Search

Ralstonia solanacearum is a notorious pathogen of bacterial wilt on Solanum lycopersicum . Most isolates from diseased tomato tissues are biovar 3, and their genomes are publicly available; however, information on biovar 4 strains is limited. Here, the complete genome sequence of R. solanacearum Bs715, a biovar 4 strain, is presented.

Confirmation of T-DNA inheritance and stability requires testing of several parameters such as phenotypic performance of transgenic lines, transgene integrity structure and expression stability for at least three consecutive generations, and Mendelian inheritance analysis, which are very important to transgenic breeding and commercialization of genetically modified crops (GMOs). In the present study, we measured nine agronomic and yield-related traits of Iksan526 and confirmed stable phenotypic performance comparing to the donor variety Dongjin over three years. Transgene integrity structure, flanking sequence, functional element copy numbers, absence of backbone sequence and transgene expression were all verified in Iksan526 over three consecutive generations, indicating a stable inheritance of the transgene. In addition, phenotypic and genotypic segregation were analyzed on F2 and F3 populations derived from a cross of Dongjin/Iksan526, and results complied with Mendel’s law of segregation based on single transgene locus. This study demonstrated detailed analyses and confirmation of transgene event Iksan526 which is crucial for commercialization of GMO.

Resveratrol, a plant phenolic compound, has potential therapeutic benefits due to its antioxidant properties. This is substantiated by previous studies that show that resveratrol derived from rice grains is an effective treatment agent for metabolic syndrome. Here, we characterized the T-DNA sequence, inserted T-DNA structure, copy number, integrity of the transgene locus, resveratrol synthase gene expression and resveratrol contents in the grains of two resveratrol transgenic rice lines, Iksan515 and Iksan526. The T-DNA transformation vector contained two expression cassettes of the resveratrol synthase gene under the control of the ubiquitin promoter and the bar selection marker gene under the control of the CaMV35S promoter. Flanking sequence analysis indicated that the T-DNAs were inserted into intergenic regions of chromosome 4 for Iksan515 and chromosome 12 for Iksan526. Two T-DNAs connected in an inverted repeat structure at a single locus of the rice genome were identified by whole genome sequencing and Southern blot hybridization in both Iksan515 and Iksan526. No novel open reading frames (ORFs) around insertion sites, sequences encoding allergenic or toxic protein, or other unintended effects by T-DNA insertion were found in either case. In addition, resveratrol synthase gene expression in leaves and resveratrol detection in brown rice grains suggested the successful expression of the inserted foreign resveratrol synthase gene in two transgenic rice lines.

A 1.28 L-batch reactor and continuous-flow stirred tank reactor (CFSTR) fed with formate and trichloroethene (TCE) were operated for 120 days and 56 days, respectively, to study the effect of formate as electron donor on anaerobic reductive dechlorination (ARD) of TCE to cis-1,2-dichloroethylene (c-DCE), vinyl chloride (VC), and ethylene (ETH). In batch reactor, injected 60 µmol TCE was completely degraded in the presence of 20% hydrogen gas (H2) in less than 8 days by anaerobic dechlorination mixed-culture (300 mg-soluble protein), Evanite Culture with ability to completely degrade tetrachloroethene (PCE) and -TCE to ETH under anaerobic conditions. Once the formate was used as electron donor instead of hydrogen gas in batch or chemostat system, the TCE-dechlorination rate decreased and acetate production rate increased. It indicates that the concentration of hydrogen produced in both systems is possibly more close to threshold for homoacetogenesis process. Soluble protein concentration of Evanite culture during the batch test increased from 300 mg to 688 mg for 120 days. Through the protein monitoring, we confirmed an increase of microbial population during the reactor operation. In CFSTR test, TCE was fed continuously at 9.9 ppm (75.38 µmol/L) and the influent formate feed concentration increased stepwise from 1.3 mmol/L to 14.3 mmol/L. Injected TCE was accumulated at 18 days of HRT, but TCE was completely degraded at 36 days of HRT without accumulation of the injected-TCE during the left of experiment period, getting H2 from fermentative hydrogen production of injected formate. Although c-DCE was also accumulated for 23 days after beginning of CFSTR operation, it reached steady-state in the presence of excessive formate. We also evaluated microbial dynamic of the culture at different chemical state in the reactor by DGGE (denaturing gradient gel electrophoresis).

Transgenic Nicotiana benthamiana plants harboring the coat protein(CP) gene of Zucchini green mottle mosaic virus(ZGMMV) were chosen as a model host for the environmental risk assessment of genetically modified plants with virus resistance. This study was focused on whether new virus type may arise during serial inoculation of one point CP mutant of ZGMMV on the transgenic plants. In vitro transcripts derived from the non-functional CP mutant were inoculated onto the virus-tolerant and -susceptible transgenic N. benthamiana plants. Any notable viral symptoms that could arise on the inoculated transgenic host plants were not detected, even though the inoculation experiment was repeated a total of ten times. This result suggests that potential risk associated with the CP-expressiing transgenic plants may not be significant. However, cautions must be taken as it does not guarantee environmental safety of these CP-mediated virus-resistant plants, considering the limited number of the transgenic plants tested in this study. Further study at a larger scale is needed to evaluate the environmental risk that might be associated with the CP-mediated virus resistant plant.

Cultivated hot pepper crops showing severe mosaic symptom were found in Korea in 2004 and their causal agent was identified as Cucumber mosaic virus (CMV). These pepper crops was resistant to the virus in the filled, and they belonged to pathotype 0 (P0) resistant pepper. Resistance screening of selected pepper plants showed that a pepper isolate of CMV was the P0 resistance-breaking virus. This P0 resistance-breaking isolate of CMV, named as Ca-P1, was isolated from leaves of the virus-infected Capsicum annuum cv. Manidda that showed systemic severe mosaic symptom. Ca-P1-CMV could induce systemic mosaic symptoms on P0-susceptible (P0-S) and P0-resistant (P0-R) cultivars whereas an ordinary strain (Fny-CMV) could not infect P0-R. This result suggests that Ca-P1-CMV can overcome P0 resistant pepper cultivars. To analyze its genome sequence, the complete nucleotide sequence of RNA3 of Ca-P1-CMV was determined from the infectious full-length cDNA clone of the virus. RNA3 of Ca-P1-CMV consisted of 2,219 nucleotides. Overall sequence homology of RNA3-encoded two viral proteins (movement protein and coat protein) revealed high similarity (75.2-97.2%) with the known CMV strains. By sequence analysis with known representative strains of CMV, Ca-P1-CMV belongs to a typical member of CMV subgroup IB. The resistance and resistance-breaking mechanisms of pepper and counterpart CMV, respectively, remain to be investigated, which will enrich the genetic resources and accelerate CMV-resistant pepper breeding programs.

To determine the pathogenicity domain and to apply cross protection, Pepper mild mottle virus (PMMoV) point-mutations in the replicase (REP) gene between the methyl-transferase and helicase domains, and deletions truncating pseudoknots in the 3′ non-coding region (NCR), were constructed. Some mutants substituting a single amino acid in REP residue 348 exhibited mild symptoms in Nicotiana benthamiana or pepper plants. Accumulation of these mutants was higher than that of other REP mutants or wild-type PMMoV. Deletion mutants in the 3′ NCR pseudoknot showed the lowest virus replication and accumulation among the mutants tested. Six attenuated mutants, which combined 3′ NCR deletions and single or double REP substitution mutations were constructed to investigate cross protection effects on pepper plants. All six of the attenuated mutants showed milder symptom development than wild-type virus. These results suggest that REP and the pseudoknot in the 3′ NCR are major pathogenicity determinants of the virus, and engineered PMMoV attenuated mutants can be useful for protection against the virus in pepper plants.

Five isolates of Cucumber mosaic virus (CMV) from Lilium sp. (lily), which were isolated from specimens in Japan, Korea and Taiwan, were unable to support satellite RNA (satRNA) accumulation. In order to map the CMV sequences that are involved in satRNA support, HL-CMV (Japanese lily isolate), Y-CMV (ordinary strain) and Y-satellite RNA (Y-sat) were used as the source material. The pseudorecombinants between Y-CMV and HL-CMV revealed that RNA1 was essential for satRNA replication in lily. The results of chimeric constructs and various mutations showed that two amino acid residues (at positions 876 and 891) in the 1a protein were the determinants for the inability of HL-CMV to support a satRNA. Specifically, Thr at position 876 had a more pronounced effect than Met at position 891. Specific changes in RNA sequence were also detected in the 3′ terminus of Y-sat and these particular alterations allowed it to be supported by HL-CMV. It is believed that, through evolution, the adaptation of CMV to lily resulted in the introduction of amino acid changes in the 1a protein, changes that coincidentally affected the ability of lily CMV to support satRNAs.

Three isolates of Cucumber mosaic virus (CMV) from lily plants showing mosaic and distortion symptoms were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) using primers specific to Cucumovirus genus namely, LK-CMV, LK4-CMV, and LK5-CMV. Restriction enzymes patterns of the RTPCR products revealed that the lily isolates belonged to subgroup IA of CMV. In terms of biological properties, the lily isolates have highly similar but distinct pathogenicity as reported in other lily strains and ordinary strains of CMV. To characterize the molecular properties, cDNAs containing coat protein (CP) gene and 3`` non-coding region (NCR) of RNA3 for the isolates were cloned and their nucleotide sequences were determined. The CP similarity (218 amino acids) was highly homologous (>97%) with that of subgroup I CMV strains. However, an additional 20-nulcleotide long segment was only present in 3`` NCR of lily isolates, which form an additional stem-loop RNA structure. By using chimeric construct exchange cDNA containing 3``NCR of LK-CMV into the full-length cDNA clone of RNA3 of Fny-CMV, this additional segment may prove to be significant in the identification and fitness of the virus in lily plants. The pathology of zucchini squash infected by F1F2L3-CMV, a pseudorecombinant virus was showed to change drastically the severe mosaic and stunting symptom into a mild chlorotic spot on systemic leave, compared with Fny-CMV. To delimit the sequence of RNA3 affected the pathology, various RNA3 chimeras were constructed between two strains of CMV. The symptom determinants of F1F2L3-CMV were mapped to the positions amino acid 234, 239, and 250 in 3a movement protein (MP). RNA3 chimeras changed the sequences encoding three amino acids were resulted in alteration of systemic symptom.

Summary Pseudomonas syringae pv. phaseolicola, the causative agent of halo blight, is the most important bacterial pathogen of bean. Both nontoxigenic (Tox-) and toxigenic (Tox+) strains of this pathogen cause halo blight in beans. However, nontoxigenic strains cannot be detected by currently available molecular and serological tools. In this study, a TaqMan probe and primer set were designed based on the phage integrase family site-specific recombinase of P. s. pv. phaseolicola 1448A because it is known that most site-specific recombinases are structurally and functionally diverse. The specificity of the probe and primers was evaluated using purified DNA from 29 isolates of 3 different pathovars of P. syringae. The probe and primer set were able to detect Tox- and Tox+ isolates of P. s. pv. Phaseolicola, but no other phytopathogenic bacteria. The assay was also able to detect at least 5 genome equivalents of cloned amplified target DNA, using purified DNA, or 7 colony forming unit (CFU) per reaction when using calibrated cell suspensions. Thus, the TaqMan real-time PCR-based method can be used for the rapid detection of both types of P. s. pv. Phaseolicola, and will potentially simplify and facilitate the diagnosis and monitoring of this pathogen, and guide plant disease management.