title: Holotrichia oblita Midgut Proteins That Bind to Bacillus thuringiensis Cry8-Like Toxin and Assembly of the H. oblita Midgut Tissue Transcriptome.
abstract: TheBacillus thuringiensisstrain HBF-18 (CGMCC 2070), containing twocrygenes (cry8-like andcry8Ga), is toxic toHolotrichia oblitalarvae. Both Cry8-like and Cry8Ga proteins are active against this insect pest, and Cry8-like is more toxic. To analyze the characteristics of the binding of Cry8-like and Cry8Ga proteins to brush border membrane vesicles (BBMVs) inH. oblitalarvae, binding assays were conducted with a fluorescent DyLight488-labeled Cry8-like toxin. The results of saturation binding assays demonstrated that Cry8-like bound specifically to binding sites on BBMVs fromH. oblita, and heterologous competition assays revealed that Cry8Ga shared binding sites with Cry8-like. Furthermore, Cry8-like-binding proteins in the midgut fromH. oblitalarvae were identified by pulldown assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, theH. oblitamidgut transcriptome was assembled by high-throughput RNA sequencing and used for identification of Cry8-like-binding proteins. Eight Cry8-like-binding proteins were obtained from pulldown assays conducted with BBMVs. The LC-MS/MS data for these proteins were successfully matched with theH. oblitatranscriptome, and BLASTX results identified five proteins as serine protease, transferrin-like, uncharacterized protein LOC658236 ofTribolium castaneum, ATPase catalytic subunit, and actin. These identified Cry8-like-binding proteins were different from those confirmed previously as receptors for Cry1A proteins in lepidopteran insect species, such as aminopeptidase, alkaline phosphatase, and cadherin.IMPORTANCEHolotrichia oblitais one of the main soil-dwelling pests in China. The larvae damage the roots of crops, resulting in significant yield reductions and economic losses.H. oblitais difficult to control, principally due to its soil-dwelling habits. In recent years, some Cry8 toxins fromBacillus thuringiensiswere shown to be active against this pest. Study of the mechanism of action of these Cry8 toxins is needed for their effective use in the control ofH. oblitaand for their future utilization in transgenic plants. Our work provides important basic data and promotes understanding of the insecticidal mechanism of Cry8 proteins againstH. oblitalarvae.
authors.affiliation: State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.|State Key Laboratory of Agrobiotechnology and National Maize Improvement Center of China, Department of Plant Genetics and Breeding, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Jian
authors.initials: J
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Changlong
authors.initials: C
authors.lastname: Shu
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
authors.forename: Mario
authors.initials: M
authors.lastname: Soberón
authors.affiliation: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Bravo
authors.affiliation: Cangzhou Academy of Agricultural and Forestry Sciences, Cangzhou, People's Republic of China.
authors.forename: Chunqing
authors.initials: C
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Fuping
authors.initials: F
authors.lastname: Song
authors.affiliation: State Key Laboratory of Agrobiotechnology and National Maize Improvement Center of China, Department of Plant Genetics and Breeding, China Agricultural University, Beijing, People's Republic of China.
authors.forename: Jinsheng
authors.initials: J
authors.lastname: Lai
authors.affiliation: State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China jzhang@ippcaas.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001517:Coleoptera / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology*; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000737:chemistry / Q000378:metabolism / Q000382:microbiology; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007814:Larva / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D011485:Protein Binding; D053719:Tandem Mass Spectrometry; D059467:Transcriptome
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: Bacillus thuringiensis; Cry8-like; Holotrichia oblita; binding proteins