The DNA damage signal transducer ortholog <Emphasis Type="Italic">Mop53BP1</Emphasis> is required for proper appressorium differentiation and pathogenicity in <Emphasis Type="Italic">Pyricularia oryzae</Emphasis>

Appressorium differentiation, one of the most important steps in pathogenesis by the rice blast fungus, Pyricularia oryzae, is strongly coordinated with the cell cycle. In this study, we identified an ortholog gene of 53BP1, which encodes a signal transducer protein that participates in G2-M cell cycle checkpoint in higher eukaryotes, in the genome of P. oryzae and characterized the phenotype of deletion and overexpression mutants. Deletion mutants showed no significant deficiency in vegetative growth compared to wild-type and complemented strains, even on the media containing DNA-damaging agents. However, these null mutants had abnormal appressoria and formed more appressoria per conidium than in the wild type and were unable to penetrate the epidermis of rice leaves. eGFP-fused Mop53BP1 and qRT-PCR analyses revealed that Mop53BP1 is expressed during the first hours of appressorium formation. In addition, in overexpression mutants, Mop53BP1 localized to nuclei during all stages of appressorium maturation and penetration, and the mutants were resistant to the microtubule inhibitor benomyl, suggesting that Mop53BP1 is nuclear protein and may have some role related to microtubules.     

Rhodococcus equi virulence plasmids recovered from horses and their environment in Jeju, Korea: 90-kb type II and a new variant, 90-kb type V

Rhodococcus equi was isolated from fecal and soil samples from four native Jeju horse farms and six Thoroughbred farms in Jeju, Korea. The isolates were examined for the presence of virulence-associated 15-17-kDa antigens (VapA) by colony blotting, using the monoclonal antibody 10G5, and for the gene encoding VapA by PCR. R. equi was isolated from all 36 soil samples collected from the 10 farms with between 5.0 x 10(2) and 7.5 x 10(4) colony-forming units (cfu) per gram of soil, and from 37 of 40 fecal samples with between 5.0 x 10(1) and 1.1 x 10 (5) cfu per gram of feces. Virulent R. equi was isolated from seven farms and appeared in 2.0% of isolates (10 of 508). Of the 10 virulent isolates, four contained a 90-kb type II plasmid, which has been found in isolates from the Kiso native horses of Japan, and the other six contained a new variant, which did not display the EcoRI and EcoT22I digestion patterns of the 10 representative plasmids already reported (85-kb types I, II, III, and IV; 87-kb types I and II; 90-kb types I, II, III, and IV). We designated the new variant as the "90-kb type V" plasmid, because its EcoRI digestion pattern is similar to that of the 90-kb type II plasmid. This is the first report of the prevalence of virulent R. equi in Jeju, Korea. The same virulence plasmid type is found in both Korean and Japanese isolates, providing insight into the origin, ancestry, and dispersal of native horses in Korea and Japan.     

Effects of oral exposure of bisphenol A on mRNA expression of nuclear receptors in murine placentae assessed by DNA microarray

Bisphenol A (BPA), a candidate endocrine disruptor (ED), is considered to bind to estrogen receptors and to regulate expressions of estrogen responsive genes. It has also shown evidence of affecting the reproductive, immunological and nervous systems of mammalian embryos. However, the effects of BPA on placentae, a central organ of feto-maternal interlocution, are still unclear. To reveal the mechanisms of BPA effects on placentae in mammals, we compared the mRNA expression of 20 nuclear receptors between placentae of vehicle controls and those of orally BPA exposed pregnant mice by a DNA microarray technique. In murine placentae, mRNAs of 11 nuclear receptors were not detected. However, greater than 1.5 fold changes in mRNA expression of nine nuclear receptors between vehicle control and BPA treated mice were noted. Moreover, remarkable changes in mRNA expression of six non-nuclear receptor proteins were induced by BPA exposure. There were various differences in the effects of BPA on the expression of these mRNAs between the placentae with male embryos and those with female embryos. Such embryo-sex dependent differences are interesting and important pointers to understanding of the endocrine disrupting effect of BPA. The present data indicate that BPA affects the expression of nuclear receptor mRNAs in placentae and may disrupt the physiological functions of placentae.     

Characterization and genetic analysis of laboratory mutants of <Emphasis Type="Italic">Cochliobolus heterostrophus</Emphasis> resistant to dicarboximide and phenylpyrrole fungicides

 Laboratory mutants of Cochliobolus heterostrophus resistant to iprodione were obtained after chemical mutageneses. All the mutants were able to grow on the medium amended with iprodione 100 μg/ml. They showed positive cross-resistance to procymidone and fludioxonil and were sensitive to high osmolarity. Crosses between the mutant and a wild-type strain revealed that the fungicide resistance and osmotic sensitivity traits were inherited by their offspring in a 1 : 1 mutant/wild type ratio, indicating that the mutant phenotypes in these strains were due to alteration at a single gene locus. Results from allelism tests indicated that three genes (Dic1, Dic2, Dic3) conferred the mutant phenotypes. Among them, Dic1 mutant strains were classified into three types on the basis of their phenotypes. The first type was moderately resistant to the fungicides and less sensitive to osmotic stress than the other Dic1 mutant strains. The second type showed moderate fungicide resistance, but growth was inhibited under lower osmotic stress (50 mM KCl). The other Dic1 mutant strains grew well on medium containing iprodione and fludioxonil even at a concentration of 100 μg/ml and were highly sensitive to osmotic stress. The Dic2 and Dic3 mutant strains had moderate resistance to the fungicides with low-level osmotic sensitivity. The Dic1 gene was epistatic to Dic2 and Dic3 for fungicide resistance and hypostatic to them for osmotic sensitivity. These results suggest that the osmoregulatory system is involved in fungicide resistance in laboratory mutants of C. heterostrophus. Received: March 14, 2002 / Accepted: August 13, 2002     

Expressions of estrogen receptors in the bovine corpus luteum: cyclic changes and effects of prostaglandin F2alpha and cytokines

Estrogen (E) exerts its function by binding to two intracellular estrogen receptors, ERalpha and ERbeta. Although ERs have been reported to be expressed in the bovine corpus luteum (CL), the mechanisms that control ER expression in the bovine CL are not fully understood. To determine the possible regulatory mechanisms of ERalpha and ERbeta that meditate distinct E functions, we examined 1) the changes in the protein expressions of ERs in the CL throughout the luteal phase and 2) the effects of prostaglandin (PG) F2alpha, tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) on the expressions of ERs in cultured bovine luteal cells. Western blot analyses revealed that ERalpha and ERbeta proteins were expressed throughout the luteal phase. The ERalpha protein level was high at the early luteal (Days 2-3 after ovulation) and mid-luteal stages (Days 8-12) and was extremely low at the regressed luteal stage (Days 19-21). The ERbeta protein level increased from the early to developing luteal stage, remained at the same level at the mid-luteal stage and decreased thereafter. The ratio of ERbeta to ERalpha was higher in the regressed stage than in the other stages. Luteal cells obtained from mid-stage CLs (Days 8-12) were incubated with PGF2alpha (0.01-1 microM), TNFalpha (0.0145-1.45 nM) or IFNgamma (0.0125-1.25 nM) for 24 h. PGF2alpha and TNFalpha inhibited ERa and ERbeta mRNA expressions. IFNgamma suppressed ERbeta mRNA expression but did not affect the expression of ERalpha mRNA. However, the ERalpha and ERbeta protein levels were not affected by any of the above treatments. These data indicate that PGF2alpha, TNFalpha and IFNgamma regulate ERalpha and ERbeta mRNA expressions in bovine luteal cells. Moreover, the changes in the ERbeta/ERalpha ratio throughout the luteal phase suggest that ERalpha is associated with luteal maintenance. Therefore, a dramatic decrease in ERalpha at the regressed luteal stage could result in progression of structural luteolysis in the bovine CL.     

Optimal germination condition by sulfuric acid pretreatment to improve seed germination of Sabina vulgaris Ant.

Understanding the germination traits of plants is important not only for understanding natural regeneration processes but also for developing seedling production techniques for planting. Sabina vulgaris Ant. is a common species used for reforestation in semi-arid areas of the Mu-Us Desert, in Inner Mongolia, China, but its extremely low germination rate, both in situ and in vivo, is a bottleneck for seedling production. Sulfuric acid pretreatment was applied to improve germination, and the germination rate was compared for different soaking time (10, 30, 60, 90, and 120 min), different temperatures (10, 15, 20, 30, and 35°C) and under different lighting conditions (dark and light). Sulfuric acid treatment gave a high germination rate, reaching 60% at 30 days after sowing. However, the non-treated seeds produced no germination. The optimal treatment time in sulfuric acid was 120 min. Germination after sulfuric acid treatment increased at incubation temperatures from 10 to 30°C, but decreased at 35°C. Incubation at 25–30°C gave maximum germination of more than 50%. Light treatment had little effect on germination. Pretreatment with sulfuric acid improved water absorption by the embryo by creating cracks and cavities in the seed coat tissue. These results indicated that S. vulgaris seeds have physical dormancy caused by their hard seed coats, which prevents absorption of water into the embryo. A combination of pretreatment with sulfuric acid and incubation at 25–30°C was most effective in improving the germination of S. vulgaris seeds.     

Distribution of the FoToml gene encoding tomatinase in formae speciales of Fusarium oxysporum and identification of a novel tomatinase from F. oxysporum f. sp. radicis-lycopersici, the causal agent of Fusarium crown and root rot of tomato

The antifungal glycoalkaloid -tomatine accumulates in tomato plants and may protect plants from fungal infection. Fusarium oxysporum f. sp. lycopersici, the causal agent of vascular wilt of tomato, produces a tomatinase (FoToml) that degrades -tomatine to the nontoxic compounds tetrasaccharide lycotetraose and tomatidine. Induction of tomatinases and the distribution of FoToml homologs were examined among 30 strains belonging to 16 formae speciales of F. oxysporum. Tomatinase activity was found in 27 strains belonging to 15 formae speciales, but FoToml homologs (>98% sequence identity) were detected in only six strains belonging to four formae speciales. To identify tomatinases other than FoToml, -tomatine-inducible proteins of another tomato pathogen F. oxysporum f. sp. radicis-lycopersici were analyzed by two-dimensional gel electrophoresis. A protein with a molecular mass of 64kDa accumulated in the -tomatine-induced culture filtrates, and the protein had tomatinase activity, degrading -tomatine to lycotetraose and tomatidine.     

Permeation of carboxylates as ionic compounds into fish meats by soaking and its osmotic dehydrating effect

Permeability of Na-gluconate, Na-malate and Na-acetate into fish-meat strips by soaking, and their osmotic dehydrating effects were investigated in comparison with those of sodium chloride (NaCl) and sorbitol. Carboxylic acids, anion species of carboxylates, were less permeable into soaked meats than chlorine of NaCl, and their permeability was similar to that of sorbitol. However, the permeation of sodium, cation species of the carboxylates and NaCl, was promoted with the concentration of free sodium ion dissociated in soaking solutions. The ratio of sodium to the anion species in soaked meats was varied, depending on the numbers of the sodium atom in the molecules as well as the dissociation degree of the ionic compounds in the soaking solution. Furthermore, the osmotic dehydrating effects of Na-gluconate and Na-malate were analogous with that of sorbitol, and higher than those of NaCl and Na-acetate. These results indicated that the permeation characteristics and the osmotic dehydrating effects of the carboxylates were associated with their ionization in soaking solution as well as their molecular weights.     

Hydathode function and changes in contents of elements in eddo exposed to zinc in hydroponic solution

To explore the zinc stress response in eddo, plants were grown for 15 d in hydroponic solutions containing 1 (control), 200, and 1000 μM zinc, and the hydathode function and changes in the contents of various elements in these plants were investigated. Under 1000 μM zinc, the dry weights of leaf blades and roots are decreased by 17 and 42%, respectively. The zinc contents in leaf blades, petioles, corms, and roots increased with increasing zinc levels in the solution. The zinc content in roots was 6.57 mg g^?1 dry weight, which was 2.8–4.3 times higher than in other plant parts under 1000 μM zinc. These results demonstrate that the severe root damage occurring under 1000 μM zinc is due to higher zinc content in the roots. Under zinc stress, the contents of iron and copper in roots increased, but the contents of magnesium and calcium in petioles, corms, and roots, iron in leaf blades and corms, and manganese in petioles and corms decreased. In the guttation fluid, the concentrations of zinc, magnesium, and potassium increased, while the iron concentration decreased under 1000 μM zinc. Thus, elemental changes occurred in the guttation fluid as well as in different plant parts in eddo. In the 200 and 1000 μM zinc treatments, the amount of zinc eliminated via guttation was 2.8 and 8.5 times higher, respectively, than in the control. The results indicate that guttation partly contributes to the excretion of excess zinc under zinc stress conditions.     

Analysis of plasmids encoding the tyrosine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to elucidate mechanisms of tyramine accumulation during fish sauce production, two tyramine-producing bacterial strains, referred to as TyrA and TyrB, were isolated from fish sauce mash accumulating over 141 mg of tyramine per 100 g of sample. Both strains were identified as Tetragenococcus halophilus based on phenotypic characterization and a 16S rRNA gene sequence analysis. Molecular analysis of the tyramine-producing gene in the two strains confirmed the presence of a ~30-kb plasmid encoding a single copy of the pyridoxal phosphate-dependent tyrosine decarboxylase gene (tdcA) along with three other genes related to tyramine synthesis (tdc cluster). The complete nucleotide sequences of plasmids extracted from the two strains indicated that both plasmids were almost identical, except for a 1.6-kb transposon sequence in the plasmid from the strain TyrB. Both plasmids had a replication region, a plasmid maintenance region, and two putative mobile genetic elements located upstream and downstream of the tdc cluster. This structure was identical to that of tetragenococcal plasmids encoding histidine decarboxylase (hdcA), which were sequenced previously. These results suggest a common origin for plasmids encoding hdcA and tdcA. In addition, the genes for both these biogenic amines are distributed among tetragenococcal species via this plasmid.