An active DNA transposon nDart causing leaf variegation and mutable dwarfism and its related elements in rice

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.     

Distribution and mapping of an active autonomous <Emphasis Type="Italic">aDart</Emphasis> element responsible for mobilizing nonautonomous <Emphasis Type="Italic">nDart1</Emphasis> transposons in cultivated rice varieties

An endogenous 0.6-kb rice DNA transposon, nDart1, has been identified as a causative element of a spontaneous mutable virescent allele pyl-v conferring pale-yellow leaves with dark-green sectors in the seedlings, due to somatic excision of nDart1 integrated into the OsClpP5 gene encoding the nuclear-coded chloroplast protease. As the transposition of nDart1 depends on the presence of an active autonomous aDart element in the genome, the plants exhibiting the leaf variegation carry the active aDart element. As several mutable alleles caused by nDart1 insertions have subsequently been identified, nDart1-promoted gene tagging has been proven to be an effective system. At present, the nDart/aDart system appears to be the only endogenous rice DNA transposon system whose transposition activity can be controlled under natural growth conditions without any artificial treatments, including tissue cultures. To apply the nDart/aDart tagging system in various cultivated rice varieties, we explored the presence and distribution of an active autonomous aDart element in 19 temperate japonica, 30 tropical japonica, and 51 indica varieties. Only eight temperate japonica varieties were found to bear a single copy of an active aDart element, and no aDart activity could be detected in the indica varieties examined. Six of seven japonica varieties appear to carry the active aDart element at the identical site on chromosome 6, whereas the remaining one contains aDart on chromosome 5. Leaf variegations in the plants with the mutable pyl-v allele and the excision frequencies of endogenous nDart1 elements indicated that the aDart element on chromosome 6 is more active than that on chromosome 5. The findings described here are an important step in the development of a new and efficient nDart1-promoted gene-tagging system in various rice cultivars. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Transposition and target preferences of an active nonautonomous DNA transposon nDart1 and its relatives belonging to the hAT superfamily in rice

The nonautonomous nDart1 element in the hAT superfamily is one of a few active DNA transposons in rice. Its transposition can be induced by crossing with a line containing an active autonomous element, aDart1, and stabilized by segregating aDart1. No somaclonal variation should occur in nDart1-promoted gene tagging because no tissue culture is involved in nDart1 activation. By transposon display analysis, we examined the activities of nDart1-related elements in the selfed progeny of a mutable virescent pyl-v plant containing aDart1. Although various nDart1-related elements are present in the rice genome, only nDart1-3 subgroup elements, nDart1-0 and nDart1-3 in particular, were found to be transposed frequently and integrated into various sites almost all over the genome, and a fraction of the transposed elements were found to be transmitted to the next generation. More than half of the newly integrated elements were identified as nDart1-0. Analysis of the newly inserted sites revealed that the nDart1-3 subgroup elements were predominantly integrated into single-copy regions. More than 60% of the transposed elements were inserted into the genic regions that comprise putative coding regions and their 0.5-kb flanking segments, and approximately two-thirds of them were within the 0.5-kb area in front of the putative initiation codons, i.e., promoter-proximal genic regions. These characteristic features of nDart1-3 subgroup elements seem to be suitable for developing an efficient and somaclonal variation-free gene tagging system for rice functional genomics.     

Activation and epigenetic regulation of DNA transposon nDart1 in rice

A large part of the rice genome is composed of transposons. Since active excision/reintegration of these mobile elements may result in harmful genetic changes, many transposons are maintained in a genetically or epigenetically inactivated state. However, some non-autonomous DNA transposons of the nDart1-3 subgroup, including nDart1-0, actively transpose in specific rice lines, such as pyl-v which carries an active autonomous element, aDart1-27, on chromosome 6. Although nDart1-3 subgroup elements show considerable sequence identity, they display different excision frequencies. The most active element, nDart1-0, had a low cytosine methylation status. The aDart1-27 sequence showed conservation between pyl-stb (pyl-v derivative line) and Nipponbare, which both lack autonomous activity for transposition of nDart1-3 subgroup elements. In pyl-v plants, the promoter region of the aDart1-27 transposase gene was more hypomethylated than in other rice lines. Treatment with the methylation inhibitor 5-azacytidine (5-azaC) induced transposition of nDart1-3 subgroup elements in both pyl-stb and Nipponbare plants; the new insertion sites were frequently located in genic regions. 5-AzaC treatment principally induced expression of Dart1-34 transposase rather than the other 38 aDart1-related elements in both pyl-stb and Nipponbare treatment groups. Our observations show that transposition of nDart1-3 subgroup elements in the nDart1/aDart1 tagging system is correlated with the level of DNA methylation. Our system does not cause somaclonal variation due to an absence of transformed plants, offers the possibility of large-scale screening in the field and can identify dominant mutants. We therefore propose that this tagging system provides a valuable addition to the tools available for rice functional genomics.     

Site specific cytosine methylation in rice nonautonomous transposable element <Emphasis Type="Italic">nDart</Emphasis>

The mobile nonautonomous element nDart, which is active in intact rice plants, exhibits locus specific transposition. Due to the high homogeneity of nDart elements, the locus specificity of nDart transposition might be controlled by factors other than genetic differences. In this study, we elucidated the regulation of the locus specificity of nDart transposition. The difference of transpositional activities in 10 nDart elements among rice varieties exhibiting nDart transposition was clearly correlated with the methylation state of nDart elements. Both hyper- and hypo-methylated nDart elements were inactive, while site specific methylation in both subterminal regions was identified in active nDart loci. The specific methylation sites contain the pentamer motif GCC/ACG. The repeated motifs in the subterminal region of nDart elements may contribute to the stable secondary structure of nDart elements with low free energy. Our results suggested that site specific cytosine methylation may loosen the stable secondary structure of the nDart element to allow it to bind TPase, which then perform the excision of nDart elements from genomic loci.     

Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Transposon display for active DNA transposons in rice

Transposon display (TD) is a powerful technique to identify the integration site of transposons in gene tagging as a functional genomic tool for elucidating gene function. Although active endogenous DNA transposons have been used extensively for gene tagging in maize, only two active endogenous DNA transposons in rice have been identified, the 0.43-kb element mPing of the MITE family and the 0.6-kb nDart element of the hAT family. The nDart transposition was shown to be induced by crossing with a line containing its autonomous element aDart and stabilized by segregating aDart under natural growth conditions, while mPing-related elements were shown to transpose in cultured cells, plants regenerated from an anther culture, and gamma-ray-irradiated plants. No somaclonal variation should occur in nDart-promoted gene tagging because no tissue culture was involved in nDart activation. As an initial step to develop an effective tagging system using nDart in rice, we tried to visualize GC-rich nDart-related elements comprising 18 nDart-related sequences of 0.6-kb and 63 nDart-related elements longer than 2 kb in Nipponbare by TD. Comparing the observed bands in TD with the anticipated virtual bands of the nDart-related elements based upon the available rice genome sequence, we have improved our TD protocol by optimizing the PCR amplification conditions and are able to visualize approximately 87% of the anticipated bands produced from the nDart-related elements. To compare the visualization efficiency of these nDart-related elements with that of 50 mPing elements and a unique Ping sequence in Nipponbare, we also tried to visualize the mPing-related elements; all mPing-related elements are easily visualized. Based on these results, we discuss the parameters affecting the visualization efficiencies of these rice DNA transposons. We also discuss the utilization of nDart elements in gene tagging for functional genomics in rice.     

Overexpression of a NAC-domain protein promotes shoot branching in rice

For a better understanding of shoot branching in rice (Oryza sativa), a rice activation-tagging library was screened for mutations in tiller development. Here, an activation-tagging mutant Ostil1 (Oryza sativa tillering1) was characterized, which showed increased tillers, enlarged tiller angle and semidwarf phenotype. Flanking sequence was obtained by plasmid rescue. RNA-interfering and overexpression transgenic rice plants were produced using Agrobacterium-mediated transformation. The mutant phenotype was cosegregated with the reallocation of Ds element, and the flanking region of the reallocated Ds element was identified as part of the OsNAC2 gene. Northern analysis showed that expression of OsNAC2 was greatly induced in the mutant plants. Transgenic rice overexpressing the OsNAC2 resulted in recapture of the mutant phenotype, while downregulation of OsNAC2 in the Ostil1 mutant through RNA interfering (RNAi) complemented the mutant phenotype, confirming that the Ostil1 was caused by overexpression of OsNAC2. Overexpression of OsNAC2 regulates shoot branching in rice. Overexpression of OsNAC2 contributes tiller bud outgrowth, but does not affect tiller bud initiation. This suggests that OsNAC2 has potential utility for improving plant structure for higher light-use efficiency and higher yield potential in rice.     

The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.     

Shotgun cloning of transposon insertions in the genome of Caenorhabditis elegans

We present a strategy to identify and map large numbers of transposon insertions in the genome of Caenorhabditis elegans. Our approach makes use of the mutator strain mut-7, which has germline-transposition activity of the Tc1/mariner family of transposons, a display protocol to detect new transposon insertions, and the availability of the genomic sequence of C. elegans. From a pilot insertional mutagenesis screen, we have obtained 351 new Tc1 transposons inserted in or near 219 predicted C. elegans genes. The strategy presented provides an approach to isolate insertions of natural transposable elements in many C. elegans genes and to create a large-scale collection of C. elegans mutants.     

Regulation of white locus expression: the structure of mutant alleles at the white locus of Drosophila melanogaster

We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDZL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus.     

Genetic interaction between 2 tillering genes, reduced culm number 1 (rcn1) and tillering dwarf gene d3, in rice

Mutant genes, reduced culm number 1 (rcn1) and bunketsuwaito tillering dwarf (d3), affect tiller number in rice (Oryza sativa L.) in opposite directions. The d3 mutant was reported to increase tiller number and reduce plant stature. Our objective was to compare the phenotype of the d3rcn1 double mutant with each single mutant and parental rice cultivar "Shiokari" and to clarify whether the Rcn1 gene interacted with the D3 gene. We recovered a new rcn1 mutant from Shiokari and developed d3rcn1 double mutant with Shiokari genetic background. A new rcn1 mutant, designated as "S-97-61" exhibited a reduction in tiller number and plant stature to about the same level as the previously reported original rcn1 mutant. Three near-isogenic lines, rcn1 mutant, d3 mutant, and d3rcn1 double mutant, were grown together with the parental Shiokari. The reduction in tillering by the rcn1 mutation was independent of the d3 genotype, and tillering number of d3rcn1 double mutant was between those of the d3 and rcn1 mutants. These results demonstrated that the Rcn1 gene was not involved in the D3-associated pathway in tillering control.     

Target site choice of the related transposable elements Tc1 and Tc3 of Caenorhabditis elegans.

We have investigated the target choice of the related transposable elements Tc1 and Tc3 of the nematode C. elegans. The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions in an 1 kbp region of the genome were determined. There was no phenotypic selection for the insertions. All insertions were into the sequence TA. Both elements have a strong preference for certain positions in the 1 kbp region. Hot sites for integration are not clustered or regularly spaced. The orientation of the integrated transposon has no effect on the distribution pattern. We tested several explanations for the target site preference. If simple structural features of the DNA (e.g. bends) would mark hot sites, we would expect the patterns of the two related transposons Tc1 and Tc3 to be similar; however we found them to be completely different. Furthermore we found that the sequence at the donor site has no effect on the choice of the new insertion site, because the insertion pattern of a transposon that jumps from a transgenic donor site is identical to the insertion pattern of transposons jumping from endogenous genomic donor sites. The most likely explanation for the target choice is therefore that the primary sequence of the target site is recognized by the transposase. However, alignment of the Tc1 and Tc3 integration sites does not reveal a strong consensus sequence for either transposon.     

水稻基因组上一个Ds切离及其双位点插入行为的分子鉴定

玉米转座元件Ac/Ds是hAT转座子家族的成员, 导入水稻基因组后具有转座活性, 尽管转座机制还不完全清楚, 但它们通常经保守的非复制型“剪切-粘贴”过程转座。研究表明, 在Ac编码的转座酶作用下, Ds从原位点切离后常优先重新插入到连锁位点。文章利用TAIL-PCR技术从水稻一个Ds插入突变体及其回复突变体中分离Ds侧翼序列, 结合生物信息学分析方法, 对Ds在突变体上插入位点、回复突变体内切离足迹和重新插入位点进行了分子鉴定。结果显示, 突变体中Ds从3号染色体切离后, 在原插入位点残留了8 bp足迹序列(CATCATGA), 引起Ds标记基因外显子和内含子数目增加, 从而影响基因结构。切离后的Ds重新插入回复突变体第2和第6号染色体上, 分别编码烟草胺氨基转移酶和衰老相关蛋白的2个基因的编码区。因此, 典型的“剪切-粘贴”机制不能完全解释Ds的转座行为, Ds转座存在“剪切-复制-粘贴”的特点。     

Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences

A two-element Activator/Dissociation (Ac/Ds) gene trap system was successfully established in rice (Oryza sativa ssp. japonica cv. Nipponbare) to generate a collection of stable, unlinked and single-copy Ds transposants. The germinal transposition frequency of Ds was estimated as an average of 51% by analyzing 4413 families. Study of Ds transposition pattern in siblings revealed that 79% had at least two different insertions, suggesting late transposition during rice development. Analysis of 2057 Ds flanking sequences showed that 88% of them were unique, whereas the rest within T-DNA. The insertions were distributed randomly throughout the genome; however, there was a bias toward chromosomes 4 and 7, which had two times as many insertions as that expected. A hot spot for Ds insertions was identified on chromosome 7 within a 40-kbp region. One-third of Ds flanking sequences was homologous to either proteins or rice expressed sequence tags (ESTs), confirming a preference for Ds transposition into coding regions. Analysis of 200 Ds lines on chromosome 1 revealed that 72% insertions were found in genic region. Anchoring of more than 800 insertions to yeast artificial chromosome (YAC)-based EST map showed that Ds transposes preferentially into regions rich in expressed sequences. High germinal transposition frequency and independent transpositions among siblings show that the efficiency of this system is suitable for large-scale transposon mutagenesis in rice.     

A Dominant Mutation in ARL2 Causes Impaired Adventitious Root Development in Rice

Adventitious roots are vital for water and nutrient assimilation by cereal crops because they comprise the bulk of the fibrous root system. We isolated and analyzed a rice mutant, adventitious rootless 2 (arl2), which failed to initiate adventitious root primordia during early development. Its seminal root produced fewer lateral roots than from the wild type. This mutant also exhibited pleiotropic phenotypes of longer and thicker seminal roots, a different morphology for the first leaf, delayed heading, and a greater tiller angle. Physiological experiments showed that exogenous auxin and ethylene could rescue adventitious root growth, a response opposite that for two previously reported mutants, arl1 and gnom1. Activity in the auxin signal pathway and the polar auxin transport system was normal for arl2. Compared with the wild type, arl2 plants showed enhanced sensitivity to ethephon but decreased sensitivity to AgNO₃, an inhibitor of ethylene. Genetics analysis demonstrated that this mutant is controlled by a single dominant gene; ARL2 was mapped within a 100-kb interval on the short arm of chromosome 2.     

Recently mobilized transposons in the human and chimpanzee genomes

Transposable genetic elements are abundant in the genomes of most organisms, including humans. These endogenous mutagens can alter genes, promote genomic rearrangements, and may help to drive the speciation of organisms. In this study, we identified almost 11,000 transposon copies that are differentially present in the human and chimpanzee genomes. Most of these transposon copies were mobilized after the existence of a common ancestor of humans and chimpanzees, approximately 6 million years ago. Alu, L1, and SVA insertions accounted for >95% of the insertions in both species. Our data indicate that humans have supported higher levels of transposition than have chimpanzees during the past several million years and have amplified different transposon subfamilies. In both species, approximately 34% of the insertions were located within known genes. These insertions represent a form of species-specific genetic variation that may have contributed to the differential evolution of humans and chimpanzees. In addition to providing an initial overview of recently mobilized elements, our collections will be useful for assessing the impact of these insertions on their hosts and for studying the transposition mechanisms of these elements.     

Natural genetic variation caused by transposable elements in humans

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25-35% of approximately 2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.