Flagellin glycosylation is ubiquitous in a broad range of phytopathogenic bacteria

Glycosylation of flagellin is known to be involved in filament stabilization, motility, and virulence in Pseudomonas syringae. Here we investigated flagellin glycosylation in other phytopathogenic bacteria. Analyses of deduced amino acid sequences, glycostaining, and molecular masses of purified flagellins revealed that flagellins from all phytopathogenic bacteria investigated were glycosylated. Furthermore, the flagellin in a glycosylation-defective mutant of Xanthomonas campestris pv. campestris (Xcc) had a reduced molecular mass, and motility and virulence of the mutant toward host leaves decreased. These results suggest that flagellin glycosylation is ubiquitous in most phytopathogenic bacteria and that flagellin glycosylation is required for virulence in Xcc.     

Occurrence of glucosamine in higher plants

Occurrence of the unknown basic ninhydrin positive substance in young plant tissue supplied with a high level of ammonium nitrogen has been reported in the previous paper (1). This substance disappeared when the supply of ammonium nitrogen was stopped or the source of nitrogen was changed from ammonium nitrogen to nitrace nitrogen, and this substance was verg scarcely detected when the level of ammonium nitrogen in culture solution was as low as 3 ppm or the foren of nitrogen source was nitrace nitrogen. It is very interesting to identify this substance, as the occurrence of this substance is a distinctive character of the plant which han been supplied with a higher level of ammonium nitrogen and has developed the symptoms of ammonia toxicity.     

Responses to aluminium of suspension-cultured tobacco cells in a simple calcium solution

In a simple solution containing 3 mM CaCl₂ and 3% sucrose (pH 4.5), tobacco cells (Nicotiana tabacum L.) at the logarithmic phase of growth remained viable at least for 24 h. In this medium, the toxic effect of aluminium (Al) on the plasma membrane was investigated for up to 24 h. After the addition of Al to the cell suspension, Al started to accumulate immediately in the cells, and a maximum value was observed at 9 h. Al induced callose deposition, but did not enhance significantly the uptake of Evans blue (a nonpermeating dye), the per oxidation of lipids and the leakage of potassium (K) ions. Furthermore, the AI-treated cells were stained with fluorescein diacetate (FDA) as much as untreated control cells. These results suggest that the accumulation of Al does not damage the membrane. The addition of Fe(II) to the cells which had been exposed to Al for 12 h resulted in immediate lipid peroxidation and Evans blue uptake several hours later. A combination of Al and Fe(II) caused the K leakage, and enhanced the deposition of callose more than Al alone. These results suggest that the accumulation of Al sensitizes the membrane to the Fe(II)-mediated peroxidation of lipids, and that the Al-enhanced per oxidation of lipids is a direct cause of the loss of integrity of the plasma membrane (or cell death) in the Ca medium.     

Evaluation of water resources by snow storage using water balance and tank model method in the Tedori River basin of Japan

We evaluate water resources as the difference between the observed and virtual discharges, defined as discharge without snow storage during the snowfall and snowmelt season, in the Tedori River basin of Ishikawa Prefecture, Japan. The virtual discharge was estimated using the Tank model, in which parameters of calculated discharge were determined when the predictions were consistent with the observed discharges except for those during snowfall and snowmelt periods. Precipitation increase factors, i.e., the ratio of precipitation in the basin to that of a Kanazawa site, ranged from 1.46 to 2.00 with an average of 1.68. Water resources by snow storage (WRSS) during 31 years from July 1976 to June 2006 were estimated to be 400–1,500 mm in depth, which could be considered a significant amount. WRSS gradually decreased over time from 1976 to 2006. We propose the water balance method to estimate WRSS and water resources by snowmelt (WRSM), which easily estimates these parameters without the need for any detailed analysis of hydrograph as that of the Tank model. If snowfall decreases due to global warming, we suggest this area would suffer an irrigation water shortage under the water right, especially in the early spring.     

Generation of transgenic rats expressing enhanced green fluorescent protein in gonadotropin-releasing hormone neurons

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons govern reproductive function by controlling the release of gonadotropins from the pituitary. To facilitate identification of living GnRH neurons, here we attempted to generate transgenic rats that express enhanced green fluorescent protein (EGFP) in GnRH neurons. About 3 kb of rat GnRH promoter region was inserted into the EGFP reporter cassette, and the expression of EGFP fluorescence was confirmed in several cell lines following transient transfection. Then we successfully generated a transgenic rat by injecting linearized GnRH-EGFP transgene into the pronuclei of fertilized oocytes. The GnRH-EGFP transgenic rats expressed EGFP in the brain, but not in the ovary, testis or thymus. Immunohistochemical examination revealed that detectable EGFP fluorescence was confined to the cell body of GnRH-immunoreactive neurons in the septum and preoptic area, while no EGFP signal was discernible in the median eminence where abundant GnRH-immunoreactive fibers were observed. The mean percentage of EGFP-positive cells in the GnRH-positive cells was 76.3%. The GnRH-EGFP transgenic rats generated in the present study will enable characterization of properties of individual GnRH neurons.     

Epizootiological study of tick-borne encephalitis virus infection in Japan

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. Rodent species that are potential hosts for TBEV are widely distributed in various regions in Japan. In this study, we carried out large-scale epizootiological surveys in rodents from various areas of Japan. A total of 931 rodent and insectivore sera were collected from field surveys. Rodents seropositive for TBEV were found in Shimane Prefecture in Honshu and in several areas of Hokkaido Prefecture. These results emphasize the need for further epizootiological and epidemiological research of TBEV and preventive measures for emerging tick-borne encephalitis in Japan.     

Isolation and identification of flavonoids accumulated in proanthocyanidin-free barley

Flavonoids accumulated in proanthocyanidin-free near-isogenic lines iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi, developed by backcross breeding using a leading cultivar, Nishinohoshi, as a recurrent parent and a proanthocyanidin-free mutant as a nonrecurrent parent in Japan, were examined. A new flavanone, (2RS)-dihydrotricin 7-O-β-D-glucopyranoside (1), known flavanones (2RS)-dihydrotricin (2) and (2RS)-homoeriodictyol (3), and known flavones chrysoeriol 7-O-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside] (4), chrysoeriol 7-O-β-D-glucopyranoside (5), tricin (6), and chrysoeriol (7) were isolated from iso ant 17 of Nishinohoshi. The structures and stereochemistries of the isolated flavonoids (1-7) were elucidated on the basis of spectroscopic analyses. The concentrations of the isolated flavonoids (1-7) in iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi were similar to each other, whereas the flavonoids 1-5 and 7 were not detected in Nishinohoshi, an old Japanese cultivar, Amaginijo, and North American cultivar Harrington. The concentration of tricin (6) in Nishinohoshi was a half those in iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi. Except for iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi, the concentration of tricin (6) was highest in Nishinohoshi, followed by Amaginijo and Harrington. Thus, tricin (6), its precursor dihydrotricin (2), and its glucopyranoside, dihydrotricin 7-O-β-D-glucopyranoside (1), as well as chrysoeriol (7) and homoeriodictyol (3) were accumulated in iso ant 13, iso ant 17, and iso ant 22 of Nishinohoshi probably by blocking at the step of flavanone 3-hydroxylase in the procyanidin biogenetic pathway, resulting in enhancement of the alternative biogenetic pathway.     

Alternaria host-selective toxins: determinant factors of plant disease

Seven diseases caused by pathotypes of Alternaria alternata, which produced host-selective toxins (HSTs), a diverse group of low-molecular-weight secondary metabolites, are known, and each HST has an essential role as a determinant of pathogenicity in all interactions between the plant host and A. alternata. Although these HST-producing pathotypes are morphologically indistinguishable, each has a distinct host range and can be distinguished by its specificity on the respective host plant, hence their designation as pathotypes of A. alternata. In 1933, Tanaka made the first discovery of a HST; fungus-free culture filtrates of A. kikuchiana (now called A. alternata Japanese pear pathotype) were toxic to susceptible cultivar Nijisseiki, but not to resistant cultivars. Over the 80 years since then, the structure of HST molecules, target sites and mode of actions of HSTs, and the molecular genetics of HST production regulating by supernumerary chromosomes encoding HST gene clusters have been studied extensively. We focus this review on studies of low-molecular-weight HSTs produced by A. alternata and give an overview of various types of HST studies.     

The TIR–NBS but not LRR domains of two novel N-like proteins are functionally competent to induce the elicitor p50-dependent hypersensitive response

The tobacco N protein recognizes the helicase domain (p50) of the Tobacco mosaic virus (TMV) replicase as an elicitor and mediates hypersensitive response (HR). We obtained two cDNA clones encoding novel N-like (NL) proteins NL-C26 and NL-B69 from Nicotiana tabacum cv. Samsun NN. NL-C26 and NL-B69 had a Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) structure and showed 78% and 73% identities to N, respectively. The NL-C26 and NL-B69 genes were also expressed in N. tabacum cv. Samsun nn, which lacks the N gene. Unlike N, NL-C26 and NL-B69, when coexpressed with p50, failed to induce HR on the sites of agroinfiltration in Samsun nn leaves. However, the elicitor-dependent HR in Samsun nn was induced efficiently by chimeric N proteins with the continuous TIR–NBS domains of NL-C26 and NL-B69. On the other hand, the efficiency of HR induction varied significantly among chimeric N proteins with either of the TIR and NBS domains of the NL proteins. In contrast, chimeras carrying the LRR domains of the NL proteins did not induce HR. Thus, the TIR–NBS domains of NL-C26 and NL-B69 could functionally adapt to the LRR domain of N, which may determine the specificity for the elicitor. We speculate that the NL genes are potential HR-inducing resistance genes for undetermined pathogens other than TMV.     

Pathogenicity of H5N1 influenza A viruses isolated in Vietnam between late 2003 and 2005

Since late 2003, highly pathogenic H5N1 influenza A viruses have spread among poultry and wild aquatic birds in Asian countries. Transmission of these viruses to humans can be lethal. Most human cases of infection with H5N1 viruses have occurred in Vietnam. Therefore, to understand the pathogenicity in mammals of these H5N1 viruses, we took viruses isolated from poultry (5 strains) and humans (2 strains) in Vietnam and tested their virulence in mice. The results showed that the H5N1 viruses from humans were pathogenic in mice and that one avian isolate was also pathogenic. These findings suggested that the H5N1 viruses circulating in poultry adapted during replication in humans or that strains pathogenic in mice were transmitted directly to humans.