoterW in Box


Miniature inverted repeat transposable elements (MITEs) are thought to be a driving force for genome evolution. Although numerous MITEs are found associated with genes, little is known about their function in gene regulation. Whereas the rice ubiquitin2 (rubq2) promoter in rice (Oryza sativa) line IR24 contains two nested MITEs (Kiddo and MDM1), that in line T309 has lost Kiddo, providing an opportunity to understand the role of MITEs in promoter function. No difference in endogenous rubq2 transcript levels between T309 and IR24 was evident using RT-PCR. However, promoter analysis using both transient and stably transformed calli revealed that Kiddo contributed some 20% of the total expression. Bisulfite genomic sequencing of the rubq2 promoters revealed specific DNA methylation at both symmetric and asymmetric cytosine residues on the MITE sequences, possibly induced by low levels of homologous transcripts. When methylation of the MITEs was blocked by 5-azacytidine treatment, a threefold increase in the endogenous rubq2 transcript level was detected in IR24 compared with that in T309. Together with the observed MITE methylation pattern, the detection of low levels of transcripts, but not small RNAs, corresponding to Kiddo and MDM1 suggested that RNA-dependent DNA methylation is induced by MITE transcripts. We conclude that, although Kiddo enhances transcription from the rubq2 promoter, this effect is mitigated by sequence-specific epigenetic modification.


Miniature inverted repeat transposable elements (MITEs) are a collection of transposable elements (TEs) that have structures typical of nonautonomous DNA transposons, including target site duplications and terminal inverted repeats (Feschotte et al., 2002). However, their small size (usually less than 500 bp) and large copy number (usually hundreds or thousands) distance them from conventional transposons, which typically have a size of several thousand base pairs but are present as only several copies. Numerous MITE families have been found in higher eukaryotic organisms. Recently, mPing, an active MITE family, was discovered in rice (Oryza sativa) (Jiang et al., 2003; Kikuchi et al., 2003; Nakazaki et al., 2003), and several other MITE families have been linked to known transposon families (Robertson, 1996; Smit and Riggs, 1996; Tu, 1997; Casacuberta et al., 1998; Surzycki and Belknap, 1999; Feschotte and Mouches, 2000; Le et al., 2001; Turcotte et al., 2001; Zhang et al., 2001; Yang and Hall, 2003a, 2003b). It is believed that MITEs are transposed by transposases produced by autonomous transposons.

It seems likely that the overwhelming presence of TEs in higher plant genomes would impose disadvantages, especially when they are newly introduced. As summarized by Bennetzen (2000), negative outcomes resulting from transposition events may cause host extinction or reshaping of the structure of the genetic system. However, all currently existing genomes represent the successful coevolution of the TEs and the host genome. In addition to their participation in the formation of essential genetic structures such as heterochromatin, centromeres, and telomeres (Biessmann et al., 1992a, 1992b; Dimitri and Junakovic, 1999; Volpe et al., 2002), conventional TEs are actively involved in gene regulation. In the long run, their regulatory functions contribute to genome evolution through promoter enhancement or repression (Errede et al., 1987; Banks et al., 1988; Banks and Fedoroff, 1989; Tanda and Corces, 1991), intron disruption (Wessler et al., 1987; Bradley et al., 1993; Greene et al., 1994), coding sequence disruption (Gierl et al., 1985; Wessler et al., 1986; Kim et al., 1987; Michaud et al., 1994), untranslated region (UTR) function disruption (Barkan and Martienssen, 1991; Chatterjee and Martin, 1997), and position effect or enhancer blockage (Dorsett, 1990; Holdridge and Dorsett, 1991; Jack et al., 1991; Geyer and Corces, 1992). Among these studies, effects attributed to class I TEs (retrotransposons) were substantially more frequently documented than those for class II TEs (transposons). It is possible that the high copy numbers of retrotransposons and their association with genic regions render them more likely to affect gene expression than is the case for conventional transposons. Because the copy number of a particular conventional DNA transposon family is limited in a genome, its insertion close to or inside a gene is an infrequent event. Thus, the overall effect of a conventional DNA transposon family on a genome is limited. By contrast, MITE families have high copy numbers with small size and often are closely associated with genes through their insertion into promoters, UTRs, or introns (Bureau and Wessler, 1992, 1994; Zhang et al., 2000; Yang et al., 2001). Thus, MITEs may have a broad impact on genome structure and function. Indeed, dramatic changes to genome structure resulting from transposition have been demonstrated in cases of MITE-associated polymorphism (Wessler et al., 2001; Park et al., 2003; Edwards et al., 2004). However, little convincing evidence has been reported for MITE function in gene regulation (Wessler et al., 1995; Casacuberta and Santiago, 2003).

We previously discovered that the rice ubiquitin2 (rubq2) promoter in indica line IR24 contains two nested MITEs: the founding member of the Kiddo family nested inside MDM1 (Figure 1A). Kiddo is a member of the IS5/Hbr/PIFa superfamily, and MDM1 is a mutator-derived element (Yang and Hall, 2003a, 2003b). These two MITEs constitute 603 bp of the 812 bp upstream of the transcription initiation site. Other than that it has lost Kiddo (Yang et al., 2001), the rubq2 promoter in japonica rice line T309 is essentially identical to that in IR24.Therefore, these two promoters provide an opportunity to study the effect of Kiddo on rubq2 promoter activity.

Figure 1.
Constructs and Expression Analysis.

We show here that, although no differences in endogenous rubq2 transcript levels were detected between T309 and IR24 by RT-PCR, analysis of reporter activity in rice calli transgenic for green fluorescent protein (gfp) driven by various promoter truncations revealed that Kiddo contributes ~20% of the total rubq2 promoter activity. To explain the discrepancy in the results obtained with these two approaches, we hypothesize that Kiddo makes positive functional contributions but that they are concealed as a result of epigenetic changes. Indeed, DNA cytosine methylation on the rubq2 promoter was observed to be restricted to the MITE sequence elements and to be associated with low levels of transcribed Kiddo and MDM1. Prevention of DNA methylation by 5-azacytidine (5-azaC) treatment resulted in a threefold increase in the endogenous rubq2 transcript level for IR24 compared with that for T309. These results indicate that Kiddo plays a two-edged role in gene regulation: on the one hand, its presence increases transcription rates; on the other hand, it can induce epigenetic modifications. The implications of this two-edged role in genome evolution are also discussed.


Contributions of Kiddo to rubq2 Promoter Activity

The rubq2 promoter in rice line IR24 contains two nested MITEs, Kiddo and MDM1, whereas the promoter in T309 contains only MDM1 (Figure 1A). Because Kiddo contains a G box and several other motifs characteristic of transcription factor binding sites (Yang et al., 2001), it appeared likely that its presence could enhance transcription. To evaluate this possibility, the endogenous activity of the two promoters was compared. RT-PCR was performed to amplify a 3′ UTR fragment using RNA from T309 and IR24. No difference in the endogenous rubq2 transcript levels was detected for the two rice lines (Figure 1B), suggesting that Kiddo may not have an effect on promoter activity.

In a separate series of experiments undertaken to dissect rubq2 promoter functions, constructs were made that contained truncated rubq2 promoters driving gfp (mGFP5-ER) (Haseloff and Amos, 1995). Initially, these plasmids were used for particle bombardment of rice calli; discrete green fluorescent spots representing individual cells were obtained (see Supplemental Figure 1 online). The fluorescence intensity of 100 randomly chosen spots was determined using a semiquantitative microscopic approach (see Methods). Taking rubq2-953 as the full-length promoter, in three independent experiments the Kiddo element was found to contribute ~20% of its activity (Figures 1C and 1D). Binary vectors containing rubq2-953 and rubq2-953(dKiddo) were used for Agrobacterium tumefaciens–mediated transformation of rice calli. Comparison of the fluorescence intensity using 150 independent stably transformed calli for each construct (Figure 1D) revealed a similar (22%) contribution of Kiddo to the rubq2 promoter to that found in the transient assays.

Kiddo and MDM1 DNA Sequences Are Targeted for Methylation

Because plant transposons and other repetitive sequences are often methylated and silenced (Liang et al., 2002; Rabinowicz et al., 2003), the possibility exists that the Kiddo and MDM1 sequences within the endogenous rubq2 promoters are methylated. Such epigenetic modification could depress the positive contribution of the Kiddo element evident in the transgenesis experiments.

To determine whether the MITEs in the rubq2 promoters are methylated, genomic DNA from T309 and IR24 was used for bisulfite genomic sequencing. Primers were designed for the top strand. After bisulfite treatment and amplification, PCR products were cloned into the TOPO cloning vector for sequencing (see Methods). PCR products from T309 and IR24 were sequenced for five and seven clones, respectively (Figures 2A and 2B). Interestingly, for both samples, methylation was confined strictly to the MITE sequences on the rubq2 promoters; virtually identical results were obtained in a second, entirely independent, experiment (see Supplemental Figure 2 online). On the IR24 rubq2 promoter, 61% of the cytosine positions were methylated for Kiddo and 41% were methylated for MDM1. For both elements, cytosine methylation was seen at symmetric (CpG and CpNpG) and asymmetric positions. However, symmetric sites were methylated at a higher frequency (74%) than were asymmetric sites (47%) (Table 1). Regions corresponding to those having high sequence similarity within the MITE families (Yang et al., 2001; Yang and Hall, 2003b) were heavily methylated. Interestingly, low levels of methylation were seen toward the ends of the MITE terminal inverted repeats. This may indicate protection from methylation, possibly resulting from the binding of transposases or other proteins.

Figure 2.
Bisulfite Genomic DNA Sequencing of the Top Strands of rubq2 Promoters in Rice Lines IR24 and T309.
Table 1.
Methylation of Kiddo and MDM1 on the rubq2 Promoter

Methylation of Kiddo Neutralizes Its Enhancement of the rubq2 Promoter

Methylation of promoter DNA sequences is known to be associated with transcriptional gene silencing (Paszkowski and Whitham, 2001). To determine whether methylation of the two MITEs caused changes in rubq2 promoter activity, IR24 and T309 seeds were germinated on 5-azaC (25 mg/L) to prevent or reduce methylation of the MITE sequences within the rubq2 promoters after mitosis. RT-PCR amplification of the 3′ UTR (Figure 1A) of RNA extracted from 5-azaC–treated and untreated IR24 and T309 seedlings revealed an increased rubq2 transcript level in IR24 treated with 5-azaC compared with the untreated IR24 sample. However, no similar increase was evident for 5-azaC–treated T309 samples (Figure 3A). The small difference among the samples may be illusory, because the RT-PCR approach used is semiquantitative in nature; because of this, RNA gel blot analysis was performed.

Figure 3.
Effect of 5-azaC Treatment on Endogenous rubq2 Transcript Levels.

When the same sets of RNA samples were used for RNA gel blot analysis, as expected, a unique band was obtained for IR24 samples using a probe targeting the rubq2 3′ UTR (Figure 3B). However, in addition to the expected band, an extra band with a higher molecular weight was seen for the T309 samples. We noted that excision of Kiddo left a TATA box–like site upstream of the original TATA box on the T309 rubq2 promoter and suspected that the extra band may represent a new transcription initiation site from the putative TATA box. When 5′ rapid amplification of cDNA ends experiments were performed to identify the initiation site(s) for rubq2 in T309, initiation was confirmed only for the original TATA box (accession number AY661468), even though the downstream primers for 5′ rapid amplification of cDNA ends were designed at the 5′ UTR, the ubiquitin coding sequence, or the 3′ UTR. When the 5′ UTR probe (Figure 1A) was used for RNA gel blot analysis, a unique band was seen for both T309 and IR24 samples. This, together with the fact that the extra band was not detected when the RNA gel blot was probed with the promoter sequence upstream of the 5′ UTR, indicated that the extra band detected with the 3′ UTR probe was not transcribed by the rubq2 promoter. Indeed, when a genomic DNA gel blot of T309 and IR24 was probed with the rubq2 3′ UTR sequences, an extra fragment was identified in T309 (see Supplemental Figure 3 online). Additionally, when the rubq2 3′ UTR sequence was used as the query for database searches, in addition to the rubq2 transcripts (AK061956 and AK101547), cDNA sequences containing a fragment that is 88% identical to the rubq2 3′ UTR were retrieved from japonica (AK067024 and AF216530) but not indica databases, giving further support to the conclusion that the upper bands were not transcribed from the rubq2 promoter. The intensities of the gene-specific bands were measured, and the ratio of the rubq2 transcript level to the elongation factor1-α (EF1-α) transcript level was defined as the relative activity of the rubq2 promoters. When compromised for methylation by exposure to 25 mg/L 5-azaC, some threefold greater transcript abundance was found for the Kiddo-containing rubq2 promoter in IR24 than for the Kiddo-less T309 rubq2 promoter (Figure 3D). As found for reactivation of methylation-associated silenced rice lines (Kumpatla et al., 1997; Kumpatla and Hall, 1998), it is possible that greater transcriptional stimulation would be attained at increased (50 to 75 mg/L) 5-azaC concentrations. Unfortunately, IR24 seeds appear to be incapable of germination at the higher 5-azaC levels (G. Yang and T.C. Hall, unpublished data). The results (Figure 3) also reveal that, although methylation of MDM1 results in a limited decrease in rubq2 promoter activity, methylation of the Kiddo element can decrease rubq2 promoter activity dramatically.

Compared with the twofold to threefold enhancement of transcription from the rubq2 promoter in 5-azaC–treated IR24 plants (Figure 3D), a much less dramatic enhancement (~20%) of rubq2 promoter activity by Kiddo was observed in transformation experiments (Figures 1C and 1D). Methylation of the MITE sequences in both experiments was presumed to be compromised. However, the promoters used in transgenic approaches did not contain the first intron sequence, an element that has been suggested to function as an enhancer (Wang et al., 2000). The higher enhancement by Kiddo in the endogenous rubq2 promoter may reflect the synergistic effects of the first intron on the promoter.

Methylation of Kiddo and MDM1 Is Associated with a Low Level of Homologous RNAs, but Not Small RNAs

Conventional DNA methylation occurs only at CG dinucleotides. The methylation patterns for Kiddo and MDM1 are unusual in that all cytosine sites (CG, CNG, and asymmetric sites) are modified. This is indicative of RNA-dependent DNA methylation (RdDM) (Wassenegger, 2000; Matzke et al., 2002, 2004). In plants, de novo methylation of cytosines is thought to be mediated by the DNA methyltransferases DRM1 and DRM2 (Cao and Jacobsen, 2002; Cao et al., 2003) and the maintenance of CG methylation by MET1 homologs (Finnegan and Kovac, 2000; Kankel et al., 2003). CNG methylation, which is plant-specific, is maintained by a special type of DNA methyltransferase, chromomethylase3 (Lindroth et al., 2001). Because no maintenance activity is known for asymmetric Cs, methylation on asymmetric Cs is lost rapidly once de novo methylation ceases. Hence, the asymmetric methylation observed for Kiddo and MDM1 can be considered an ongoing de novo methylation (Aufsatz et al., 2002a, 2002b; Matzke et al., 2004). For the de novo methylation at asymmetric Cs of Kiddo and MDM1 to persist, a constitutive homologous RNA would be predicted to induce RdDM.

It has been speculated that Kiddo and MDM1 are possibly cotranscribed with genes because some members of these MITE families reside in genic 5′ UTRs, introns, or 3′ UTRs (Bureau and Wessler, 1992, 1994; Zhang et al., 2000; Yang et al., 2001). Perhaps surprisingly, when the Kiddo sequence was used to probe RNA gel blots of T309 and IR24 seedling RNA, no specific hybridization signal was detected (Figure 4A). However, when RT-PCR was used to amplify Kiddo from the RNA samples, a trace amount of Kiddo transcript was detected (Figure 4B). Although only a very faint band was detected in IR24 and IR24/azaC samples for MDM1 by RT-PCR (Figure 4B), a weak signal was detected in RNA gel blot analysis for all four samples (Figure 4A). Because the RT-PCR primers were designed to amplify full-length elements, this indicated the presence of transcripts containing partial MDM1 sequences and provides an explanation for the failure to amplify full-length MDM1 transcripts in T309 and T309/azaC samples. The faint RT-PCR bands for Kiddo or MDM1 may come from a trace amount of unstable spliced introns bearing the MITEs, because the RT-PCR bands would be much stronger than those shown in Figures 4B and 4C. These MITEs were present on messenger or other relatively stable RNAs.

Figure 4.
Low Levels of Kiddo and MDM1 Transcripts in T309 and IR24 Cells.

Small RNAs (smRNAs) homologous with and resulting from the presence of double-stranded RNAs (dsRNAs) are reported to be associated with RdDM and have been speculated to cause RdDM (Mette et al., 2000). Because both Kiddo and MDM1 have internal hairpin structures, the low levels of Kiddo and MDM1 transcripts may reflect the remnant RNAs after massive degradation of Kiddo and MDM1 transcripts by Dicer homologs in rice. If this is the case, the presence of smRNAs corresponding to the MITEs would be expected. RNA gel blot analysis using fractionated low molecular weight RNA samples (see Methods) was performed. Strong hybridization was detected for an internal control microRNA (mir160) probe (Reinhart et al., 2002), but no significant signal was detected using Kiddo or MDM1 probes (Figure 4C). Intriguingly, a very faint signal was present at approximately nucleotide 26 to 27 (Figure 4C, Kiddo). However, increasing the amount of RNA loaded or changing the hybridization temperature (42 and 40°C) did not improve the intensity of the bands, suggesting that the signal may be nonspecific. These results indicated that dsRNAs may be needed for the methylation of Kiddo and MDM1 elements and that smRNAs do not play a major role in inducing methylation at these elements.


Evolutionary Implications of the Role of MITEs in Higher Genomes

At first sight, TEs appear to be disruptive forces for the integrity of a genome. However, once a host mechanism (e.g., homology-dependent gene silencing) is in place that counters their disruptive activities, coexistence of the eukaryotic host and the TEs is possible. Indeed, MITEs are found mainly in higher eukaryotic organisms, and it may be speculated that some correlation exists between the abundance of MITEs and genome complexity. The biggest benefit brought to organisms such as rice and maize (Zea mays) by TEs is probably their contribution to genome dynamics.

Dynamic genomes are capable of ready adaptation to changing environments. The transposition frequency of a TE partly depends on its size. The larger the size, the less frequent the transposition for a given level of transposase (Izsvak et al., 2000; Karsi et al., 2001). To fully benefit from TE-based dynamics, it is preferable to have elements that are small in size and present in a high copy number. In fact, among class I TEs, short interspersed elements successfully proliferated in higher eukaryotes (Schmidt, 1999; Shedlock and Okada, 2000). For class II elements, autonomous transposons can give rise to shorter nonautonomous elements and extreme forms of transposon truncations may result, so that only the structures and sequences necessary for efficient transposition are retained. MITEs, therefore, may represent the ultimate forms of transposons. Because they are small and apparently competent to transpose, as demonstrated in their high copy number, they are easy to mobilize. Additionally, they are probably less disruptive than are large canonical transposons when inserting into genic regions.

Several opportunities to study the consequences of MITE insertion on gene expression have arisen (reviewed in Wessler et al., 1995); however, to the best of our knowledge, no detailed evidence for the involvement of MITEs in gene regulation has been reported. It is possible that no major effect of MITEs on gene regulation was immediately evident, and the prevailing wisdom is that MITEs are neutral in gene regulation (Wessler et al., 2001). Indeed, using RT-PCR, we initially observed no difference in endogenous rubq2 transcript levels between the T309 and IR24 rice lines. However, this apparently neutral situation may reflect compensation of their contribution to quick adaptation of the rubq2 promoter and, by extension, of the genome.

The difference in transcriptional enhancement observed between transgenic calli (Figures 1C and 1D) and wild-type plants (Figure 3) from the presence of Kiddo on the rubq2 promoter may arise from several causes. For example, rubq2-gfp expression could be augmented or diminished by a position effect, reflecting the insertion site of the transgene. However, accurate estimation of activity is expected through our use of average values of expression from a large pool of samples (100 spots for biolistic experiments, and 150 independent stably transformed calli). Studies of transcription from the phaseolin promoter in transgenic plants (Frisch et al., 1995; Li et al., 1998) showed that its properties, such as strict spatial confinement, can be dramatically different when it is organized into chromatin than when it is present as naked DNA. A parallel exists for our studies in that the biolistically introduced rubq2 transgene is not present as chromatin, at least initially. Even when present in stably transformed callus, the rubq2 transgene promoter may not have attained the state established by the endogenous rubq2 promoter, which is marked by DNA sequence methylation (Figure 2) that may lead to repression of its activity. This epigenetic repression may mitigate the potential positive contribution of Kiddo to the rubq2 promoter, giving the appearance that the MITE is neutral in promoter function. Thus, whereas the contribution of Kiddo to transcriptional activity may be difficult to discern in nature, its immature status when introduced by transformation permits experimental detection of its contribution.

Under normal conditions, when the TEs are tightly controlled, the presence of MITEs does not affect normal gene expression patterns and their effect on gene regulation is not obvious. When under certain circumstances, for example genome stress, the efficacy of TE control mechanisms (e.g., methylation of repeats) is compromised, autonomous and nonautonomous TEs are mobilized. A good example of epigenetic regulation of transposons is seen in studies of the Suppressor-mutator element. Methylation of its promoter and the GC-rich downstream control region correlates with the inactivation of both transposase transcription and the transposition of this element. This autoregulation of Suppressor-mutator is mediated by TnpA, a protein produced by the transposon itself (McClintock, 1958, 1961; Banks et al., 1988; Banks and Fedoroff, 1989; Fedoroff, 1989; Schläppi et al., 1993, 1994). Under similar circumstances, the activity of MITEs may also be correlated with their methylation status. More interestingly, the abundance of MITEs in higher genomes, together with their ability to change the expression patterns of genes with which they are associated, may result in their being major contributors to the evolution of an organism. The two-edged role of MITEs, such as Kiddo, to both enhance and repress gene expression may have participated in the rapid and widespread evolution and adaptation of grass plants (Zhang et al., 2000).

dsRNAs versus smRNAs in RNA-Dependent DNA Methylation

Methylation of repeat sequences is a well-known phenomenon. Nevertheless, the participation of RNA in the methylation of repeat sequences was discovered only recently. In an elegant article, Mette et al. (2000) revealed that dsRNAs are potent signals for homologous DNA sequence methylation by showing that highly expressed dsRNA of a promoter sequence can induce the efficient methylation and silencing of homologous DNA sequences. At the same time, smRNAs corresponding to the promoter sequences were detected, but it remains unclear whether the dsRNAs or the smRNAs direct DNA methylation. In a recent study of Arabidopsis thaliana API genes (Melquist and Bender, 2003), homologous DNA sequence methylation by a weakly transcribed homologous RNA signal was detected, but no corresponding smRNAs were identified. It was concluded that either unprocessed dsRNAs or low levels of smRNAs directed the methylation of homologous DNA sequences. Our results demonstrate the association of MITE sequence methylation with trace levels of transcribed MITEs. The presence of low levels of MITE transcript may explain our observations (G. Yang, Y.-H. Lee, and T.C. Hall, unpublished data) that the rubq2 promoters are gradually silenced over a period of 3 months in stably transformed rice calli.

As MITEs have inverted repeat structures, when transcribed they are likely to yield dsRNAs. However, no smRNAs were detected for either Kiddo or MDM1, suggesting that dsRNAs, rather than smRNAs, play a major role in inducing DNA methylation. Because dsRNAs can have complementary binding to both strands of target DNA sequences, they may have a higher affinity than do single-stranded homologous RNAs to their target DNA sequences. Compared with dsRNAs transcribed from an inverted repeat structure, separately produced sense and antisense RNAs may have a lesser chance to form dsRNA duplexes, resulting in decreased efficiency in DNA methylation induction. It is also possible that when Kiddo or MDM1 is present within introns and cospliced with them, they induce DNA methylation inside the nucleus.


Plasmid Construction

rubq2 promoters with and without Kiddo were amplified from rice (Oryza sativa) lines IR24 and T309, respectively (Yang et al., 2001). The PCR products were cloned between HindIII and XbaI sites of the intermediate plasmid pBJ81 (a derivative of pUC18), in which the uidA reporter was replaced with an mGFP5-ER reporter. For stable transformation of rice calli, binary vectors of pRubq2-953 and pRubq2-953(dKiddo) were constructed by cloning the rubq2 promoter and gfp fragments into pJD7 (Dong et al., 2001) between HindIII and NsiI.

Callus Induction, Biolistic Experiments, and Agrobacterium tumefaciens–Mediated Transformation

Mature seeds from T309 were dehulled and rinsed with 70% ethanol for 1 min, then soaked in 50% (v/v) bleach for 45 min on a shaker at 120 rpm. The sterilized seeds were washed five times with sterile distilled water. The seeds were then placed on N6 medium (Chu et al., 1975) with the embryo side face up for 2 weeks at 28°C. Induced calli were subcultured on N6 medium for 10 to 14 d. Actively growing calli were selected for bombardment. The selected calli were treated on high-osmolarity N6 medium supplemented with mannitol and sorbitol (0.3 M each) for 4 h before bombardment. Gold particles (1.0 μm, 600 μg) were coated with 1 μg of plasmid DNA and used for bombardment with a model PDS-1000 biolistic particle delivery system using 1300-p.s.i. rupture discs (E. I. du Pont de Nemours, Wilmington, DE). Bombarded calli were incubated in the dark (26°C) and examined after 48 h.

For Agrobacterium-mediated transformation, calli were initiated from dehulled mature seeds of T309 as described above. Embryogenic calli, selected from actively growing calli essentially as described by Hiei et al. (1994) (1997), were cocultivated with the Agrobacterium-containing binary plasmids pRubq2-953 or pRubq2-953(dKiddo) in the dark at 21°C for 3 d. After cocultivation, infected calli were rinsed with sterile distilled water containing cefotaxime (250 mg/L) before being transferred to N6 selection medium containing 2,4-D (2 mg/L), hygromycin (50 mg/L), and cefotaxime (250 mg/L). Calli were transferred onto fresh selection medium every 2 weeks. After 4 weeks on selection medium, calli expressing GFP were selected using a fluorescence microscope and placed on fresh N6 selection medium. After 1 week, the intensity of GFP green fluorescence of the stably transformed calli was measured.

Measurement of Green Fluorescence Intensity and Data Processing

In transient assays, for each plasmid used, 100 green fluorescent spots were imaged using an AxioCam HR camera (Carl Zeiss, Jena, Germany) attached to a Zeiss SV11 microscope. The exposure time was adjusted so that none of the spot images was saturated in pixels, and the mean value of green fluorescence for each spot was measured using the interactive measurement module of AxioVision 3.0 (Carl Zeiss). Each reading was normalized by subtracting the nearby background fluorescence reading. The normalized readings from 100 spots for each plasmid were used to calculate average values. Measurement of 150 stably transformed rice calli was performed using a similar approach to that in the transient experiments, except that the readings were normalized by subtracting readings from wild-type calli. Average values and standard errors were calculated with Microsoft Excel (Redmond, WA).

Bisulfite Genomic DNA Methylation Sequencing

Genomic DNAs extracted from rice lines T309 and IR24 were digested by HindIII and purified with phenol chloroform. A total of 500 to 750 ng of purified genomic DNA was used for bisulfite treatment for 14 h using the EZ DNA methylation kit from Zymo Research (Orange, CA). After elution of the treated samples, 2 to 4 μL of the elute was used for subsequent PCR amplification with AmpliTaq Gold DNA polymerase (Foster City, CA). PCRs were performed according to the manufacturer's recipe with an optimized MgCl2 concentration of 4 mM. The cycling parameters were as follows: 94°C for 2 min; four cycles of 94°C for 1 min, 60°C for 2 min, and 72°C for 4 min; 39 cycles of 94°C for 45 s, 60°C for 2 min, and 72°C for 2 min; and 72°C for 10 min. The primers to amplify the rubq2 promoter top strand DNA were 5′-AGYGAATAYGGAGGYGYGGGGTTGA-3′ and 5′-ATCTARCAACAAAACAACCTCACCCRTCC-3′. PCR products were cloned with the TOPO TA cloning kit (Invitrogen, Carlsbad, CA), and plasmids containing inserts of the correct size were sequenced using T7 primer.

RNA Isolation and Analysis

One-week-old rice seedlings growing on MS medium with and without 5-azaC (25 mg/L) were harvested and ground in liquid nitrogen. Trizol reagent (Invitrogen, Carlsbad, CA) (20 mL) was added to 2 g of ground tissue, and RNA was extracted according to the manufacturer's instructions. Low molecular weight RNAs and high molecular weight RNAs were fractionated from total RNAs using the method of Hamilton and Baulcombe (1999).

For RNA gel blot analyses, samples loaded on 1% formaldehyde denaturing agarose gel were run at 5 V/cm for 2 to 3 h in 1× 3-(N-morpholino)-propanesulfonic acid buffer. RNA was transferred using a Trans-Blot Cell (Bio-Rad, Hercules, CA) for ~1 h at 40 V. Probes were made from DNA templates using the DECAprimeII system (Ambion, Austin, TX), and hybridization was performed with Ultrahyb buffer (Ambion). Densities of hybridization signals were measured with MacBas software (Fujifilm, Tokyo, Japan), and background signals were subtracted from the reading. For smRNA detection, 60 μg of low molecular weight RNAs was separated on a 16% polyacrylamide gel containing 7 M urea. Gels were transferred onto Hybond+ nylon membranes (Amersham, Piscataway, NJ), and hybridization was performed using Ultrahyb-Oligo buffer (Ambion) at 42 and 40°C. Full-length Kiddo and MDM1, as well as synthesized DNA oligonucleotides corresponding to their double-stranded regions, were used as probes.

Total RNA samples were treated with DNase I before RT-PCR amplification using the One-Step RT-PCR kit (Promega, Madison, WI). The optimized annealing temperature used for rubq2 3′ UTR and Kiddo amplification was 63°C; 58°C was used for MDM1. Primers for rubq2 3′ UTR amplification were 5′-GTCTGATCTTCGCTGGCAAGCAGC-3′ and 5′-GCATACTGCTGTCCCACAGGAAACTG-3′; those for rice EF1-α were 5′-GCCCATGGTTGTGGAGACCTTCTC-3′ and 5′-TCATTTCTTCTTGGCGGCAGCCTTG-3′. Primers for Kiddo were 5′-GGGGCTGTTTGGTTCCCAGCCATAC-3′ and 5′-GGGTGTGTTTGGTTCTAAGCCACACTTTGC-3′; those for MDM1 were 5′-AGTAAATTGCACTTTGACCCACC-3′ and 5′-AGTGAATTACGCTTTGGACCAC-3′. For DNA contamination controls in RT-PCR, RT enzyme mix was replaced by Taq DNA polymerase from Invitrogen.

Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY661468.

Supplementary Material

[Supplemental Data]


We thank M. Matzke and M. Mette for providing a genomic DNA bisulfite sequencing protocol. This study was supported in part by National Science Foundation Grant MCB-0110477. Y.-H.L. was supported by the Korea Science and Engineering Foundation and the National Institute of Agricultural Biotechnology, Rural Development Administration, Korea. S.K. was supported by the Office of the Commission for Higher Education, Ministry of Education, Thailand.


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