An insertional mutation in the rice PAIR2 gene, the ortholog of Arabidopsis ASY1, results in a defect in homologous chromosome pairing during meiosis.

To elucidate the genetic system that establishes homologous chromosome pairing in monocot plants, we have isolated an asynaptic mutant of rice, designated pair2 (homologous pairing aberration in rice meiosis 2), in which 24 completely unpaired univalents are observed at pachytene and diakinesis. The mutation was caused by an insertion of the retrotransposon Tos17, as demonstrated by complementation of the mutation by transformation with the corresponding wild-type gene. The gene in which the element was inserted is orthologous to the ASY1 gene of Arabidopsis thaliana and the HOP1 gene of Saccharomyces cerevisiae. Mature PAIR2 mRNA and several splicing variants were found to be highly expressed in wild-type reproductive tissues, and lower expression was also detected in vegetative tissues. In situ hybridization and BrdU incorporation experiments revealed that PAIR2 expression is specifically enhanced in male and female meiocytes, but not in those at pre-meiotic S phase or in the pollen maturation stages. The results obtained in this study suggest that the PAIR2 gene is essential for homologous chromosome pairing in meiosis, as in the case of the genes ASY1 and HOP1. The study also suggested the possibility that a highly homologous copy of the PAIR2 gene located on a different chromosome is in fact a pseudogene.

Silencing of transposable elements in plants.

Plant genomes contain many transposable elements, most of which are inactivated or 'silenced'. Recent studies have brought significant new insights into the regulation of transposable elements. In Caenorhabditis elegans, they are silenced post-transcriptionally, whereas transposable elements in Arabidopsis are silenced by a chromatin-remodelling factor, one of the components of transcriptional gene silencing. These observations provide the functional correlation between gene silencing and the suppression of transposable elements, and have major implications for our understanding of the maintenance of genomic integrity.

Linear DNA intermediates of the Tto1 retrotransposon in Gag particles accumulated in stressed tobacco and Arabidopsis thaliana.

The active transcription of some plant retrotransposons under diverse stress conditions suggests active transposition. However, transposition has been demonstrated only during tissue/cell culture. To examine whether transposition is activated under conditions other than tissue/cell culture, DNA intermediates for retrotransposition of the tobacco retrotransposon Tto1 were analysed. Using transgenic Arabidopsis callus expressing high levels of Tto1 RNA in a ddm1 hypomethylation mutant background, the existence of extrachromosomal Tto1 linear DNA molecules in a Gag-particle fraction was demonstrated. By combination with ligation-mediated PCR amplification, we detected Tto1 linear DNA molecules in particle fractions from callus and methyl jasmonate-treated leaves of tobacco, but not from non-stressed leaves. Tto1 DNA intermediates could not be detected in the tobacco corolla where Tto1 is expressed. These results indicate that the transcriptional activation of Tto1 by defence-related stresses leads to the synthesis of DNA intermediates, whereas post-transcriptional suppression of Tto1 activity is suggested in the corolla.

The rice retrotransposon Tos17 prefers low-copy-number sequences as integration targets.

The rice retrotransposon Tos17 is highly activated by tissue culture. To evaluate the impact of transposition of Tos17 on the rice genome and examine its utility for insertional mutagenesis, more than 100 sequences flanking newly transposed Tos17 copies were characterised. The 5-bp target-site duplications flanking Tos17 did not show any consensus sequence, and preferred nucleotides, A/T and G/C, were only found at the second and third nucleotides from both ends of the target site duplications, respectively, indicating that Tos17 has relatively low target-site specificity at the nucleotide sequence level. Integration targets were widely distributed over the chromosomes; however, preferential integration into the sucrose synthase 2 gene and into Tos17 itself was demonstrated by PCR screening using pooled DNA prepared from the mutant population. Hybridisation studies indicated that Tos17 preferentially integrates into low-copy-number regions of the genome. In agreement with this result, about 30% of flanking sequences examined showed significant homology to known genes. Taken together, these results show that Tos17 can have a significant impact on the rice genome and can be used as a tool for efficient insertional mutagenesis.

Isolation and characterization of rice phytochrome A mutants.

To elucidate phytochrome A (phyA) function in rice, we screened a large population of retrotransposon (Tos17) insertional mutants by polymerase chain reaction and isolated three independent phyA mutant lines. Sequencing of the Tos17 insertion sites confirmed that the Tos17s interrupted exons of PHYA genes in these mutant lines. Moreover, the phyA polypeptides were not immunochemically detectable in these phyA mutants. The seedlings of phyA mutants grown in continuous far-red light showed essentially the same phenotype as dark-grown seedlings, indicating the insensitivity of phyA mutants to far-red light. The etiolated seedlings of phyA mutants also were insensitive to a pulse of far-red light or very low fluence red light. In contrast, phyA mutants were morphologically indistinguishable from wild type under continuous red light. Therefore, rice phyA controls photomorphogenesis in two distinct modes of photoperception--far-red light-dependent high irradiance response and very low fluence response--and such function seems to be unique and restricted to the deetiolation process. Interestingly, continuous far-red light induced the expression of CAB and RBCS genes in rice phyA seedlings, suggesting the existence of a photoreceptor(s) other than phyA that can perceive continuous far-red light in the etiolated seedlings.

Identification of YAC clones containing the mutable slender glume locus slg in rice (Oryza sativa L.).

A mutable slender glume gene slg, which often reverts to the wild-type state, was induced by gamma-ray irradiation of seeds of the japonica rice cultivar 'Gimbozu'. The final goal was to understand whether the slender glume mutation was associated with the insertion of a transposable element, utilizing map-based cloning techniques. The RFLP (restriction fragment length polymorphism) analysis revealed that the slg locus was located between two RFLP loci, XNpb33 and R1440, on chromosome 7 with recombination values of 3.1% and 1.0%, respectively. Using these two RFLP loci as probes, five YAC (yeast artificial chromosome) clones containing either of these two loci were selected from a YAC library. Subsequently, both end fragments of these YAC clones, amplified by the inverse PCR (IPCR) method, were used to select new YAC clones more closely located to the slg locus. After repeating such a procedure, we successfully constructed a 6-cM YAC contig, and identified four overlapping YAC clones, Y1774, Y3356, Y5124, and Y5762, covering the slg locus. The chromosomal location of the slg was narrowed down to the region with a physical distance of less than 280 kb between the right-end fragments of Y1774 and Y3356.

Contribution of the Tos17 retrotransposon to rice functional genomics.

The ongoing international efforts of the Rice Genomic Sequencing Project have already generated a large amount of sequence data. The next important challenge will be to construct saturation mutant lines for the functional analysis of all of the genes revealed by this effort in the context of the rice plant as a whole. Recently, the endogenous retrotransposon Tos17 has been shown to be an efficient insertional mutagen. Considering the ease of mutagenesis with Tos17 and its multiple-copy nature, saturation mutagenesis with this retrotransposon should be feasible in rice. Ongoing reverse-genetics studies, such as the PCR-screening of mutants and cataloguing of mutants by sequencing Tos17-insertion sites, as well as traditional forward-genetics studies, have clearly demonstrated that the Tos17 system can significantly contribute to the functional genomics of rice.

Applications of retrotransposons as genetic tools in plant biology.

Retrotransposons are mobile genetic elements that accomplish transposition via an RNA intermediate that is reverse transcribed before integration into a new location within the host genome. They are ubiquitous in eukaryotic organisms and constitute a major portion of the nuclear genome (often more than half of the total DNA) in plants. Furthermore, they are dispersed as interspersed repetitive sequences throughout most of the length of all host chromosomes. These unique properties of retrotransposons have been exploited as genetic tools for plant genome analysis. Major applications are in determining phylogeny and genetic diversity and in the functional analyses of genes in plants. Here, recent advances in molecular markers, gene tagging and functional genomics technologies using plant retrotransposons are described.

Screening of the rice viviparous mutants generated by endogenous retrotransposon Tos17 insertion. Tagging of a zeaxanthin epoxidase gene and a novel ostatc gene.

The rice (Oryza sativa) retrotransposon Tos17 is one of a few active retrotransposons in plants and its transposition is activated by tissue culture. Here, we present the characterization of viviparous mutants of rice induced by tissue culture to demonstrate the feasibility of the use of retrotransposon Tos17 as an endogenous insertional mutagen and cloning of the tagged gene for forward genetics in unraveling the gene function. Two mutants were shown to be caused by the insertion of Tos17. Osaba1, a strong viviparous mutant with wilty phenotype, displayed low abscisic acid level and almost no further increase in its levels upon drought. The mutant is shown to be impaired in the epoxidation of zeaxanthin. On the other hand, Ostatc, a mutant with weak phenotype, exhibited the pale green phenotype and slight increase in abscisic acid levels upon drought. Deduced amino acids of the causative genes of Osaba1 and Ostatc manifested a significantly high homology with zeaxanthin epoxidase isolated from other plant species and with bacterial Sec-independent translocase TATC protein, respectively. This is the first example of transposon tagging in rice.

Efficient insertion mutagenesis of Arabidopsis by tissue culture-induced activation of the tobacco retrotransposon Tto1.

The tobacco retrotransposon Tto1 is one of a few active retrotransposons in plants. Its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. Here we demonstrate that Tto1 introduced in Arabidopsis is also activated by tissue culture. Transcription of Tto1 was induced by tissue culture and driven by its LTR promoter. Transposed copies of Tto1 were observed in almost all of the plants regenerated from the explants cultured for only 1 week. A total of 255 independent regenerated lines have been produced, and the average copy number of transposed Tto1 in these lines is estimated to be 3.2. Sequences flanking Tto1 were amplified by thermal asymmetric interlaced (TAIL)-PCR. Of 165 independent amplified products, 123 showed significant homology to known genes or hypothetical protein genes. The insertion sites of Tto1 are spread over all chromosomes and the target site sequence shows moderate consensus. Taken together, these results indicate that Tto1 can be used as a tool for efficient insertion mutagenesis of Arabidopsis which is especially suitable as a reverse genetics system.

Silencing of retrotransposons in arabidopsis and reactivation by the ddm1 mutation.

Gene silencing associated with repeated DNA sequences has been reported for many eukaryotes, including plants. However, its biological significance remains to be determined. One important function that has been proposed is the suppression of transposons. Here, we address transposon suppression by examining the behavior of the tobacco retrotransposon Tto1 and endogenous retrotransposons in Arabidopsis. After an initial increase in copy number because of active transposition in the Arabidopsis genome, Tto1 became silent. The amount of transcript was reduced, and the inactivated Tto1 became methylated. This silencing correlated with an increase in copy number. These phenomena mimic repeat-induced gene silencing. The homozygous ddm1 (for decrease in DNA methylation) mutation of Arabidopsis results in genomic DNA hypomethylation and the release of silencing in repeated genes. To investigate the role of DNA methylation and the gene-silencing machinery in the suppression of Tto1, we introduced the ddm1 mutation into an Arabidopsis line carrying inactivated Tto1 copies. In the homozygous ddm1 background, Tto1 became hypomethylated and transcriptionally and transpositionally active. In addition, one of the newly isolated endogenous Arabidopsis retrotransposon families, named Tar17, also became hypomethylated and transcriptionally active in the ddm1 mutant background. Our results suggest that the inactivation of retrotransposons and the silencing of repeated genes have mechanisms in common.

MYB-related transcription factor NtMYB2 induced by wounding and elicitors is a regulator of the tobacco retrotransposon Tto1 and defense-related genes.

Transposition of the tobacco retrotransposon Tto1 is regulated mainly by transcription from the long terminal repeat (LTR). Functional analysis of the LTR showed that the 13-bp motif is a cis-regulatory element involved in activation by tissue culture, wounding, and treatment with elicitors. The 13-bp motif contains a conserved motif (L box) that has been implicated in the expression of phenylpropanoid synthetic genes in response to defense-related stresses. To gain further insight into the regulatory mechanism of the retrotransposon and defense-related genes, cDNAs encoding four different proteins binding to the 13-bp motif have been isolated and characterized. One protein is identical to the previously reported NtMYB1, the RNA for which is induced by virus infection; the others are also MYB-related factors. One of these factors, NtMYB2, was analyzed in detail. NtMYB2 mRNA was induced by wounding and by treatment with elicitors. NtMYB2 activated expression from the promoter with the 13-bp motif and from the promoter of the phenylalanine ammonia lyase gene (Pv-PAL2) in tobacco protoplasts. Overexpression of NtMYB2 cDNA in transgenic tobacco plants induced expression of Tto1 and a PAL gene. Together, these results indicate that NtMYB2 is involved in the stress response of the retrotransposon and defense-related genes.

A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors.

The tobacco Tto1 is one of the few active LTR-retrotransposons of plants, and its transposition is activated by tissue culture and is primarily regulated at the transcriptional level. The expression of Tto1 RNA can also be activated by various stresses, including viral infection, wounding, and treatment with jasmonate, a signal molecule of plant defence responses. It is shown here that the Tto1 LTR promoter is responsible for a high level of expression in cultured tissues of transgenic tobacco plants. We demonstrate that a 13-bp repeated motif (TGGTAGGTGAGAT) in the LTR functions as a cis-regulatory element, which confers the responsiveness to tissue culture, wounding and methyl jasmonate. Fungal elicitors also activate the promoter containing multiple copies of the 13-bp motif. Expression mediated by the 13-bp motif is activated markedly by okadaic acid and moderately by K252a, so that both phosphorylation and dephosphorylation of proteins are possibly involved in the signalling pathways. Interestingly, the 13-bp motif contains a conserved motif, Box L (also called AC-I or H-box like sequence) which has been shown to be involved in the expression of phenylpropanoid synthetic genes. Moreover, extended homologies are found between promoters of Tto1 and an asparagus defence gene, AoPR1, suggesting a possibility that the ancient insertion of an ancestral Tto1-related retrotransposon has provided some of the promoter/regulatory sequences, including the 13-bp motif-related sequence, of the AoPR1 gene. Based on the structural and functional similarity between the two promoters, a possible evolutionary role of the regulatory sequences of LTR-retrotransposons is discussed.

Identification of mutable slender glume gene in rice (Oryza sativa L.).

The segregation pattern and chromosomal location of a slender glume mutation, induced by gamma-ray irradiation, was investigated. The mutation is genetically unstable: in the selfed progenies of slender glumed plants, not only plants with normal glumes but also plants that are chimeric for glume shape almost always appear at low frequency. The results showed that the mutation is controlled by a single recessive, mutable mutant gene slg. The frequency of reversion of slg to its wild-type state was little affected by crossing, back-crossing, genetic background or cytoplasmic factors. Conventional trisomic and linkage analyses revealed that the slg locus was located close to the rfs (rolled fine stripe leaf) locus on chromosome 7. In a subsequent RFLP analysis, slg was found to be located between the two RFLP loci XNpb20 and XNpb33, with recombination values of 3.0 and 3.2%, respectively. Southern analysis indicated that the mutability of slg is caused by none of the known transposable elements in rice. From these results, we infer that slg has a novel transposable DNA insert in its vicinity, which was possibly activated by gamma-ray irradiation.

Meiotically and mitotically stable inheritance of DNA hypomethylation induced by ddm1 mutation of Arabidopsis thaliana.

In contrast to mammalian epigenetic phenomena, where resetting of gene expression generally occurs in each generation, epigenetic states of plant genes are often stably transmitted through generations. The Arabidopsis mutation ddm1 causes a 70% reduction in genomic 5-methylcytosine level. We have previously shown that the ddm1 mutation results in an accumulation of a variety of developmental abnormalities by slowly inducing heritable changes in other loci. Each of the examined ddm1-induced developmental abnormalities is stably transmitted even when segregated from the potentiating ddm1 mutation. Here, the inheritance of DNA hypomethylation induced by ddm1 was examined in outcross progeny by HPLC and Southern analyses. The results indicate that (i) DDM1 gene function is not necessary during the gametophyte stage, (ii) ddm1 mutation is completely recessive, and (iii) remethylation of sequences hypomethylated by the ddm1 mutation is extremely slow or nonexistent even in wild-type DDM1 backgrounds. The stable transmission of DNA methylation status may be related to the meiotic heritability of the ddm1-induced developmental abnormalities.

Loss-of-function mutations in the rice homeobox gene OSH15 affect the architecture of internodes resulting in dwarf plants.

The rice homeobox gene OSH15 (Oryza sativa homeobox) is a member of the knotted1-type homeobox gene family. We report here on the identification and characterization of a loss-of-function mutation in OSH15 from a library of retrotransposon-tagged lines of rice. Based on the phenotype and map position, we have identified three independent deletion alleles of the locus among conventional morphological mutants. All of these recessive mutations, which are considered to be null alleles, exhibit defects in internode elongation. Introduction of a 14 kbp genomic DNA fragment that includes all exons, introns and 5'- and 3'- flanking sequences of OSH15 complemented the defects in internode elongation, confirming that they were caused by the loss-of-function of OSH15. Internodes of the mutants had abnormal-shaped epidermal and hypodermal cells and showed an unusual arrangement of small vascular bundles. These mutations demonstrate a role for OSH15 in the development of rice internodes. This is the first evidence that the knotted1-type homeobox genes have roles other than shoot apical meristem formation and/or maintenance in plant development.

Transcriptional activation of the tobacco retrotransposon Tto1 by wounding and methyl jasmonate.

The tobacco retrotransposon Tto1, one of a few active retrotransposons of plants, has been shown to be activated by tissue culture. Its transposition is regulated mainly at the transcriptional level. It is shown here that expression of Tto1 can be induced in leaves of tobacco by wounding stress. Exogenous supply of methyl jasmonate, which is known to be a potent inducer of certain wound-responsive genes in plants, also induces Tto1 RNA expression. Tto1 RNA was detected within 2 to 4 h after wounding/cutting treatment, and increased levels of Tto1 RNA were observed during subsequent incubation periods for 48 h. Expression of Tto1 RNA after cutting treatment was induced more significantly in young expanding leaves rather than in older mature leaves, suggesting that developmental or physiological factors may be required for the strong response to Tto1 transcription to wounding stimuli. Experiments with transgenic tobacco plants carrying the Tto1-LTR: beta-glucuronidase fusion gene (LTR:GUS) revealed that Tto1 actually contains cis-regulatory regions in response to wounding and methyl jasmonate. These findings are discussed in relation to the mechanism of transcriptional activation and the evolutionary role played by retrotransposons.

Retrotransposons of rice: their regulation and use for genome analysis.

Retrotransposons were extensively surveyed in rice using two molecular methods. The total copy number of retrotransposons in the rice genome was estimated to be about 1000 and 32 families were isolated, showing that retrotransposons are a major class of transposable elements in rice. Although these retrotransposons appear inactive during normal growth conditions, 5 out of 32 families were active under tissue culture conditions. The most active element, Tos17, was studied in detail. Its activity was show to be regulated mainly at the transcriptional level. The analysis of target sites of transposition indicated that activation of Tos17 is an important cause of tissue culture-induced mutations in rice. Tissue culture-induced activation of Tos17 was used to develop the site-selected mutagenesis system, in which mutants carrying a Tos17 insertion in the gene of interest can be identified among rice plants regenerated from tissue culture by the PCR using one primer for the ends of Tos17 and another for the gene of interest. This system will contribute to understanding the functions of rice genes whose sequences are being determined by the rice genome project.

A mutation study of the DNA binding domain of human papillomavirus type11 E2 protein.

A site-specific mutation study was performed on the C-terminal domain, containing a cloned DNA binding region, of the human papillomavirus type11 (HPV11) E2 protein to determine the specific properties of residues directly involved in the DNA binding. The effect of a point mutations on the DNA binding was assessed by means of a gel mobility shift assay. The mutagenesis was concentrated on the residues in the third helix from the N-terminal, that is known as the "recognition helix," in the crystal structure of the bovine papillomavirus (BPV) E2 protein. Most point mutations caused a great decrease in the DNA binding activity. The leucine repeat in the DNA binding region was proved not to be a leucine prerequisite, as the leucines could be substituted by valine without significant loss of the DNA binding ability. Substitution of Leu for Glu caused a significant decrease in the DNA binding, indicating that the hydrophobicity of the residue at this position is important. The results suggest that the individual contribution of each amino acid residue in the DNA binding region is essential for the DNA binding.

The complete nucleotide sequence and characterization of the linear DNA plasmid pRS64-2 from the plant pathogenic fungus Rhizoctonia solani.

The complete nucleotide sequence of one of three linear DNA plasmids (pRS64-2) from the plant pathogenic fungus Rhizoctonia solani was determined. The pRS64-2 DNA consisted of 2877 nucleotides. The nucleotide sequences of the middle 2.2-kb regions of the other two plasmids (pRS64-1 and pRS64-3) were also determined. Comparison of the nucleotide sequences among the three plasmid DNAs indicated the presence of four regions with more than 86% sequence homology, suggesting the development of three plasmid DNAs from a common ancestor. A computer-based study of the pRS64-2 DNA-folding at both termini predicted hairpin loop structures. The hairpin loops consisted of the left- and right-hand terminal 113 and 105 nucleotides, respectively, and had no sequence homology. They had not undergone flip-flop inversion. The hairpins form cruciform base-paired structures.