Rapid and Sensitive Detection of the Causal Agents of Postharvest Kiwifruit Rot, Botryosphaeria dothidea and Diaporthe eres, Using a Recombinase Polymerase Amplification Assay.

The occurrence of postharvest kiwifruit rot has caused great economic losses in major kiwifruit-producing countries. Several pathogens are involved in kiwifruit rot, notably Botryosphaeria dothidea, and Diaporthe species. In this study, a recombinase polymerase amplification (RPA) assay was developed for the rapid and sensitive detection of the pathogens responsible for posing significant threats to the kiwifruit industries. The RPA primer pairs tested in this study were highly specific for detection of B. dothidea and D. eres. The detection limits of our RPA assays were approximately two picograms of fungal genomic DNA. The optimal conditions for the RPA assays were determined to be at a temperature of 39°C maintained for a minimum duration of 5 min. We were able to detect the pathogens from kiwifruit samples inoculated with a very small number of conidia. The RPA assays enabled specific, sensitive, and rapid detection of B. dothidea and D. eres, the primary pathogens responsible for kiwifruit rots in South Korea.

A RING finger E3 ligase gene, Oryza sativa Delayed Seed Germination 1 (OsDSG1), controls seed germination and stress responses in rice.

Seed germination is an important character for plant growth and seed quality. We identified a rice mutant that was delayed in its germination. There, T-DNA was inserted into Oryza sativa Delayed Seed Germination 1 (OsDSG1), causing a recessive null mutation. Overexpression of the gene enhanced seed germination. OsDSG1 is most similar to Arabidopsis AIP2, an E3 ligase targeting ABI3.Yeast two-hybrid experiments showed that our OsDSG1 binds to OsABI3, indicating that OsDSG1 is a rice ortholog of AIP2. Self-ubiquitination assay indicated that bacterially expressed OsDSG1 protein has E3 ubiquitin ligase activity. Real-time PCR analysis revealed that OsDSG1 was expressed in leaves and roots, and strongly in developing seeds. In addition to the delayed-germination phenotype, mutant plants were shorter and had greater tolerance to high-salt and drought stresses. In the osdsg1 mutant, transcript levels of ABA signaling genes and ABA responsive genes were significantly increased. By contrast, expressions of OsGAMYB and its downstream genes that encode hydrolytic enzymes were markedly reduced. These observations support that OsDSG1 is a major regulator of ABA signaling in germinating seeds. Finally, we observed that the germination rates of various rice cultivars depended upon the transcript levels of OsDSG1 and other ABA-signaling genes.

Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice.

We have generated 47,932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41,234 lines, we obtained 27,621 flanking sequence tags (FSTs), among which 12,505 were integrated into genic regions and 15,116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T(1) or T(2) plants will be provided to the scientific community.