Gang-Seob Lee

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals

Transcriptional reprogramming is regulated by Nod factor-induced ethylene signaling. The legume-rhizobium symbiosis is initiated through the activation of the Nodulation (Nod) factor-signaling cascade, leading to a rapid reprogramming of host cell developmental pathways. In this work, we combine transcriptome sequencing with molecular genetics and network analysis to quantify and categorize the transcriptional changes occurring in roots of Medicago truncatula from minutes to days after inoculation with Sinorhizobium medicae. To identify the nature of the inductive and regulatory cues, we employed mutants with absent or decreased Nod factor sensitivities (i.e. Nodulation factor perception and Lysine motif domain-containing receptor-like kinase3, respectively) and an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant (sickle). This unique data set encompasses nine time points, allowing observation of the symbiotic regulation of diverse biological processes with high temporal resolution. Among the many outputs of the study is the early Nod factor-induced, ET-regulated expression of ET signaling and biosynthesis genes. Coupled with the observation of massive transcriptional derepression in the ET-insensitive background, these results suggest that Nod factor signaling activates ET production to attenuate its own signal. Promoter:β-glucuronidase fusions report ET biosynthesis both in root hairs responding to rhizobium as well as in meristematic tissue during nodule organogenesis and growth, indicating that ET signaling functions at multiple developmental stages during symbiosis. In addition, we identified thousands of novel candidate genes undergoing Nod factor-dependent, ET-regulated expression. We leveraged the power of this large data set to model Nod factor- and ET-regulated signaling networks using MERLIN, a regulatory network inference algorithm. These analyses predict key nodes regulating the biological process impacted by Nod factor perception. We have made these results available to the research community through a searchable online resource.

Map-based Cloning and Characterization of the BPH18 Gene from Wild Rice Conferring Resistance to Brown Planthopper (BPH) Insect Pest

Brown planthopper (BPH) is a phloem sap-sucking insect pest of rice which causes severe yield loss. We cloned the BPH18 gene from the BPH-resistant introgression line derived from the wild rice species Oryza australiensis. Map-based cloning and complementation test revealed that the BPH18 encodes CC-NBS-NBS-LRR protein. BPH18 has two NBS domains, unlike the typical NBS-LRR proteins. The BPH18 promoter::GUS transgenic plants exhibited strong GUS expression in the vascular bundles of the leaf sheath, especially in phloem cells where the BPH attacks. The BPH18 proteins were widely localized to the endo-membranes in a cell, including the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and prevacuolar compartments, suggesting that BPH18 may recognize the BPH invasion at endo-membranes in phloem cells. Whole genome sequencing of the near-isogenic lines (NILs), NIL-BPH18 and NIL-BPH26, revealed that BPH18 located at the same locus of BPH26. However, these two genes have remarkable sequence differences and the independent NILs showed differential BPH resistance with different expression patterns of plant defense-related genes, indicating that BPH18 and BPH26 are functionally different alleles. These findings would facilitate elucidation of the molecular mechanism of BPH resistance and the identified novel alleles to fast track breeding BPH resistant rice cultivars.

SNP-based analysis of genetic diversity in anther-derived rice by whole genome sequencing

BackgroundAnther culture has advantage to obtain a homozygous progeny by induced doubling of haploid chromosomes and to improve selection efficiency for invaluable agronomical traits. Therefore, anther culturing is widely utilized to breed new varieties and to induce genetic variations in several crops including rice. Genome sequencing technologies allow the detection of a massive number of DNA polymorphism such as SNPs and Indels between closely related cultivars. These DNA polymorphisms permit the rapid identification of genetic diversity among cultivars and genomic locations of heritable traits. To estimate sequence diversity derived from anther culturing, we performed whole-genome resequencing of five Korean rice accessions, including three anther culture lines (BLB, HY-04 and HY-08), their progenitor cultivar (Hwayeong), and an additional japonica cultivar (Dongjin).ResultsA total of 1,165 × 106 raw reads were generated with over 58× coverage that detected 1,154,063 DNA polymorphisms between the Korean rice accessions and Nipponbare. We observed that in Hwayeong and its progenies, 0.64 SNP was found per one kb of Nipponbare genome, while Dongjin, bred by a conventional breeding method, had a lower number of SNPs (0.45 SNP/kb). Among 1,154,063 DNA polymorphisms, 29,269 non-synonymous SNPs located on 30,013 genes and these genes were functionally classified based on gene ontology (GO). We also analyzed line-specific SNPs which were estimated 1 ~ 3% of the total SNPs. The frequency of non-synonymous SNPs in each accession ranged from 26 SNPs in Hwayeong to 214 SNPs in HY-04.ConclusionsThe genetic difference we detected between the progenies derived from anther culture and their mother cultivar is due to somaclonal variation during tissue culture process, such as karyotype change, chromosome rearrangement, gene amplification and deletion, transposable element, and DNA methylation. Detection of genome-wide DNA polymorphisms by high-throughput sequencer enabled to identify sequence diversity derived from anther culturing and genomic locations of heritable traits. Furthermore, it will provide an invaluable resource to identify molecular markers and genes associated with diverse traits of agronomical importance.

A database for allergenic proteins and tools for allergenicity prediction.

The AllergenPro database has developed a web-based system that will provide information about allergen in microbes, animals and plants. The database has three major parts and functions:(i) database list; (ii) allergen search; and (iii) allergenicity prediction. The database contains 2,434 allergens related information readily available in the database such as on allergens in rice microbes (712 records), animals (617 records) and plants (1,105 records). Furthermore, this database provides bioinformatics tools for allergenicity prediction. Users can search for specific allergens by various methods and can run tools for allergenicity prediction using three different methods. Availability The database is available for free at http://www.niab.go.kr/nabic/

Current status of Ac/Ds mediated gene tagging systems for study of rice functional genomics in Korea

Abstract Rice is the staple food of more than 50% of the worlds population. Cultivated rice has the AA genome (di-ploid, 2n=24) and small genome size of only 430 megabase (haploid genome). As the sequencing of rice genome was completed by the International Rice Genome Sequencing Project (IRGSP), many researchers in the world have been working to explore the gene function on rice genome. Insertional mutagenesis has been a powerful strategy for assessing gene function. In maize, well characterized trans-posable elements have traditionally been used to clone genes for which only phenotypic information is available. In rice endogenous mobile elements such as MITE and Tos (Hiro-chika. 1997) have been used to generate gene-tagged popu-lations. To date T-DNA and maize transposable element systems has been utilized as main insertional mutagens in rice. A main drawback of a T-DNA scheme is that Agro-bacteria-mediated transformation in rice requires extensive facilities, time, and labor. In contrast, the Ac/Ds system offers the advantage of generating new mutants by secondary trans-position from a single tagged gene. Revertants can be uti-lized to correlate phenotype with genotype. To enhance the efficiency of gene detection, advanced gene-tagging systems (i.e. activation, gene or enhancer trap) have been employed for functional genomic studies in rice. Internationally, there have been many projects to develop large scales of inser-tionally mutagenized populations and databases of insertion sites has been established. Ultimate goals of these projects are to supply genetic materials and informations essential for functional analysis of rice genes and for breeding using agronomically important genes. In this report, we summarize the current status of Ac/Ds-mediated gene tagging systems that has been launched by collaborative works from 2001 in Korea.

Whole-Genome Resequencing and Transcriptomic Analysis to Identify Genes Involved in Leaf-Color Diversity in Ornamental Rice Plants

Rice field art is a large-scale art form in which people design rice fields using various kinds of ornamental rice plants with different leaf colors. Leaf color-related genes play an important role in the study of chlorophyll biosynthesis, chloroplast structure and function, and anthocyanin biosynthesis. Despite the role of different metabolites in the traditional relationship between leaf and color, comprehensive color-specific metabolite studies of ornamental rice have been limited. We performed whole-genome resequencing and transcriptomic analysis of regulatory patterns and genetic diversity among different rice cultivars to discover new genetic mechanisms that promote enhanced levels of various leaf colors. We resequenced the genomes of 10 rice leaf-color accessions to an average of 40× reads depth and >95% coverage and performed 30 RNA-seq experiments using the 10 rice accessions sampled at three developmental stages. The sequencing results yielded a total of 1,814 × 106 reads and identified an average of 713,114 SNPs per rice accession. Based on our analysis of the DNA variation and gene expression, we selected 47 candidate genes. We used an integrated analysis of the whole-genome resequencing data and the RNA-seq data to divide the candidate genes into two groups: genes related to macronutrient (i.e., magnesium and sulfur) transport and genes related to flavonoid pathways, including anthocyanidin biosynthesis. We verified the candidate genes with quantitative real-time PCR using transgenic T-DNA insertion mutants. Our study demonstrates the potential of integrated screening methods combined with genetic-variation and transcriptomic data to isolate genes involved in complex biosynthetic networks and pathways.

Characterization and fine mapping of a shortened uppermost internode mutant in rice

Internode elongation is an important agronomic trait in rice that is associated with lodging, yield, flooding adaptation, and hybrid seed production. We identified a novel rice mutant line showing a shortened uppermost internode among the rice Ac/Ds insertional mutant population and named it shortened uppermost internode 4 (sui4). Interestingly, T1 plants of this line segregated into three phenotypes: extremely shortened uppermost internode-type, normal wild-type, and intermediate-type. The phenotypes of F1 plants and F2 plants from the cross of sui4 with its original variety, Dongjin, indicated that the SUI4 gene shows incomplete dominance or semidominance. Because the Ds genotypes did not co-segregate with the sui4 phenotypes, we performed mapping of this gene with 273 F2 plants from a cross between sui4 and Milyang23. Primary mapping revealed that the SUI4 locus was located between the S07012 and S07015 markers on rice chromosome 7. Further fine mapping with 36 F3 lines derived from F2 plants that have recombination in this region narrowed down the location of SUI4 to the 1.1-Mbp interval of RM1253–S07015.

Multi-Layered Screening Method Identification of Flavonoid-Specific Genes, using Transgenic Rice

ABSTRACT We used microarray analysis to identify 6 730 genes potentially related to flavonoid biosynthesis by comparing their expression between high- and low-flavonoid cultivars at three stages of seed development. The genes included 167 transcription factors, 104 pathway-network genes, and 1 207 potential orthologs with flavonoid-related functions. We compared the transcription factors, pathway genes, and orthologs and identified seven conserved hypothetical genes that are involved in flavonoid biosynthesis. We then verified the seven genes experimentally by reverse transcription-polymerase chain reaction. These conserved genes most likely play regulatory roles either in rice-flavonoid production or flavonoid signaling during seed development. Our study illustrates the effectiveness of microarray analysis followed by multi-layered screening for the identification of flavonoid-related genes in rice.

Analysis of germinating seed stage expressed sequence tags in Oryza sativa L.

Abstract Seed germination is the important stage to ex-press many genes for regulation of energy metabolism, starch degradation and cell division from seed dormancy state. For the functional analysis of seed germination mech-anisms, we were analyzed the rice cDNA clones ( Oryza sativa cultivar Ilpum ) obtained from seed imbibition during 48 hours. Total number of 18,101 Expressed Sequence Tags (ESTs) were clustered using SeqMan program. Among them, 8,836 clones were identified as unique clones. We identified the chitinase gene specifically expressed in seed germination and amylase gene involved to starch degradation from the full length cDNA analysis, and several genes were registered to NCBI GeneBank. To analyzed the commonly expressed genes between inmature seed and germinated seed, 25,668 inmature ESTs and 18,101 germinated ESTs were clustered using SeqMan program and identified 2,514 clones as com-monly expressed unigene. Among them, alpha-glubulin and alcohol dehydrogenase I were supposed to LEA genes only expressed in the immature and germinated seed stages.For the clustering of orthologous group genes, we further analyzed the 8,836 EST clones from germinating seeds using NCBI clusters of orthologous groups database. Among the clones, 5,076 clones were categorized into information storage and processing, cellular processes and signaling, metabolism and poorly characterized genes, proportioning 783 (14.29%), 1,484 (27%), 1,363 (24.8%) and 1,869 (34%) clones to the previous four categories, respectively.

Structural analysis of expressed sequence tags inimmature seed of Oryza sativa L.

Abstract Rice (Oryza sativa) is the most important staple crop in Korea. With its small genome size of 389Mb, rice is a model plant for genome research. We analyzed expressed sequence tag (EST) clones from immature seeds of rice (cv. Ilpum) at 20 days after heading. The 25,668 EST clones were clustered by using SeqMan program and 7,509 clones were selected as unique clones. We compared the 7,509 unique genes with KOME database including the 32,127 FL-cDNA in rice. Finally, 4,990 clones were homologous and 2,519 clones non-homologous to FL-cDNA clones. In addition, we mapped the 7,509 cDNA clones by using TIGR rice pseudomolecule version 5. Ultimately, 7,347 clones were matched to be significant clones related to the TIGR rice pseudomolecules, but 162 clones were unmapped. For the clustering of orthologous group genes, we further analyzed the 7,509 EST clones from immature seeds using NCBI clusters of orthologous groups database. Among the clones, 4,968 clones were categorized into information storage and processing, cellular processes and signaling, metabolism and poorly characterized genes, proportioning 799 (14.89%), 1,536 (28.3%), 1,148 (21.2%) and 1,936 (35.7%) clones to the previous four categories, respectively.