• The Korean Journal of Mycology
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• v.42 no.1
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• pp.50-56
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• 2014

Transposable elements (TEs) are mobile DNA elements that often cause mutations in genes and alterations in the chromosome structure. In order to identify and characterize transposable elements (TEs) in Pleurotus eryngii, a TE-enriched library was constructed using two sets of TE-specific degenerated primers, which target conserved sequences of RT and RVE domains in fungal LTR retrotransposons. A total of 256 clones were randomly chosen from the library and their insert sequences were determined. Comparative investigation of the insert sequences with those in repeat element database, Repbase, revealed that 71 of them were found to be TE-related fragments with significant similarity to LTR retrotransposons from other species. Among the TE sequences, the 70 TEs were Gypsy-type LTR retrotransposons, including 20 of MarY1 from Tricholoma matsutake, 26 of Gypsy-8_SLL from Serpula lacrymans, and 16 of RMER17D_MM from mouse, whereas a single sequence, Copia-48-PTR, was found as only Copia-type LTR retrotransposon. Southern blot analysis of the HindIII-digested P. eryngii genomic DNA showed that the retrotransposon sequences similar to MarY1 and Gypsy-8_SLL were contained as high as 14 and 18 copies per genome, respectively, whereas other retrotransposons were remained low. Moreover, both of the two Gypsy retrotransposons were expressed in full length mRNA as shown by Northern blot analysis, suggesting that they were functionally active retrotransposons.

• The Plant Pathology Journal
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• v.16 no.2
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• pp.105-109
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• 2000

The Pi-b is the rice gene conferring race specific resistance to the blast fungus Magnaporthe grisea race having a corresponding avirulence gene, AVR-Pi-b. All resistant cultivars have two copies of the Pi-b gene, but susceptible cultivars have a single copy of the gene. About 1 Kbp insertion sequence was detected in the open reading frame of the Pi-b gene from the susceptible cv. Nipponbare. The nature of insertion sequence was identified as a solo long terminal repeat (LTR) of new rice Tyl-copia-like retrotransposon. LTR was widely distributed in the rice genome. Various types of different patterns of restriction fragment length polymorphism of LTR were detected in indica cultivars, whereas a single type was detected from japonica cultivars. The insertion of LTR sequence in the Pi-b gene in the susceptible cultivar suggested that retrotransposon-mediated insertional mutation might played an important role in the resistance breakdown as well as evolution of resistance genes in rice.

• Parasites, Hosts and Diseases
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• v.41 no.4
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• pp.221-231
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• 2003

To gain information on retrotransposons in the genome of Paragonimus westermani, PCR was carried out with degenerate primers, specific to protease and reverse transcriptase (rt) genes of long-terminal-repeat (LTR) retrotransposons. The PCR products were cloned and sequenced, after which 12 different retrotransposon-related sequences were isolated from the trematode genome. These showed various degrees of identity to the polyprotein of divergent retrotransposon families. A phylogenetic analysis demonstrated that these sequences could be classified into three different families of LTR retrotransposons, namely, Xena, Bel, and Gypsy families. Of these, two mRNA transcripts were detected by reverse transcriptase-PCR, showing that these two elements preserved their mobile activities. The genomic distributions of these two sequences were found to be highly repetitive. These results suggest that there are diverse retrotransposons including the ancient Xena family in the genome of P. westermani, which may have been involved in the evolution of the host genome.

• The Plant Pathology Journal
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• v.19 no.1
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• pp.57-63
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• 2003

An insertion sequence identified as a solo long terminal repeat (LTR) of a new rice copia-like retrotransposon was detected in the ORE of the Pi-b gene from the rice cv. Nipponbare, and was designated as Osr1. Osr1 consists of a 6386 bp nucleotide sequence including 965 bp LTRs on both ends with an 82％ nucleotide sequence identity to the wheat Tarl retrotransposon on reverse transcriptase. Nucleotide divergence was noted among the individual LTRs, as well as the coding region of Osr1. Various restriction fragment length polymorphism (RFLP) of LTR were detected in indica cultivars, whereas, only a few could be detected in the japonica cultivars. The population of Osr1 is lower in the wild-type rice compared with that in the domesticated cultivars. The insertion of LTR sequence in the h-b gene in the susceptible cultivar suggested that retro-tyansposon-mediated insertional mutation might play an important role in the resistance breakdown, as well as in the evolution of resistance genes in rice.

• Parasites, Hosts and Diseases
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• v.41 no.4
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• pp.209-219
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• 2003

The evolutionary course of the CsRn1 long-terminal-repeat (LTR) retrotransposon was predicted by conducting a phylogenetic analysis with its paralog LTR sequences. Based on the clustering patterns in the phylogenetic tree, multiple CsRn1 copies could be grouped into four subsets, which were shown to have different integration times. Their differential sequence divergences and heterogeneous integration patterns strongly suggested that these subsets appeared sequentially in the genome of C. sinensis. Members of recently expanding subset showed the lowest level of divergence in their L TR and reverse transcriptase gene sequences. They were also shown to be highly polymorphic among individual genomes of the trematode. The CsRn1 element exhibited a preference for repetitive, agenic chromosomal regions in terms of selecting integration targets. Our results suggested that CsRn1 might induce a considerable degree of intergenomic variation and, thereby, have influenced the evolution of the C. sinensis genome.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.20-20
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• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.97-97
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• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.

• BMB Reports
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• v.53 no.10
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• pp.521-526
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• 2020

Endogenous retroviruses (ERVs) are retrotransposons present in various metazoan genomes and have been implicated in metazoan evolution as well as in nematodes and humans. The long terminal repeat (LTR) retrotransposons contain several regulatory sequences including promoters and enhancers that regulate endogenous gene expression and thereby control organismal development and response to environmental change. ERVs including the LTR retrotransposons constitute 8% of the human genome and less than 0.6% of the Caenorhabditis elegans (C. elegans) genome, a nematode genetic model system. To investigate the evolutionarily conserved mechanism behind the transcriptional activity of retrotransposons, we generated a transgenic worm model driving green fluorescent protein (GFP) expression using Human endogenous retroviruses (HERV)-K LTR as a promoter. The promoter activity of HERV-K LTR was robust and fluorescence was observed in various tissues throughout the developmental process. Interestingly, persistent GFP expression was specifically detected in the adult vulva muscle. Using deletion constructs, we found that the region from positions 675 to 868 containing the TATA box was necessary for promoter activity driving gene expression in the vulva. Interestingly, we found that the promoter activity of the LTR was dependent on che-1 transcription factor, a sensory neuron driver, and lin-15b, a negative regulator of RNAi and germline gene expression. These results suggest evolutionary conservation of the LTR retrotransposon activity in transcriptional regulation as well as the possibility of che-1 function in non-neuronal tissues.

• Mycobiology
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• v.36 no.3
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• pp.161-166
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• 2008

The retrotransposon marY1 is a gypsy family retroelement, which is detected ubiquitously within the fungal taxonomic groups in which mushrooms are included. To utilize marY1 as a molecular marker for the DNA fingerprinting of mushrooms, oligonucleotides marY1-LTR-L and marY1-LTR-R were designed on the basis of highly conserved regions from the multiple sequence alignment of 30 marY1 sequences retrieved from a nucleotide sequence database. In accordance with $\underline{Re}trotransposon$ $\underline{M}icrosatellite$ $\underline{A}mplified$ $\underline{P}olymorphism$ (REMAP) fingerprinting methodology, the two oligonucleotides were utilized together with the short sequence repeat primers UBC807 and UBC818 for polymerase chain reaction using templates from different mushroom genomic DNAs. Among the tested oligonucleotides, the marY1-LTR-L and UBC807 primer set yielded the greatest amount of abundance and variation in terms of DNA band numbers and patterns. This method was successfully applied to 10 mushroom species, and the primer set successfully discriminated between different commercial mushroom cultivars of the same strains of 14 Pleurotus ostreatus and 16 P. eryngii. REMAP reproducibility was superior to other popular DNA fingerprinting methodologies including the random amplified polymorphic DNA method.

• BMB Reports
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• v.51 no.2
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• pp.55-56
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• 2018

Long terminal repeat retrotransposons (LTR-Rs) are major elements creating new genome structure for expansion of plant genomes. However, in addition to the genome expansion, the role of LTR-Rs has been unexplored. In this study, we constructed new reference genome sequences of two pepper species (Capsicum baccatum and C. chinense), and updated the reference genome of C. annuum. We focused on the study for speciation of Capsicum spp. and its driving forces. We found that chromosomal translocation, unequal amplification of LTR-Rs, and recent gene duplications in the pepper genomes as major evolutionary forces for diversification of Capsicum spp. Specifically, our analyses revealed that the nucleotide-binding and leucine-rich-repeat proteins (NLRs) were massively created by LTR-R-driven retroduplication. These retoduplicated NLRs were abundant in higher plants, and most of them were lineage-specific. The retroduplication was a main process for creation of functional disease-resistance genes in Solanaceae plants. In addition, 4-10% of whole genes including highly amplified families such as MADS-box and cytochrome P450 emerged by the retroduplication in the plants. Our study provides new insight into creation of disease-resistance genes and high-copy number gene families by retroduplication in plants.