Hidetaka Hori

A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeid beetles

A spore‐forming bacterium isolated from the soil of Japan was assigned to Bacillus thuringiensis serovar japonensis (flagellar antigen 23). Parasporal inclusions of this isolate were spherical to ovoid in shape and exhibited high larvicidal activity against coleopterous scarabaeid beetles, the cupreous chafer. Anomala cuprea, the soybean beetle, Anomala rufocuprea, and the Japanese beetle. Popillia japonica. No toxicity was shown by this isolate against larvae of Lepidoptera, Diptera, and Orthoptera, and adults of a chrysomelid coleopteran.

Identification of QTLs that control clubroot resistance in Brassica oleracea and comparative analysis of clubroot resistance genes between B. rapa and B. oleracea.

To perform comparative studies of CR (clubroot resistance) loci in Brassica oleracea and Brassica rapa and to develop marker-assisted selection in B. oleracea, we constructed a B. oleracea map, including specific markers linked to CR genes of B. rapa. We also analyzed CR-QTLs using the mean phenotypes of F3 progenies from the cross of a resistant double-haploid line (Anju) with a susceptible double-haploid line (GC). In the nine linkage groups obtained (O1-O9), the major QTL, pb-Bo(Anju)1, was derived from Anju with a maximum LOD score (13.7) in O2. The QTL (LOD 5.1) located in O5, pb-Bo(GC)1, was derived from the susceptible GC. Other QTLs with smaller effects were found in O2, O3, and O7. Based on common markers, it was possible to compare our finding CR-QTLs with the B. oleraceaCR loci reported by previous authors; pb-Bo(GC)1 may be identical to the CR-QTL reported previously or a different member contained in the same CR gene cluster. In total, the markers linked to seven B. rapaCR genes were mapped on the B. oleracea map. Based on the mapping position and markers of the CR genes, informative comparative studies of CR loci between B. oleracea and B. rapa were performed. Our map discloses specific primer sequences linked to CR genes and includes public SSR markers that will promote pyramiding CR genes in intra- and inter-specific crosses in Brassica crops. Five genes involved in glucosinolates biosynthesis were also mapped, and GSL-BoELONG and GSL-BoPro were found to be linked to the pb-Bo(Anju)1 and Bo(GC)1 loci, respectively. The linkage drag associated with the CR-QTLs is briefly discussed.

Processing of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juices of various insect larvae

Midgut juices were prepared from Adoxophyes sp., smaller tea tortrix (STT); Bombyx mori, silkworm (SW); Spodoptera litura, common cutworm (CCW); Plutella xylostella, diamondback moth (DBM); and Musca domestica, housefly (HF) and immobilized onto Sepharose 4B. delta-Endotoxins (ICPs) from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 were digested by these immobilized gut juice proteases. All gut juices tested derived relatively proteolytic resistant cores from ICP. The molecular sizes of these cores, about 55 kDa in SDS-PAGE, were resulted. In the case of CCW, however, digestion was very strong and only 1/20 concentration of core protein remained relative to other digests. The N-terminal amino acid sequencing of the core proteins showed that they were truncated at the very end of the N-terminus of protoxin, CryIA, at different sites. Although housefly larvae were completely insensitive to active toxin, the gut juice produced the core, suggesting that the housefly may lack the binding sites for the core-active toxin.

Characterization of a novel plasma membrane protein, expressed in the midgut epithelia of Bombyx mori, that binds to Cry1A toxins.

ABSTRACT We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and Kd constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.

Insecticidal spectrum of a novel isolate of Bacillus thuringiensis serovar japonensis

Abstract A newly isolated strain, designated Buibui, belonging to Bacillus thuringiensis serovar japonensis had potent larvicidal activity against the scarabaeids Anomala cuprea Hope and Popillia japonica Newman. Preliminary tests indicated that other scarabaeid larvae including A. albopilosa Hope, A. rufocuprea Motschulsky, A. daimiana Harold, A. schonfeldti Ohaus, Mimela splendens (Gyllenhal), and Blitopertha orientalis (Waterhouse) were equally susceptible to strain Buibui. However, the strain showed no insecticidal activity against larval lepidopteran insects including Bombyx mori (L.) (Bombycidae), Plutella xylostella (L.) (Plutellidae), Spodoptera litura Fabricius, S. exigua Hubner (Noctuidae), and Adoxophyes sp. (Tortricidae) and coleopterans, Allomyrina dichotoma L. (Scarabaeidae) and Henosepilachna vigintioctopunctata Fabricius (Coccinellidae). At 14 days post-treatment, the LC50 and LC95 of the δ-endotoxin against the cupreous chafer, A. cuprea, were estimated by probit analysis as 0.098 ± 0.019 and 0.29 ± 0.086 μg protein/g compost, respectively. Strain Buibui showed no β-exotoxin activity.

Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects.

RecombinantEscherichia coli strains harboring pAG1, pAG2, pKBB100, and pKBB101 were cloned by using antiserum constructed against 130-kDa crystal protein antigen ofBacillus thuringiensis serovarjaponensis strain Buibui. DNAs in the recombinant strains hybridized to the 26-base oligonucleotide probe corresponding to N-terminal amino acids of the 130-kDa crystal protein of strain Buibui. Cultures of the recombinant strains were toxic to larvae of the cupreous chafer,anomala cuprea. Furthermore, the production of the 130-kDa polypeptide was demonstrated in the cells harboring pAG1 and pAG2 by immunoblot analysis with antiserum against the 130-kDa crystal protein. Southern hybridization analysis showed that the 130-kDa crystal protein gene is located on the chromosomal DNA of strain Buibui. On the other hand, DNA probes derived fromcryIA(a) andcryIIIA genes did not hybridize to the DNA of strain Buibui.

Inhibition of processing of plant N-linked oligosaccharides by castanospermine

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits lysosomal alpha- and beta-glucosidase. It also inhibits processing of influenza viral glycoproteins by inhibiting glucosidase I and leads to altered glycoproteins with Glc3Man7GlcNAc2 structures. Castanospermine was tested as an inhibitor of glycoprotein processing in suspension-cultured soybean cells. Soybean cells were pulse-labeled with [2-3H]mannose and chased for varying periods in unlabeled medium. In normal cells, the initial glycopeptides contained oligosaccharides having Glc3Man9GlcNAc2 to Glc1Man9GlcNAc2 structures and these were trimmed during the chase to Man9GlcNac2 to Man7GlcNAc2 structures. In the presence of castanospermine, no trimming of glucose residues occurred although some mannose residues were apparently still removed. Thus, the major oligosaccharide in the glycopeptides of castanospermine-incubated cells after a 90-min chase was a Glc3Man7GlcNAc2 structure. Smaller amounts of Glc3Man6GlcNAc2 and Glc3Man5GlcNAc2 were also identified. Thus, in plant cells, castanospermine also prevents the removal of the outermost glucose residue.

GalNAc pretreatment inhibits trapping of Bacillus thuringiensis Cry1Ac on the peritrophic membrane of Bombyx mori.

Bombyx mori (Shunrei × Shogetsu) is sensitive to Cry1Aa and resistant to Cry1Ac, both insecticidal proteins of Bacillus thuringiensis. Cry1Aa passed through the peritrophic membrane (PM) much faster (0.37 μg/mm2 PM/h) than Cry1Ac (0.05 μg/mm2 PM/h) during the initial observation period. Both Cry1Aa and Cry1Ac bound to the PM but only the binding of Cry1Ac was specifically inhibited by N‐acetylgalactosamine (GalNAc). When Cry1Ac was pretreated with GalNAc, Cry1Ac permeated the PM much faster. These results suggested that Cry1Ac bound a PM protein via GalNAc on a sugar side chain. The role of the PM on Cry1Ac resistance of B. mori was briefly discussed.

Separation of distinct compartments of rice Golgi complex by sucrose density gradient centrifugation

Abstract We studied the separation of distinct compartments of the Golgi complex in rice and tobacco employing the discontinuous sucrose density gradient centrifugation technique with EDTA or MgCl2. In the presence of EDTA, rice Golgi marker enzymes, nucleoside diphosphatase and N-acetylglucosaminyltransferase were fractionated in the 28 and 33% sucrose fractions, respectively. Peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 that are shown to be located at Golgi complex were broadly distributed around a 31.5% sucrose density. Under a MgCl2-supplemented condition, the peak of nucleoside diphosphatase was shifted to 29 and 37% sucrose and N-acetylglucosaminyltransferase was distributed in the 37% sucrose fraction. The distribution of peanut lectin-recognized glycoproteins and reversibly glycosylated polypeptide-1 were also shifted to the 34.5% sucrose fraction. Furthermore, glucan synthase-I and UDP-glucose pyrophosphorylase were fractionated in the 26.5% sucrose fraction, regardless of the presence or absence of MgCl2. These results indicate that there exist at least four distinct compartments of the rice Golgi complex. The tobacco Golgi marker enzymes and Golgi-localized proteins were distributed in the 30–31% sucrose fractions in the presence of EDTA and slightly shifted to the 32–33% sucrose fractions under a MgCl2-supplemented condition. These results suggest that the sedimentation behavior of compartments of the rice Golgi complex in the gradients is specific for rice cells.

Lipids of brush border membrane vesicles (BBMV) from Plutella xylostella resistant and susceptible to Cry1Ac δ-endotoxin of Bacillus thuringiensis

Plutella xylostella (PX) that were 130000-fold more resistant to Cry1Ac were selected from the susceptible strain and maintained in the laboratory. The LC50 of the susceptible strain (PXS) was 0.38 microg toxin/g diet, whereas that of the resistant strain (PXR) was 4800 microg toxin/g diet. Brush border membrane vesicles (BBMV) were prepared from both PXS and PXR. In ligand blot analysis, Cry1Ac bound to a 120-kDa protein of BBMV; however, the intensity of the band was almost equal in both strains of insect. Hence, we analyzed the lipid components of BBMV from PXS and PXR. BBMV lipids were fractionated into non-polar lipid, phospholipid, neutral glycolipid and acidic glycolipid. Neutral glycolipid content was substantially lower in the BBMV of PXR than of PXS. The same trend was observed when lipids were extracted from whole midgut instead of BBMV. Thin layer chromatography of midgut neutral glycolipids revealed the presence of more than seven components. Among the midgut neutral glycolipids, a possible hexasaccharylceramide and a possible trisaccharylceramide of PXR were less than half the level found in PXS. The other lipid fractions in PXR and PXS were similar to each other.