Eui-Joon Kil

Tomato yellow leaf curl virus (TYLCV-IL): a seed-transmissible geminivirus in tomatoes

Tomato yellow leaf curl virus (TYLCV) is one of the most well-known tomato-infecting begomoviruses and transmitted by Bemisia tabaci. Seed transmission has previously been reported for some RNA viruses, but TYLCV has not previously been described as a seed-borne virus. In 2013 and 2014, without whitefly-mediated transmission, TYLCV was detected in young tomato plants germinated from fallen fruits produced from TYLCV-infected tomato plants in the previous cultivation season. In addition, TYLCV-Israel (TYLCV-IL) was also detected in seeds and their seedlings of TYLCV-infected tomato plants that were infected by both viruliferous whitefly-mediated transmission and agro-inoculation. The seed infectivity was 20–100%, respectively, and the average transmission rate to seedlings was also 84.62% and 80.77%, respectively. TYLCV-tolerant tomatoes also produced TYLCV-infected seeds, but the amount of viral genome was less than seen in TYLCV-susceptible tomato plants. When tomato plants germinated from TYLCV-infected seeds, non-viruliferous whiteflies and healthy tomato plants were placed in an insect cage together, TYLCV was detected from whiteflies as well as receiver tomato plants six weeks later. Taken together, TYLCV-IL can be transmitted via seeds, and tomato plants germinated from TYLCV-infected seeds can be an inoculum source of TYLCV. This is the first report about TYLCV seed transmission in tomato.

Advanced loop-mediated isothermal amplification method for sensitive and specific detection of Tomato chlorosis virus using a uracil DNA glycosylase to control carry-over contamination

In 2013, Tomato chlorosis virus (ToCV) was identified in symptomatic tomato plants in Korea. In the present study, a loop-mediated isothermal amplification (LAMP) method was developed using four specific primers designed against ORF6 in ToCV RNA2 to detect ToCV rapidly and with high sensitivity. The optimized reaction involved incubation of a reaction mixture containing 2U Bst DNA polymerase and 4mM MgSO4 for 1h at 60-62 °C. Although specific and rapid detection of ToCV by LAMP was confirmed, false-positive reactions caused by carry-over contamination sometimes occurred because of the high sensitivity of LAMP compared with other detection methods. To prevent false-positive reactions, dUTP was substituted for dTTP and uracil-DNA glycosylase (UDG) was added to the LAMP reaction. First, the LAMP reaction was conducted successfully with substitution of dUTP for dTTP. Before the next reaction, LAMP products with incorporated dUTP were cleaved selectively by UDG without any effect on thymine-containing DNA (template DNA). This modified LAMP method complemented with UDG treatment to prevent carry-over contamination offers a potentially powerful method for detecting plant viruses.

Seasonal Dynamics of Marine Microbial Community in the South Sea of Korea

High-resolution 16S rRNA tag pyrosequencing was used to obtain seasonal snapshots of the bacterial diversity and community structure at two locations in Gosung Bay (South Sea, Korea) over a one year period. Seasonal sampling from the water column at each site revealed highly diverse bacterial communities containing up to 900 estimated Operational Taxonomic Units (OTUs). The Alphaproteobacteria and Gammaproteobacteria were the most abundant groups, and the most frequently recorded OTUs were members of Pelagibacter and Glaciecola. In particular, it was observed that Arcobacter, a genus of the Epsilonproteobacteria, dominated during summer. In addition, Psedoalteromonadaceae, Vibrionaceae and SAR11-1 were predominant members of the OTUs found in all sampling seasons. Environmental factors significantly influenced the bacterial community structure among season, with the phosphate and nitrate concentrations contributing strongly to the spatial distribution of the Alphaproteobacteria; the Gammaproteobacteria, Flavobacteria, and Actinobacteria all showed marked negative correlations with all measured nutrients, particularly silicon dioxide and chlorophyll-a. The results suggest that seasonal changes in environmental variables contribute to the dynamic structure of the bacterial community in the study area.

Lamium amplexicaule (Lamiaceae): a weed reservoir for tomato yellow leaf curl virus (TYLCV) in Korea

After the first identification of tomato yellow leaf curl virus (TYLCV) in the southern part of Korea in 2008, TYLCV has rapidly spread to tomato farms in most regions of Korea. From 2008 to 2010, a survey of natural weed hosts that could be reservoirs of TYLCV was performed in major tomato production areas of Korea. About 530 samples were collected and identified as belonging to 25 species from 11 families. PCR and Southern hybridization were used to detect TYLCV in samples, and replicating forms of TYLCV DNA were detected in three species (Achyranthes bidentata, Lamium amplexicaule, and Veronica persica) by Southern hybridization. TYLCV transmission mediated by Bemisia tabaci from TYLCV-infected tomato plants to L. amplexicaule was confirmed, and TYLCV-infected L. amplexicaule showed symptoms such as yellowing, stunting, and leaf curling. TYLCV from infected L. amplexicaule was also transmitted to healthy tomato and L. amplexicaule plants by B. tabaci. The rate of infection of L. amplexicaule by TYLCV was similar to that of tomato. This report is the first to show that L. amplexicaule is a reservoir weed host for TYLCV.

Loop-mediated isothermal amplification for the rapid detection of Chrysanthemum chlorotic mottle viroid (CChMVd)

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, sensitive, and convenient diagnosis of infectious diseases. Chrysanthemum chlorotic mottle viroid (CChMVd) causes one of the most serious viral diseases in chrysanthemum in Korea. A sensitive LAMP assay was developed for rapidly detecting CChMVd infection. The assay was based on a set of four primers matching the specific region of the CChMVd genome. The CChMVd LAMP primer sets were designed using the sequences from nonsymptomatic and symptomatic CChMVd isolates in Korea. The efficiency and specificity of this method were optimized using Bst DNA polymerase, which allowed for increased viroid detection sensitivity. The reaction was carried out at 65 °C for 90 min, and was improved by adding SYBR Green I dye to the inside of the reaction tube lid prior to amplification. The results indicate that this LAMP method will be useful for chrysanthemum viroid disease monitoring and detecting CChMVd infectious disease.

Oxaliplatin-Induced Peripheral Neuropathy via TRPA1 Stimulation in Mice Dorsal Root Ganglion Is Correlated with Aluminum Accumulation.

Oxaliplatin is a platinum-based anticancer drug used to treat metastatic colorectal, breast, and lung cancers. While oxaliplatin kills cancer cells effectively, it exhibits several side effects of varying severity. Neuropathic pain is commonly experienced during treatment with oxaliplatin. Patients describe symptoms of paresthesias or dysesthesias that are triggered by cold (acute neuropathy), or as abnormal sensory or motor function (chronic neuropathy). In particular, we found that aluminum levels were relatively high in some cancer patients suffering from neuropathic pain based on clinical observations. Based on these findings, we hypothesized that aluminum accumulation in the dorsal root ganglion (DRG) in the course of oxaliplatin treatment exacerbates neuropathic pain. In mice injected with oxaliplatin (three cycles of 3 mg/kg i.p. daily for 5 days, followed by 5 days of rest), we detected cold allodynia using the acetone test, but not heat hyperalgesia using a hot plate. However, co-treatment with aluminum chloride (AlCl3∙6H2O; 7 mg/kg i.p. for 14 days: equivalent 0.78 mg/kg of elemental Al) and oxaliplatin (1 cycle of 3 mg/kg i.p. daily for 5 days, followed by 5 days of rest) synergistically induced cold allodynia as well as increased TRPAl mRNA and protein expression. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis showed a significant increase in aluminum concentrations in the DRG of mice treated with aluminum chloride and oxaliplatin compared to aluminum chloride alone. Similarly, in a mouse induced-tumor model, aluminum concentrations were increased in DRG tissue and tumor cells after oxaliplatin treatment. Taken together, these findings suggest that aluminum accumulation in the DRG may exacerbate neuropathic pain in oxaliplatin-treated mice.

Identification of natural weed hosts of Tomato chlorosis virus in Korea by RT-PCR with root tissues

Tomato chlorosis virus (ToCV) is a whitefly-transmitted crinivirus that causes interveinal chlorosis and bronzing on tomato leaves. Although outbreaks of ToCV have been reported in many countries, the virus was not reported in Korea until 2013. To identify weed hosts of ToCV that may serve as virus reservoirs, we analyzed various weeds that were growing together with ToCV-infected tomatoes and viruliferous Bemisia tabaci in a tomato greenhouse. We performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of root samples from 148 samples of 61 species from 24 families of plants grown in the greenhouse to avoid possible ToCV contamination by whiteflies on leaves. Seventeen weed species were identified as ToCV hosts based on RT-PCR results of root samples: Conyza canadensis, Erigeron annuus, Sonchus asper, Youngia japonica, Trigonotis peduncularis, Cardamine flexuosa, Cerastium glomeratum, Stellaria media, Chenopodium album, Ipomoea hederacea, Quamoclit coccinea, Vicia angustifolia var. segetilis, V. tetrasperma, Phytolacca americana, Mazus pumilus, Solanum americanum and S. nigrum. The amplicons obtained by RT-PCR were confirmed as ToCV by sequence analysis. Life cycle analysis of the weeds indicated that each weed could play an important role as a “green bridge” or virus reservoir between tomato cultivation seasons. This is the first report documenting the possible occurrence of ToCV in tomato plants based on weed life cycles.

Fatty acid methyl ester profiles and nutritive values of 20 marine microalgae in Korea

OBJECTIVE To screen the fatty acid (FA) composition of 20 marine microalgae species, including seven Diophyceae, six Bacillariophyceae, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species. METHODS Microalgal cells cultured at the Korea Institute of Ocean Science & Technology were harvested during the late exponential growth phase and the FA composition analyzed. RESULTS The FA composition of microalgae was species-specific. For example, seven different species of Dinophyceae were composed primarily of C14:0, C16:0, C18:0, C20:4n-6, C20:5n-3 and C22:6n-3, while C14:0, C16:0, C16:1, C18:0, C20:5n-3 and C22:6n-3 were abundant FAs in six species of Bacillariophyceae. In addition, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species all contained a high degree of C16:1n-7 [(9.28-34.91)% and (34.48-35.04)%], C14:0 [(13.34-25.96)%] and [(26.69-28.24)%], and C16:0 [(5.89-29.15)%] and [(5.70-16.81)%]. Several factors contribute to the nutritional value of microalgae, including the polyunsaturated FA content and n-3 to n-6 FA ratio, which could be used to assess the nutritional quality of microalgae. CONCLUSIONS This study is the first comprehensive assessment of the FA composition and nutritional value of microalgae species in South Korea, and identifies the potential utility of FAs as species-specific biomarkers.

Sweet pepper confirmed as a reservoir host for tomato yellow leaf curl virus by both agro-inoculation and whitefly-mediated inoculation

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.

An amino acid residue in the middle of the fingers subdomain is involved in Neq DNA polymerase processivity: enhanced processivity of engineered Neq DNA polymerase and its PCR application

Neq DNA polymerase is the first archaeal family B DNA polymerase reported to lack uracil recognition function and successfully utilize deaminated bases. We have focused on two amino acid residues (Y515, A523) in the fingers subdomain of Neq DNA polymerase, which were predicted to be located in the middle of the fingers subdomain, based on amino acid sequence alignment of the Neq DNA polymerase with structurally determined archaeal DNA polymerases. Those two residues were replaced by site-directed mutagenesis, and the enzymatic properties of the mutants were analyzed. Here, we show that the A523 residue located in the middle of the fingers subdomain affects the processivity of Neq DNA polymerase. Mutational analysis has allowed us to enhance the protein function as well as understand the function of the residues. One mutant protein, Neq A523R DNA polymerase, exhibited a roughly 3-fold enhanced processivity and extension rate compared to wild type, enabling more efficient PCR. In the presence of uracil, Neq A523R DNA polymerase outperformed Taq DNA polymerase with enhanced specificity and sensitivity. These results suggest that Neq A523R DNA polymerase could be most effectively utilized in real-time PCR using uracil-DNA glycosylase without the risk of carry-over contamination.