Kazuhiro Iiyama

Burkholderia gladioli associated with symptoms of bacterial grain rot and leaf-sheath browning of rice plants

Rice plants with bacterial leaf-sheath browning and grain rot were observed in Fukuoka Prefecture in Japan during the autumn seasons of 1995 and 1996. Burkholderia spp. were consistently isolated from the infected leaf sheaths and grains. These isolates were pathogenic and induced symptoms of seedling rot, grain rot, and leaf-sheath browning in rice plants, as well as in some orchidaceous plants (cymbidium, dendrobium, and oncidium leaves), gladiolus leaves, and onion bulbs. On the basis of morphological, physiological and pathological tests, and species-specific polymerase chain reaction, the isolates were identified as belonging to either Burkholderia glumae or Burkholderia gladioli. B. gladioli, as well as B. glumae, attacked rice plants after artificial inoculation and reproduced the symptoms similar to those after natural infections. We confirmed that rice is an additional natural host of B. gladioli. It is clarified that bacterial grain rot of rice is caused not only by B. glumae but also by B. gladioli.

Effect of Superoxide Dismutase Gene Inactivation on Virulence of Pseudomonas aeruginosa PAO1 toward the Silkworm, Bombyx mori

ABSTRACT To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm, Bombyx mori, mutants of Pseudomonas aeruginosa PAO1 lacking manganese-SOD (PAO1sodM), iron-SOD (PAO1sodB), or both (PAO1sodMB) were generated. The mutants were injected into the hemocoel of B. mori. The virulence decreased in the order PAO1 = PAO1sodM > PAO1sodB > PAO1sodMB. In particular, PAO1sodMB was avirulent at a dose of 105 cells or less. The sod double mutant PAO1sodMB was then complemented with either pSodM or pSodB in trans. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of P. aeruginosa against B. mori. The results of growth in B. mori hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in sod mutants. In conclusion, the SODs are required for full virulence of P. aeruginosa against B. mori and Fe-SOD is more important than Mn-SOD in the infection process.

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.

Phylogenetic analysis of Metarhizium spp. isolated from soil in Japan

As a result of analyzing the internal transcribed spacer (ITS) and 5′ end of the EF-1α sequence of 145 isolates of Metarhizium spp. isolated from soil in Japan using selective agar medium, eight species were identified. ITS sequence analysis divided the isolates into three clades. Two were identified as M. flavoviride var. pemphigi and M. lepidiotae, respectively. EF-1α sequence analysis identified the other clades as six species: M. anisopliae, M. brunneum, M. guizhouense, M. majus, M. pingshaense and M. robertisii. The distribution of M. flavoviride var. pemphigi was restricted to forest or wood soil, and conidial sizes of M. guizhouense and M. majus were incongruent with the phylogeny based on the sequence of the 5′ end of EF-1α.

Expression, purification, and characterization of endo-β-N- acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system

AbstractEndo-β-N-acetylglucosaminidase (Endo H) from Streptomyces plicatus hydrolyzes the core di-GlcNAc units of asparagine-linked oligosaccharides and is regarded as an important tool for glycobiology research. In the present study, we established a large-scale system to produce secreted Endo H using a silkworm-baculovirus expression system (silkworm-BES). The recombinant Endo H purified from silkworm hemolymph had activity comparable to that from recombinant Escherichia coli. As well as its well-characterized substrate RNase B, the Endo H from silkworm-BES was able to deglycosylate the high-mannose glycoproteins from silkworm hemolymph. Interestingly, the secretion amount of recombinant Endo H was significantly varied among the different silkworm strains, which could provide valuable information for larger-scale protein productions from silkworm-BES.

Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1

RNA interference (RNAi) is a biological phenomenon that silences the expression of genes of interest. Passive double-stranded RNA (dsRNA) uptake has been uniquely observed in Caenorhabditis elegans due to the expression of systemic RNAi defective-1 (SID-1). We report that ectopic expression of CeSID-1 endows the Sf9 cells with a capacity for soaking RNAi. Soaking the Sf9-SID1 with dsRNA corresponding to either exogenous or endogenous target genes induced a significant decrease in the amount of mRNA or protein. These results enabled us to modify the target proteins of baculovirus expression vector system in both quantities and posttranslational modifications. The current low-cost and high-efficiency RNAi system is useful for high-throughput gene function analysis and mass production of recombinant protein.

Bombyx Mori Strains Useful for Efficient Recombinant Protein Production Using a Baculovirus Vector

The silkworm-baculovirus expression system is known as one of the most efficient methods for mass production of recombinant eukaryotic proteins. Many protein species, such as secretory, membrane, and nuclear proteins, are successfully expressed in this system and used for scientific research and industry. Several recent studies have greatly contributed to reducing the amount of time and labor required to generate recombinant viruses. Additionally, modifications of virus vectors have made this system a more attractive alternative for protein scientists. However, there are few reports on the screening and improvement of the silkworm, Bombyx mori, as a host insect. In this review, we describe an overview of the silkworm-baculovirus expression system and the improvement of silkworm as a host insect by the screening of silkworm bioresources and transgenic silkworm technology.

Role of Nitric Oxide-Detoxifying Enzymes in the Virulence of Pseudomonas aeruginosa against the Silkworm, Bombyx mori

Pseudomonas aeruginosa has two nitric oxide (NO)-detoxification enzymes, NO reductase and flavohemoglobin, which catalyze the reduction and oxygenation of NO respectively. In this study, the NO reductase-deficient mutant showed decreased virulence against the silkworm Bombyx mori, but the flavohemoglobin-deficient mutant did not, indicating that NO-reduction is important to the full virulence of P. aeruginosa against B. mori.

Specific Oligonucleotide Primers Based on Sequences of the 16S-23S rDNA Spacer Region for the Detection of Burkholderia gladioli by PCR

Specific primers were designed based on the sequences of the spacer region between the 16S and 23S ribosomal DNA (rDNA) for direct, rapid and specific detection of Burkholderia gladioli. These primers were named GLA-f and GLA-r. PCR performed on boiled bacterial suspensions yielded an amplification product of approximately 300 bp. No products from other bacterial species, including B. glumae were amplified, even after complete DNA extraction by the cetyltrimethyl-ammonium bromide (CTAB) method. Using the specific primers designed in this study, the PCR method can detect B. gladioli in plant samples within 6 hr. These data demonstrate the potential of specific PCR for the detection of B. gladioli.

Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell

The double-stranded RNA (dsRNA) mediated RNA interference (RNAi) is widely employed in silkworm and its tissue-derived cell lines for gene function analysis. Baculovirus expression vector system (BEVS) has an advantage for large-scale protein expression. Previously, combining these useful tools, we improved traditional AcMNPV-Sf9 BEVS to produce modified target glycoproteins, where the ectopic expression of Caenorhabditis elegans systemic RNAi defective-1 (SID-1) was found to be valuable for soaking RNAi. In current study, we applied CeSID-1 protein to a Bombyx mori NPV (BmNPV)-hypersensitive Bme21 cell line and investigated its properties both in soaking RNAi ability and recombinant protein expression. The soaking RNAi-mediated suppression in the Bme21 cell enables us to produce modified glycoproteins of interest in BmNPV–Bme21 BEVS.