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.

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.

Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana.

Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.

Ascorbic acid alleviates toxicity of paclitaxel without interfering with the anticancer efficacy in mice.

Paclitaxel is used extensively as a chemotherapeutic agent against a broad range of tumors but often leads to the early termination of treatment due to severe toxic side effects. In this study, we hypothesized that ascorbic acid could reduce the toxic side effects without interfering with the anticancer effect of paclitaxel. To demonstrate this, we examined the effect of the combinational treatment of ascorbic acid and paclitaxel using H1299 (a non-small cell lung cancer cell line) and BALB/c mice implanted with or without sarcoma 180 cancer cells. In H1299 cells, the anticancer effects of the combinational treatment with paclitaxel and ascorbic acid were up to 1.7-foldhigher than those of single-agent paclitaxel treatment. In addition, it was shown that the viability of the HEL299 normal cells was up to 1.6-fold higher with the combinational treatment than with paclitaxel treatment alone. In vivo mouse experiments also showed that mice co-treated with paclitaxel and ascorbic acid did not exhibit the typical side effects induced by paclitaxel, such as a reduction in the numbers of white blood cells and red blood cells and the level of hemoglobin (P < .05). The analysis of cancer-related gene expression by quantitative real-time polymerase chain reaction and immunohistochemistry revealed that the combinational treatment suppressed cancer cell multiplication. Taken together, these results suggest that combinational chemotherapy with ascorbic acid and paclitaxel not only does not block the anticancer effects of paclitaxel but also alleviates the cytotoxicity of paclitaxel in vivo and in vitro.

The Arabidopsis thaliana homeobox gene ATHB12 is involved in symptom development caused by geminivirus infection.

BACKGROUND: Geminiviruses are single-stranded DNA viruses that infect a number of monocotyledonous and dicotyledonous plants. Arabidopsis is susceptible to infection with the Curtovirus, Beet severe curly top virus (BSCTV). Infection of Arabidopsis with BSCTV causes severe symptoms characterized by stunting, leaf curling, and the development of abnormal inflorescence and root structures. BSCTV-induced symptom development requires the virus-encoded C4 protein which is thought to interact with specific plant-host proteins and disrupt signaling pathways important for controlling cell division and development. Very little is known about the specific plant regulatory factors that participate in BSCTV-induced symptom development. This study was conducted to identify specific transcription factors that are induced by BSCTV infection. METHODOLOGY/PRINCIPAL FINDINGS: Arabidopsis plants were inoculated with BSCTV and the induction of specific transcription factors was monitored using quantitative real-time polymerase chain reaction assays. We found that the ATHB12 and ATHB7 genes, members of the homeodomain-leucine zipper family of transcription factors previously shown to be induced by abscisic acid and water stress, are induced in symptomatic tissues of Arabidopsis inoculated with BSCTV. ATHB12 expression is correlated with an array of morphological abnormalities including leaf curling, stunting, and callus-like structures in infected Arabidopsis. Inoculation of plants with a BSCTV mutant with a defective c4 gene failed to induce ATHB12. Transgenic plants expressing the BSCTV C4 gene exhibited increased ATHB12 expression whereas BSCTV-infected ATHB12 knock-down plants developed milder symptoms and had lower ATHB12 expression compared to the wild-type plants. Reporter gene studies demonstrated that the ATHB12 promoter was responsive to BSCTV infection and the highest expression levels were observed in symptomatic tissues where cell cycle genes also were induced. CONCLUSIONS/SIGNIFICANCE: These results suggest that ATHB7 and ATHB12 may play an important role in the activation of the abnormal cell division associated with symptom development during geminivirus infection.

Phylogenetic lineage of Tobacco leaf curl virus in Korea and estimation of recombination events implicated in their sequence variation.

New strains of Tobacco leaf curl virus (TbLCV) were isolated from tomato plants in four different local communities of Korea, and hence were designated TbLCV-Kr. Phylogenetic analysis of the sequences of the whole genome and of individual ORFs of these viruses indicated that they are closely related to the Tobacco leaf curl Japan virus (TbLCJV) cluster, which includes Honeysuckle yellow vein virus (HYVV), Honeysuckle yellow vein mosaic virus (HYVMV), and TbLCJV isolates. Four putative recombination events were recognized within these virus sequences, suggesting that the sequence variations observed in these viruses may be attributable to intraspecific and interspecific recombination events involving some TbLCV-Kr isolates, Papaya leaf curl virus (PaLCV), and a local isolate of Tomato yellow leaf curl virus (TYLCV).

C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis.

The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development.

Phylogenetic analysis and inflow route of Tomato yellow leaf curl virus (TYLCV) and Bemisia tabaci in Korea.

Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus of the family Geminiviridae, members of which are characterized by closed circular single-stranded DNA genomes of 2.7-2.8 kb in length, and include viruses transmitted by the Bemisia tabaci whitefly. No reports of TYLCV in Korea are available prior to 2008, after which TYLCV spread rapidly to most regions of the southern Korean peninsula (Gyeongsang-Do, Jeolla-Do and Jeju-Do). Fifty full sequences of TYLCV were analyzed in this study, and the AC1, AV1, IR, and full sequences were analyzed via the muscle program and bayesian analysis. Phylogenetic analysis demonstrated that the Korea TYLCVs were divided into two subgroups. The TYLCV Korea 1 group (Masan) originated from TYLCV Japan (Miyazaki) and the TYLCV Korea 2 group (Jeju/Jeonju) from TYLCV Japan (Tosa/Haruno). A B. tabaci phylogenetic tree was constructed with 16S rRNA and mitochondria cytochrome oxidase I (MtCOI) sequences using the muscle program and MEGA 4.0 in the neighbor-joining algorithm. The sequence data of 16S rRNA revealed that Korea B. tabaci was closely aligned to B. tabaci isolated in Iran and Nigeria. The Q type of B. tabaci, which was originally identified as a viruliferous insect in 2008, was initially isolated in Korea as a non-viruliferous insect in 2005. Therefore, we suggest that two TYLCV Japan isolates were introduced to Korea via different routes, and then transmitted by native B. tabaci.

Expression analysis of proline metabolism-related genes from halophyte Arabis stelleri under osmotic stress conditions.

Arabis stelleri var. japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana. Using an A. thaliana microarray chip, we determined changes in the expression of approximately 2 800 genes between A. stelleri plants treated with 0.2 M mannitol versus mock-treated plants. The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system, stimulus response, stress response, chemical stimulus response, and defense response. The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A. stelleri var. japonica seedlings treated with 0.2 M mannitol, 0.2 M sorbitol, and 0.2 M NaCl. The expression of Δ¹ -pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol. The proline dehydrogenase gene, which is known to be repressed by dehydration stress and induced by free L-proline, was induced at an early stage by mannitol treatment, but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl. The level of free L-proline accumulation increased progressively in response to treatments with mannitol, sorbitol, and NaCl. Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol. These findings demonstrate that the osmotic tolerance of the novel halophyte, Arabis stelleri, is associated with the accumulation of L-proline.

ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene.

Arabidopsis thaliana homeobox 12 (ATHB12) is rapidly induced by ABA and water stress. A T-DNA insertion mutant of ATHB12 with a reduced level of ATHB12 expression in stems had longer inflorescence stems and reduced sensitivity to ABA during germination. A high level of transcripts of gibberellin 20-oxidase 1 (GA20ox1), a key enzyme in the synthesis of gibberellins, was detected in athb12 stems, while transgenic lines overexpressing ATHB12 (A12OX) had a reduced level of GA20ox1 in stems. Consistent with these data, ABA treatment of wild-type plants resulted in decreased GA20ox1 expression whereas ABA treatment of the athb12 mutant gave rise to slightly decreased GA20ox1 expression. Retarded stem growth in 3-week-old A12OX plants was rescued by exogenous GA(9), but not by GA(12), and less GA(9) was detected in A12OX stems than in wild-type stems. These data imply that ATHB12 decreases GA20ox1 expression in stems. On the other hand, the stems of A12OX plants grew rapidly after the first 3 weeks, so that they were almost as high as wild-type plants at about 5 weeks after germination. We also found changes in the stems of transgenic plants overexpressing ATHB12, such as alterations of expression GA20ox and GA3ox genes, and of GA(4) levels, which appear to result from feedback regulation. Repression of GA20ox1 by ATHB12 was confirmed by transfection of leaf protoplasts. ABA-treated protoplasts also showed increased ATHB12 expression and reduced GA20ox1 expression. These findings all suggest that ATHB12 negatively regulates the expression of a GA 20-oxidase gene in inflorescence stems.

Biological and molecular responses of Chironomus riparius (Diptera, Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid).

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10microgL(-1) of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10microgL(-1) of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1microgL(-1) of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10microgL(-1) concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.

Ecotoxicological multilevel-evaluation of the effects of fenbendazole exposure to Chironomus riparius larvae.

Veterinary antibiotics may find their way into the aquatic environment through direct or indirect pathways due to their widespread use. Fenbendazole is a benzimidazole anthelmintic that is widely used in veterinary medicine. To evaluate the potential ecological risk of fenbendazole, we examined the molecular and biochemical responses of biomarker genes such as heat shock proteins (HSPs), cytochrome P450 (CYP450), glutathione S-transferases (GSTs) and hemoglobins (Hbs) in Chironomus riparius for long periods. The expression of HSP70, HSP40, HSP90 and CYP450 in C. riparius increased significantly after exposure to all concentrations of fenbendazole evaluated, while the levels of GST and HbA only increased in C. riparius exposed to relatively high concentrations of fenbendazole (30 microg L(-1)). HbB expression did not differ significantly between the control and treatment groups. Exposure to 30 microg L(-1) fenbendazole had significant effects on the survival, growth, sex balance of emergent adults and development of mouthpart deformity in C. riparius. These results should constitute an important contribution to the understanding of the toxicology of fenbendazole in C. riparius. Moreover, the responses of the biomarker genes also provide valuable information that will aid in understanding the effects of fenbendazole in aquatic ecosystems.

Arabidopsis leaf necrosis caused by simulated acid rain is related to the salicylic acid signaling pathway.

Arabidopsis leaves treated with simulated acid rain (SiAR) showed phenotypes similar to necrotic lesions caused by biotic stresses like Pseudomonad infiltration. Exposure of Arabidopsis to SiAR resulted in the up-regulation of genes known to be induced by the salicylic acid (SA)-mediated pathogen resistance response. The expression of enhanced disease susceptibility (EDS), nonexpressor of PR (NPR) and pathogen-related 1 (PR1), all of which are involved in the salicylic acid signaling pathway, were increased after SiAR exposure. However, vegetative storage protein (VSP), a member of the jasmonic acid pathway did not show a significant change in transcript level. SiAR treatment of transgenic plants expressing salicylate hydroxylase (Nah-G), which prevents the accumulation of salicylic acid, underwent more extensive necrosis than wild-type plants, indicating that the signaling pathway activated by SiAR may overlap with the SA-dependent, systemic acquired resistance pathway. Both Col-0 and Nah-G plants showed sensitivity to SiAR and sulfuric SiAR (S-SiAR) by developing necrotic lesions. Neither Col-0 plants nor Nah-G plants showed sensitivity to nitric SiAR (N-SiAR). These results suggest that SiAR activates at least the salicylic acid pathway and activation of this pathway is sensitive to sulfuric acid.