An insecticidal protein produced by Bacillus sphaericus A3-2 was purified to elucidate its structure and mode of action. The active principle purified from the culture broth of A3-2 was a protein with a molecular mass of 53 kDa that rapidly intoxicated German cockroaches (Blattela germanica) at a dose of about 100 ng when injected. The insecticidal protein sphaericolysin possessed the undecapeptide motif of cholesterol-dependent cytolysins and had a unique N-terminal sequence. The recombinant protein expressed in Escherichia coli was equally as potent as the native protein. Sphaericolysin-induced hemolysis resulted from the protein's poreforming action. This activity as well as the insecticidal activity was markedly reduced by a Y159A mutation. Also, coapplication of sphaericolysin with cholesterol abolished the insecticidal action, suggesting that cholesterol binding plays an important role in insecticidal activity. Sphaericolysin-lysed neurons dissociated from the thoracic ganglia of the German cockroaches. In addition, sphaericolysin's activity in ganglia was suppressed by the Y159A mutation. The sphaericolysin-induced damage to the cockroach ganglia was greater than the damage to the ganglia of common cutworms (Spodoptera litura), which accounts, at least in part, for the higher sensitivity to sphaericolysin displayed by the cockroaches than that displayed by cutworms.Using entomopathogens as biopesticides can reduce the use of synthetic pesticides. Entomopathogens have been isolated from soils and the carcasses of insects, although the natural resources of the pathogens are not limited to these. The larvae of Myrmeleontidae insects, referred to as ant lions, suck out the body fluids of their prey. Because ant lions can kill prey larger than themselves, it was postulated that they used toxins. From the regurgitated fluid of Myrmeleon bore larvae, we have purified an insecticidal protein with a molecular mass of 170 kDa (19) and found that it was produced in the larval region from the thorax to the abdomen (33). In addition to the toxin produced by ant lions, insecticidal proteins were found to be produced by bacterial pathogens isolated from M. bore larvae (21, 34). Thus, we have further isolated bacteria from the ant lion's crop, which serves as a reservoir of regurgitated fluid, to evaluate their toxicity to insects. It was found that some bacterial species obtained from the crop exhibit insecticidal actions against Spodoptera litura cutworms when injected (22). Of these, the A3-2 isolate was closely related to Bacillus sphaericus subgroup IIA on the basis of 16S rRNA gene sequencing and DNA-DNA hybridization tests. This Bacillus species is currently used as a biopesticide with mosquitocidal action involving protein toxins, notably, binary toxin, Mtx1, and Mtx2 (2, 6). However, it is not clear whether A3-2 uses such mosquitocidal toxins to kill insects other than mosquitoes.The aim of this study is to determine an insecticidal factor produced by A3-2 and elucidate its mode of action. Here we report that th...

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Ion beams are powerful mutagens that can induce novel mutants in plants. We previously established a system for producing a mutant population of soybean via ion-beam irradiation, isolated plants that had chlorophyll deficiency, and maintained their progeny via self-fertilization. Here we report the characterization of the progeny plants in terms of chlorophyll content, flowering time and isoflavone content in seeds. Chlorophyll deficiency in the leaf tissues was linked with reduced levels of isoflavones, the major flavonoid compounds accumulated in soybean seeds, which suggested the involvement of metabolic changes associated with the chlorophyll deficiency. Intriguingly, flowering time was frequently altered in plants that had a reduced level of chlorophyll in the leaf tissues. Plant lines that flowered either earlier or later than the wild-type plants were detected. The observed coincidental changes were presumed to be attributable to the following origins: structural changes of DNA segments leading to the loss of multiple gene functions, or indirect effects of mutations that affect one of these traits, which were manifested as phenotypic changes in the background of the duplicated composition of the soybean genome.

Retrotransposons constitute a large portion of plant genomes. The chromosomal distribution of a wide variety of retrotransposons has been analyzed using genome sequencing data in several plants, but the evolutionary profile of transposition has been characterized for a limited number of retrotransposon families. Here, we characterized 96 elements of the SORE-1 family of soybean retrotransposons using genome sequencing data. Insertion time of each SORE-1 element into the genome was estimated on the basis of sequence differences between the 5' and 3' long terminal repeats (LTRs). Combining this estimation with information on the chromosomal location of these elements, we found that the insertion of the existing SORE-1 into gene-rich chromosome arms occurred on average more recently than that into gene-poor pericentromeric regions. In addition, both the number of insertions and the proportion of insertions into chromosome arms profoundly increased after 1 million years ago. Solo LTRs were detected in these regions at a similar frequency, suggesting that elimination of SORE-1 via unequal homologous recombination was unbiased. Taken together, these results suggest the preference of a recent insertion of SORE-1 into chromosome arms comprising euchromatic regions. This notion is contrary to an earlier view deduced from an overall profiling of soybean retrotransposons and suggests that the pattern of chromosomal distribution can be more diverse than previously thought between different families of retrotransposons.

Transcription of soybean retrotransposon SORE-1 was temporally upregulated during ovule development. This transcriptional pattern was under intrinsic control conferred by the long terminal repeat of SORE-1. Transcriptionally active retrotransposons are capable of inducing random disruption of genes, providing a powerful tool for mutagenesis. Activation of retrotransposons in reproductive cells, in particular, can lead to heritable changes. Here, we examined developmental control of transcription of soybean retrotransposon SORE-1. Transgenic Arabidopsis plants that contain β-glucuronidase (GUS) reporter gene fused with the SORE-1 long terminal repeat (LTR) had GUS staining in the ovule. Quantitative analysis of transcripts in plants with this DNA construct and those with the full-length SORE-1 element indicated a temporal upregulation of SORE-1 transcription during ovule development. A comparable phenomenon was also observed in soybean plants that had a recent insertion of this element in the GmphyA2 gene. These results provide evidence that the temporal upregulation of SORE-1 in the reproductive organ is sufficiently controlled by its LTR and indicate that the intrinsic expression pattern of SORE-1 is consistent with its mutagenic property.