Mosquitocidal toxin-like islands in Bacillus thuringiensis S2160-1 revealed by complete-genome sequence and MS proteomic analysis.

Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

Identification and analysis of toxins in novel Bacillus thuringiensis strain Bt S3076-1 against Spodoptera frugiperda and Helicoverpa armigera (Lep.: Noctuidae).

Despite the successful application of toxins from Bacillus thuringiensis as biological control agents against pests, pests are showing resistance against an increasing number of Bacillus thuringiensis toxins due to evolution; thus, new toxins with higher toxicity and broad-spectrum activity against insects are being increasingly identified. To find new toxins, whole genome sequencing of the novel B. thuringiensis strain Bt S3076-1 was performed, and ten predicted toxic genes were identified in this study, including six cry genes, two tpp genes, one cyt gene and one vip gene, among which six were novel toxins. Subsequently, SDS‒PAGE analysis showed that the major proteins at the spore maturation stage were approximately 120 kDa, 70 kDa, 67 kDa, 60 kDa and 40 kDa, while active proteins after trypsin digestion (approximately 70 kDa and 40 kDa) exhibited LC50 values of 149.64 µg/g and 441.47 µg/g against Spodoptera frugiperda and Helicoverpa armigera larvae, respectively. Furthermore, pathological observation results showed that the peritrophic membrane of Spodoptera frugiperda and Helicoverpa armigera larvae was degraded. These findings will provide an experimental reference for further research on the insecticidal activity, toxicity spectrum and synergism of these toxins in Bt S3076-1.

Cry80Aa1, a novel Bacillus thuringiensis toxin with mosquitocidal activity to Culex pipiens pallens.

Bacillus thuringiensis (Bt) can synthesize insecticides to efficiently control insects. In this study, Bt strain S3580-1 with mosquitocidal activity was subjected to whole genome sequencing using an Illumina HiSeq 2000 system. A novel toxin, Cry80Aa1, was identified based on the resulting data. A conserved domain analysis revealed that Cry80Aa1 includes the Ricin_B_lectin domain (located at 10-150) and the Toxin_10 domain (located at 155-353). Phylogenetic tree analysis showed that Cry80Aa1 was in a distinct clade significantly distinguished from the known Cry proteins containing the Toxin_10 domain. Bioassays demonstrated that the Cry80Aa1 protein exhibited toxicity to third instar larvae of Culex pipiens pallens (LC50: 71.9 µg/mL; 95% FL: 59.5-122.7 µg/mL).

Bacillus thuringiensis novel toxin Epp is toxic to mosquitoes and prodenia litura larvae.

As a pathogenic bacterium, Bacillus thuringiensis (Bt) has become an alternative to chemical insecticides in commercial agricultural to control forestry pests and mosquitoes. To prevent pest resistance, many novel Bt strains have been isolated. Strain S3580-1 (WGS: VHPX0000000) used in this research and originally isolated from Hainan Qixianling National Forest Park (China) showed significant toxicity to Culex pipiens pallens. Here, using whole genome sequencing, assembly, and bioinformatics analysis, the predicted S3580-1CG_5163 (GenBank Accession No. MK124137) gene-encoded protein was found to share low homology with known toxins designated by the Bt toxin nomenclature system. It was considered to be an ETX/MTX2-type toxin and was designated Epp. Bioinformatics analysis showed that the predicted S3580-1CG_5163 gene-encoded protein Epp shared low identity with other known toxic protein sequences containing Cry-ETX/MTX conserved domains at the amino acid level, but significant similarity at the structural level. In addition, bioassays showed that Epp was toxic against Spodoptera litura (LC50 296.133 µg/mL; 95% FL 200.555-471.318 µg/mL) and Cx. pipiens pallens (LC50 322.193 µg/mL; 95% FL 238.217-477.243 µg/mL). On pathological observation, the peritrophic membrane of Cx. pipiens pallens larvae was degraded causing the midgut structure to become incomplete, resulting in larval death. Further bioassays are required to fully elucidate the insecticidal spectrum of the ETX/MTX2-type toxin Epp, and thereby provide future research directions.

Computational identification and evolutionary analysis of toxins in Mosquitocidal Bacillus thuringiensis strain S2160-1.

Mosquitocidal Bacillus thuringiensis (Bt) strain S2160-1 was proposed to be an alternative to Bacillus thuringiensis subsp. israelensis (Bti). Discovering and validating a toxic gene by experimentation was a complex and time-consuming task, which can benefit from high-throughput sequencing analysis. In this research, we predicted and identified toxic proteins in the strain S2160-1 based on the draft whole genome sequence data. Through a local BLASP, 46 putative toxins were identified in S2160-1 genome, by searching against a customized B. thuringiensis toxin proteins database containing 653 protein or peptide sequences retrieved from public accessible resources and PCR/clone results in our laboratory (e value = 1e - 5). These putative toxins consist of 42 to 1216 amino acids. The molecular weights are ranged from 4.86 to 137.28 kDa. The isoelectric point of these candidate toxins varied from 4.3 to 10.06, and 16 out of which had a pH greater than 7.0. The analysis of tertiary structure and PFAM domain showed that 12 potential plasmid toxins may share higher similarity (9/12 QMEAN4 score > 0.3) with known Bt toxins. In addition, functional annotation indicated that these 12 potential toxins were involved in "sporulation resulting in formation of a cellular spore" and "toxin activity". Moreover, multiple alignment and phylogenetic analysis were carried out to elucidate the evolutionary relationship among 101 known crystal or toxin proteins from public database and them with MEGA 6.0. It indicated that PS2160P2_1 and PS2160P2_153 may be potential Cry4-like toxins in Bt S2160-1. This research may lay the foundation for future functional analysis of Bt S2160-1 toxin proteins to reveal their biological roles.

Phytohormones signaling and crosstalk regulating leaf angle in rice.

Leaf angle is an important agronomic trait in rice (Oryza sativa L.). It affects both the efficiency of sunlight capture and nitrogen reservoirs. The erect leaf phenotype is suited for high-density planting and thus increasing crop yields. Many genes regulate leaf angle by affecting leaf structure, such as the lamina joint, mechanical tissues, and the midrib. Signaling of brassinosteroids (BR), auxin (IAA), and gibberellins (GA) plays important roles in the regulation of lamina joint bending in rice. In addition, the biosynthesis and signaling of BR are known to have dominant effects on leaf angle development. In this review, we summarize the factors and genes associated with the development of leaf angle in rice, outline the regulatory mechanisms based on the signaling of BR, IAA, and GA, and discuss the contribution of crosstalk between BR and IAA or GA in the formation of leaf angle. Promising lines of research in the transgenic engineering of rice leaf angle to increase grain yield are proposed.

Identification of a mosquitocidal toxin from Bacillus thuringiensis using mass spectrometry.

The Bacillus thuringiensis strain S2160-1 has previously been identified as being highly toxic to mosquito larvae and a viable alternative to strains currently used commercially to control these insects. A PCR approach had identified the presence of four putative insecticidal toxin genes (cry30Ea, cry30 Ga, cry50Ba and cry54Ba) in this strain, but did not identify the genes that encoding three of the main crystal toxin proteins of size 140 and 130 and 30 kDa. In this study we used mass spectrometry to identify the 130 kDa toxin as a rare Cry4 toxin (Cry4Cb3). The gene encoding this toxin was cloned and expressed and the toxin shown to have mosquitocidal activity against Culex quinquefasciatus.

Characterization of a new highly mosquitocidal isolate of Bacillusthuringiensis--an alternative to Bti?

The mosquito is a very important vector involved in the worldwide transmission of disease-causing viruses and parasites. Controlling the mosquito population remains one of the best means for preventing the serious infectious diseases of malaria, yellow fever, dengue, filariasis and so on and there has been an increasing interest in developing biopesticides as a useful substitute to chemical insecticides. As a result, Bacillus thuringiensis subsp. israelensis (Bti) has been extensively used due to its specificity and high toxicity to a variety of mosquito larvae. However it is prudent to seek alternatives to Bti with alternative spectra of mosquitocidal activity or that are able to overcome any resistance that might develop against Bti. The Bt S2160-1 strain was isolated from soil samples collected from Southern China and found to have a comparable mosquitocidal activity to Bti. However there were significant differences in terms of their plasmid profiles, crystal proteins produced and cry gene complement. A PCR-restriction fragment length polymorphism identification system was developed and used in order to identify novel cry-type genes and four such genes (cry30Ea, cry30Ga, cry50Ba and cry54Ba) were identified in Bt S2160-1. In conclusion, Bt S2160-1 has been identified as a potential alternative to Bti, which could be used for the control of mosquito populations in order to reduce the incidence of mosquito-borne diseases.

Fine mapping and in silico isolation of the EUI1 gene controlling upper internode elongation in rice.

Upper internode elongation in rice is an important agronomic trait. Well-known mutants with an elongated uppermost internode (eui) are important germplasms for developing unsheathed-panicle male-sterile lines in hybrid rice breeding. We finely mapped the eui1 gene and identified its candidate gene using in silico analysis based on previous research work and rice genomic sequence data. The rice eui1 gene was mapped to two overlapping BAC clones, OSJNBa0095J22 and OSJNBb0099O15, between the markers AC40 and AC46, that were 0.64 cM apart and spanned approximately 152 kb. A simple sequence repeat (SSR) marker AC41 that cosegregated with eui1 was located in an intron of a putative cytochrome P450-related gene. In silico analysis suggested that this encoded the cytochrome CYP714D1. Allelic sequencing confirmed that EUI1 corresponded to this P450 gene. A gamma ray-induced eui1 mutant carried a deletion in exon II of the EUI1 gene, and resulted in a frame-shift deletion that produced a truncated polypeptide. We conclude that the EUI1 gene controlling the upper internode elongation in rice is 9,804 bp long, and comprises two exons and one intron. The length of the cDNA is 1,931 bp containing a 1,734 bp ORF, a 110 bp 5'-UTR and a 87 bp 3'-UTR. The ORF encodes an unknown 577 amino acid functional protein, that appears to be a member of the cytochrome P450 family.

[Genetic effects on seed traits in soybean].

The genetic effects of seed traits in soybean, including 100-seed weight, seed length, seed width, seed thickness, length/width, length/thickness and width/thickness, were analyzed using an incomplete diallel cross of eight varieties with its F1 and F2 populations. The results showed that the above seven traits were controlled by direct genetic effects of seed and affected to different extent of maternal and cytoplasmic effects simultaneously. Among the traits, the inheritance of 100-seed weight, seed length, length/width, length/thickness, and width/thickness were mainly controlled by cytoplasmic effects, while those of seed width and thickness were mainly by maternal effects. Both the seed direct heritabilities and the cytoplsmic heritabilities of 100-seed weight, seed length, length/width and width/thickness were medium-sized. The individual selection and seed selection of above four traits at late generation may create good results. The maternal heritabilities of seed width and thickness were pretty high. To increase these two traits, an individual maternal selection should be done at early generation. Our results showed that varieties P2 and P7 could be used as ideal parents for improvements of 100-seed weight, seed length/width, length/thickness and width/thickness, while varieties P1, P4 and P6 are the ideal parents for increasing seed length, width and thickness respectively.