Jang-Yong Lee

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices.

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.

G7H, a new Soybean mosaic virus strain: its virulence and nucleotide sequence of CI gene.

A new Soybean mosaic virus (SMV) strain was isolated in Korea and designated as G7H. Its virulence on eight differentials and 42 Korean soybean cultivars was compared with existing SMV strains. G7H caused the same symptoms as G7 did on the eight differential cultivars. However, it caused different symptoms on the G7-immune Korean soybean cultivars; G7H caused necrosis in Suwon 97 (Hwangkeumkong) and Suwon 181 (Daewonkong), and a mosaic symptom in Miryang 41 (Duyoukong), while G7 caused only local lesions on those varieties. The nucleotide sequence of the cylindrical inclusion region of G7H was determined and compared with other SMV strains. G7H shared 96.3 and 91.3% nucleotide similarities with G2 and G7, respectively; whereas G7 shared 95.6% nucleotide similarity with G5H.

Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (Perilla frutescens)

Increasing vitamin E activity in economically important oil crops such as perilla will enhance the nutritional value of these crops. Perilla (Perilla frutescens Britt) represents an important oil crop in Asian countries, including Korea. Using Agrobacterium-mediated transformation, we have engineered perilla with the γ-tocopherol methyltransferase (γ-TMT) gene under the control of seed-specific vicillin promoter. Molecular characterization including PCR, Southern and Northern blots confirmed that the γ-TMT transgene was successfully inherited to and expressed in the progeny plants. The γ -TMT transgene was specifically expressed in immature seeds of transgenic plants, leading to efficient conversion of γ-tocopherol to α-tocopherol and dramatic increase in seed α-tocopherol content, as detected by high performance liquid chromatography analysis. We also showed that such a high α-tocopherol content phenotype was transmitted to the progeny plants. In addition, there was no significant change in fatty acid composition in transgenic seeds as compared with untransformed control Yeupsil cultivar, suggesting the lack of interplay between the fatty acid and tocopherol biosynthesis pathways. This was the first report on over expression of the γ-TMT gene in transgenic perilla displaying desirable high α-tocopherol content phenotype. Since α-tocopherol has the highest vitamin E activity, the transgenic perilla with high α-tocopherol content in seeds developed in this study will benefit both human and animal health.

Genistein production in rice seed via transformation with soybean IFS genes

To produce genistein in rice, the isoflavone synthase (IFS) genes, SpdIFS1 and SpdIFS2 were cloned from the Korean soybean cultivar, Sinpaldalkong II as it has a higher genistein content than other soybean varieties. SpdIFS1 and SpdIFS2 show a 99.6% and 98.2% identity at the nucleotide level and 99.4% and 97.9% identity at the amino acid level, respectively, with IFS1 and IFS2 from soybean (GenBank accession Nos. AF195798 and AF195819). Plant expression vectors were constructed harboring SpdIFS1 or SpdIFS2 under the control of a rice globulin promoter that directs seed specific expression, and used to transform two rice varieties, Heugnam, a black rice, and Nakdong, a normal rice cultivar without anthocyanin pigment. Because naringenin, the substrate of SpdIFS1 and SpdIFS2, is on the anthocyanin biosynthesis pathway, the relative production rate of genistein was compared between SpdIFS-expressing transgenic Heugnam and Nakdong. Southern blot analysis of eight of the resulting transgenic rice plants revealed that the T0 plants had one to three copies of the SpdIFS1 or SpdIFS2 gene. The highest level of genistein content found in rice seeds was 103 μg/g. These levels were about 30-fold higher in our transgenic rice lines than the genistein aglycon content of a non-leguminous IFS-expressing transgenic tobacco petal, equaling about 12% of total genistein content of Sinpaldalkong II. There were no significant differences found between the genistein content in Heugnam and Nakdong transgenic rice plants.

First Report of Bacterial Black Spot Disease in Watermelon Caused by Acidovorax valerianellae in Korea

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai), an important member of the Cucurbitaceae family, is cultivated on 21,000 ha that produces 850,000 t in Korea. In April 2011, we received grafted watermelon with necrotic leaf spots from a commercial watermelon grower in Andong, Korea. Black spots were observed on cotyledons of the plants in seedbeds, and approximately 9% of watermelon plants were infected with the disease. Initial symptoms on the seedling were black, greasy spots sometimes surrounded by a halo of discoloration. Younger leaves usually showed symptoms later than cotyledons. Bacteria isolated from the infected plants were gram-negative, motile, straight rods with a single flagellum and 0.84 to 0.89 μm wide and 1.54 to 1.69 μm long. They formed rough colonies with a white-cream color after 48 h of incubation on Luria-Bertani (LB) agar at 28°C. Colonies of isolates were nonfluorescent, smooth, and white on Kings medium B. On YBGA (7 g of yeast extract, 7 g of bactopeptone, 7 g of glucose, 15 g of agar, 1,000 ml of distilled water; pH 7.2) colonies are circular, raised with an entire margin, and white to cream. Pathogenicity tests were conducted with potted, greenhouse-grown watermelon plants. Bacterial colonies grown on LB medium for 48 h at 28°C were suspended in sterile distilled water, and the suspension (1.0 × 108 CFU/ml) was infiltrated into mesophyll of watermelon leaves with a syringe as previously described (2). Inoculated plants were maintained at 28°C and 90% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. Five plants were used for inoculation. Sterilized distilled water was used as a control. The bacterial isolates induced necrosis in the infiltrated area within 3 to 5 days. Typical water-soaked spots appeared after 3 days of incubation and became gray to black after 6 days. The bacterium was successfully reisolated from the diseased lesions, thus completing Kochs postulates. A cell suspension (50 μl of 1 × 106 CFU/ml) was infiltrated with a syringe into the intercellular spaces of tobacco leaves to determine the hypersensitive reaction (HR). A typical HR developed 20 h after leaf infiltration. The 16S rDNA region of the isolates, amplified by using universal PCR primers, shared 99% sequence identity with an Acidovorax valerianellae strain (GenBank Accession No. AJ431731) (1). The resulting sequences of 1,424 bp were deposited in GenBank (Accession No. JN983471). The isolates we obtained in this study clustered with A. valerianellae on a phylogenetic tree generated by the neighbor-joining method implemented in MEGA Version 4.1. In the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), all isolates were 63 to 77% similar with a match probability of 100% to A. konjaci. Fatty acid composition analysis of isolates based on the MIDI Library version TSBA 5.0 and Library Generation system software version 5.0 showed that the isolates were 52 and 72% similar to an Acidovorax sp., respectively. To our knowledge, this is the first report of bacterial black spot disease in watermelon caused by A. valerianellae in Korea. A. valerianellae is a causal agent of bacterial black spot in corn salad and is transmitted by inoculated seeds (3). Further studies are required to determine whether it is seed transmitted in watermelon. References: (1) L. Gardan et al. Int. J. Syst. Evol. Microbiol. 53:795, 2003. (2) C. Grondeau et al. Plant Pathol. 56:302, 2007. (3) C. Grondeau et al. Plant Pathol. 58:846, 2009.

The Effects of Genetically Modified Crops on Soil Microbial Community

Abstract BACKGROUND: Genetically modified (GM) crops must receive relevant regulator’s authorization before they can be sold as seed or used food, feed and processing. Before approving any GM crop, the relevant government ministries are required to examine environmental risk assessment to make scientifically sound and socially acceptable decisions. But one of the least studied and understood areas in the environmental risk assessment of GM crops are their impact on soil microbial community. METHODS AND RESULTS: Recently, advanced methods have been developed to characterize the soil microbial community in various environments. In this study, the culture-dependent and culture -independent technical appr-oaches for profiling soil microbial communities are summa-rized and their applicability to assess GM crops are discussed. CONCLUSION(S): We concluded that the effect of GM crops on soil microbial community need to be assessed on a case by case basis. The combination of culture-dependent and culture-independent method was necessary for reliable and detailed assessment of effect of GM crops on soil microbial community.

Effects of Transgenic Soybean Cultivation on Soil Microbial Community in the Rhizosphere

BACKGROUND: Soybean [Glycine max (L.) Merrill] is a legume and an important oil crop worldwide. This study was conducted to evaluate the possible impact of transgenic soybean cultivation on the soil microbial community. METHODS AND RESULTS: Microorganisms were isolated from the rhizosphere soils . Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with transgenic and non-transgenic soybeans were similar to each other, and there was no significant difference between transgenic and non-transgenic soybeans. Dominant bacterial phyla in the rhizosphere soils cultivated with transgenic or non-transgenic soybeans were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in transgenic and non-transgenic soybean soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed the different patterns, but didn’t show significant difference to each other at 0.05 significance level. DNAs were isolated from soils cultivating transgenic or non-transgenic soybeans and analyzed for persistence of transgenes in the soil by using PCR. PCR analysis revealed that there were no amplified γ-tmt and bar gene in soil DNA. CONCLUSION(S): The results of this study suggested that microbial community of soybean field were not significantly affected by cultivation of the transgenic soybeans.