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. 2013 May 31;8(8):e25169. doi: 10.4161/psb.25169

The copy number of rice CACTA DNA transposons carrying MIR820 does not correlate with MIR820 expression

Misuzu Nosaka 1, Aiko Ishiwata 1, Sae Shimizu-Sato 1, Akemi Ono 1, Kiyoe Ishimoto 1, Yusaku Noda 1, Yutaka Sato 1,*
PMCID: PMC3999066  PMID: 23733074

Abstract

miR820 is a small RNA species (22 and 24 nucleotides), produced from transcripts originated from a region inside CACTA DNA transposons in rice. Because MIR820 is a transposon gene, its expression may depend on the transposon copy number. Here, we investigated the copy number of MIR820 and its expression levels in various cultivars and wild species of rice. We found no correlation between copy number and expression level, suggesting that MIR820 transcription is regulated not by the copy dosage but by the epigenetic state of each copy.

Keywords: transposon, Oryza sativa, DNA methyltransferase, miR820, OsDRM2


Transposable elements (TEs) and their remnants are the major components of eukaryotic genomes.1 TEs have increased their copy number in the host genome because they replicate faster than the host genome. Thus, TEs are referred to as ultimate parasites that proliferate selfishly in the genome.2,3 TE transposition induces insertion mutations and chromosome aberrations, causing instability of the host genome. Therefore, most TEs are kept silent by their host. Small RNA-mediated RNA silencing participates in suppression of TEs; this mechanism has a similar role in plants and animals.4-6

The prominent presence of TEs in the host genome suggests the existence of a running battle between the host defense machinery suppressing transposition of TEs and TEs’ countermeasures against host-mediated silencing. However, little is known about the strategies that TEs have escaped the host silencing. We have previously shown that members of the microRNA820 (miR820) family negatively regulate OsDRM2 (a de novo DNA methyltransferase gene), which allows transposons to escape silencing by the host. We also found a dramatic proliferation of CACTA transposons carrying MIR820 in some wild rice accessions, such as BB- and BBCC-genome species.7 Therefore, we assumed that MIR820 expression would be extremely high in these species.

To test this, we conducted northern blot analysis to detect miR820 in three different cultivated rice accessions (Oryza sativa) and one wild rice species (Oryza punctata, accession W1514) (Fig. 1A). Surprisingly, no expression was detected in W1514 despite the presence of more than 18 copies of MIR820.7 Accordingly, no cleavage of OsDRM2 by miR820 was detected in W1514 (Fig. 1B). Next, we determined the levels of pre-miR820 by quantitative RT-PCR (qRT-PCR) in two O. sativa cultivars (AA) and four wild rice accessions belonging to two species containing the BB or BBCC genome, in which CACTA carrying MIR820 is highly amplified (Fig. 1C).7 The qRT-PCR revealed that the absence of miR820 in W1514 is due to a reduced level of the pre-miR820 transcript, rather than to its reduced processing. This implies that the high copy number of MIR820 in wild rice accession with BB or BBCC genome may cause a stronger MIR820 silencing in these species.

graphic file with name psb-8-e25169-g1.jpg

Figure 1. Expression and functional analysis of miR820 in cultivated rice and wild rice species. (A) Northern blot analysis of miR820 expression in AA and BB Oryza species. (B) Detection of miR820-cleaved OsDRM2 mRNA by RNA ligation-mediated 5′-RACE (upper panel) in AA and BB species. The same cDNA templates were used for PCR to amplify uncleaved OsDRM2 (middle panel) and OsACTIN (bottom panel) as controls. (C) The relative expression levels of pre-MIR820 measured by qRT-PCR in AA, BB and BBCC Oryza species and calculated by subtracting the values with RT reaction by the values without RT reaction, then normalized by OsACTIN. Values are means of triplicate experiments, with bars showing standard errors. Probes and primers are described in our previous work.7

To explore whether the expression level of MIR820 depends on its copy number, we also analyzed rice accessions with low or moderate copy numbers, as determined by Southern hybridization. We used 45 cultivars from the Japanese Rice Core Collection (JRC) and 56 cultivars from the World Rice Core Collection (WRC) (Tables 1 and 2).8,9 Among the JRC cultivars, the average copy number of MIR820 was 4.5 (minimum: 2; maximum: 6). Among the WRC cultivars, the average copy number was 6.3 (minimum: 3; maximum: 11) (Fig. 2A). Then, we determined the levels of pre-miR820 by qRT-PCR in the same accessions and found no correlation between the MIR820 copy number and pre-miR820 levels (Fig. 2B).

Table 1. Copy numbers of MIR820 in the Japanese Rice Core Collection (JRC).

JRC No. Name Origin Copy number
JRC01 Gaisen Mochi Japan (unknown) 4
JRC03 Hinode Kinki 5
JRC04 Senshou Tokyo 4
JRC05 Yamada Bake Kagoshima 4
JRC06 Kaneko B Kantou Touzan 5
JRC07 Iruma Nishiki Saitama 5
JRC08 Okka Modoshi Japan (unknown) 4
JRC10 Hirayama Tokyo 4
JRC11 Kahei Kagoshima 4
JRC12 Oiran Kumamoto 5
JRC13 Bouzu Mochi Ooita 5
JRC17 Akage Akita 4
JRC19 Wataribune Shiga 3
JRC20 Hosogara Aomori 4
JRC21 Akamai Kouchi 5
JRC22 Mansaku Nagano 5
JRC23 Ishijiro Toyama 5
JRC24 Joushuu Yamagata 5
JRC25 Dango Japan (unknown) 5
JRC26 Aikoku Fukui 5
JRC27 Ginbouzu Ishikawa 5
JRC28 Shinriki Mochi Kumamoto 5
JRC29 Shichimenchou Mochi Japan (unknown) 5
JRC30 Morita Wase Yamagata 5
JRC31 Kameji Shimane 6
JRC32 Omachi Okayama 5
JRC33 Shinriki Hyougo 5
JRC34 Kyoutoasahi Kyoto 5
JRC35 Kabashiko Miyazaki 5
JRC37 Shinyamadaho 2 Hyougo 5
JRC38 Nagoya Shiro Akita 2
JRC39 Shiroine Tokushima 4
JRC40 Akamai Nagasaki 5
JRC41 Akamai Tokushima 3
JRC42 Touboshi Kagoshima 5
JRC43 Akamai Kantou Touzan 3
JRC44 Karahoushi Kagoshima 3
JRC46 Fukoku Hokkaido 5
JRC47 Okabo Japan (unknown) 4
JRC48 Hakamuri Kagoshima 4
JRC49 Rikutou Rikuu 2 Japan (unknown) 5
JRC51 Shinshuu Nagano 5
JRC52 Aichiasahi Aichi 3
JRC53 Raiden Kantou Touzan 5
JRC54 Houmanshinden Ine Kagoshima 4

Table 2. Copy numbers of MIR820 in the World Rice Core Collection (WRC).

WRC No. Name Origin Copy number
WRC01 Nipponbare Japan 5
WRC02 Kasalath India 6
WRC03 Bei Khe Cambodia 6
WRC04 Jena 035 Nepal 5
WRC05 Naba India 8
WRC06 Puluik Arang Indonesia 3
WRC07 Davao 1 Philippines 3
WRC09 Ryou Suisan Koumai China 5
WRC10 Shuusoushu China 9
WRC11 Jinguoyin China 5
WRC12 Dahonggu China 7
WRC13 Asu Bhutan 6
WRC14 IR 58 Philippines 4
WRC15 Co 13 India 7
WRC16 Vary Futsi Madagascar 7
WRC17 Keiboba China 11
WRC18 Qingyu (Seiyu) China 8
WRC19 Deng Pao Zhai China 4
WRC20 Tadukan Philippines 6
WRC21 Shwe Nang Gyi Myanmar 6
WRC22 Calotoc Philippines 8
WRC23 Lebed Philippines 5
WRC24 Pinulupot 1 Philippines 5
WRC25 Muha Indonesia 10
WRC26 Jhona 2 India 10
WRC27 Nepal 8 Nepal 9
WRC28 Jarjan Bhutan 7
WRC29 Kalo Dhan Nepal 7
WRC30 Anjana Dhan Nepal 5
WRC31 Shoni Bangladesh 6
WRC32 Tupa 121–3 Bangladesh 6
WRC33 Surjamukhi India 9
WRC34 ARC 7291 India 9
WRC35 ARC 5955 India 9
WRC36 Ratul India 7
WRC37 ARC 7047 India 8
WRC39 Badari Dhan Nepal 8
WRC40 Nepal 555 India 6
WRC41 Kaluheenati Sri Lanka 5
WRC42 Local Basmati India 5
WRC43 Dianyu 1 China 4
WRC44 Basilanon Philippines 6
WRC45 Ma sho Myanmar 6
WRC46 Khao Nok Laos 5
WRC47 Jaguary Brazil 8
WRC48 Khau Mac Kho Vietnam 8
WRC49 Padi Perak Indonesia 8
WRC50 Rexmont USA 5
WRC51 Urasan 1 Japan 7
WRC52 Khau Tan Chiem Vietnam 3
WRC53 Tima Bhutan 5
WRC55 Tupa729 Bangladesh 7
WRC57 Milyang 23 Korea 4
WRC98 Deejiaohualuo China 4
WRC99 Hong Cheuh Zai China 5
WRC100 Vandaran Sri Lanka 5

graphic file with name psb-8-e25169-g2.jpg

Figure 2. Copy numbers and expression levels of MIR820 in the Japanese (JRC) and World (WRC) rice collections. (A) Distribution of the copy number of CACTA transposons carrying MIR820 determined by Southern blot analysis. Genomic DNAs from the NIAS JRC and WRC rice core collections were digested with EcoRI and EcoRV and probed with the pre-miR820 DNA fragment. “Total” is the combination of both collections. (B) The relative expression levels of MIR820 measured by qRT-PCR as the same method mentioned before. The expression levels in all JRC and WRC accessions, in WRC accessions with less than six copies and those with six or more copies of CACTA are indicated. Numbers below JRC all or WRC all indicate the average copy numbers. Values are means of triplicate experiments, with bars showing standard errors. The numbers below JRC or WRC indicate the copy number of MIR820. Probes and primers are described in our previous work.7

Previously, we have shown that miR820 downregulates the expression of de novo DNA methyltransferase, responsible for transposon inactivation.7 Therefore, one would assume that once miR820 effectively suppresses OsDRM2, silencing of transposons (including CACTA carrying MIR820) would be released, resulting in an increased transcription of MIR820. This would lead to a feed-forward loop, reinforcing the function of miR820. However, in this study, we found that the expression of MIR820 is relatively constant in many cultivated rice cultivars, despite its copy number varying from 2 to 11. This suggests that MIR820 transcription is regulated not by copy dosage, but rather by the epigenetic state of each locus. This mechanism may have evolved during the “arms race” between the host and the parasite and may allow the host to inhibit the feed-forward loop triggered by miR820 and thus to prevent the overwhelming victory of the parasites.

Acknowledgments

We thank Ms Tomoko Atsumi for technical assistance. The wild rice accessions and Core Collections of rice cultivars used in this study were obtained from the National Institute of Genetics, supported by the National Bioresource Project, MEXT and the Genebank at the National Institute of Agrobiological Sciences (NIAS), Japan. This work was supported by JSPS KAKENHI Grant 23658006 to Y.S.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed

Footnotes

References

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