Spiroplasma citri Spiralin Acts In Vitro as a Lectin Binding to Glycoproteins from Its Insect Vector Circulifer haematoceps

ABSTRACT In order to understand the molecular mechanisms underlying transmission of Spiroplasma citri by the leafhopper Circulifer haematoceps, we screened leafhopper proteins as putative S. citri-binding molecules using a spiroplasma overlay assay of protein blots (Far-western assay). Insect proteins were separated by one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, blotted, and probed with S. citri proteins. In this in vitro assay, we found that spiroplasma proteins exhibited affinity for seven leafhopper proteins. The interactions between S. citri proteins and insect proteins with molecular masses of 50 and 60 kDa were found to be sugar sensitive. These insect proteins were identified as high mannose N-glycoproteins, which support an interaction of glycoprotein-lectin type with S. citri proteins. Lectin detection in S. citri has revealed only one protein of 24 kDa. Using a leafhopper protein overlay assay on an S. citri protein blot, one spiroplasma protein with a similar molecular mass of 24 kDa was shown to display an insect protein-binding capacity. This protein was identified as the spiralin, which is the most abundant membrane protein of S. citri. Far-western experiments performed with purified spiralin and insect glycoproteins confirmed the binding of spiralin to the insect glycoproteins of 50 and 60 kDa. Thus, the spiralin could play a key role in the transmission of S. citri by mediating spiroplasma adherence to epithelial cells of insect vector gut or salivary gland.     

Spiroplasma citri Movement into the Intestines and Salivary Glands of Its Leafhopper Vector, Circulifer tenellus

ABSTRACT Spiroplasma citri, a helical, wall-less prokaryote in the class Molli-cutes, is transmitted by the beet leafhopper, Circulifer tenellus. Invasion of leafhopper tissues and cytopathological effects by S. citri were investigated by transmission electron microscopy. All eight cell types of the principle salivary glands, as well as the adjacent muscle cells and the cells of the accessory salivary glands, were colonized by the spiroplas-mas. In both midgut epithelia and salivary gland cells, spiroplasmas usually occurred in membrane-bound cytoplasmic vesicles that often were located near the cell periphery. In several salivary gland cells, spiroplas-mas were also observed within membranous pockets apparently formed by invagination of the plasmalemma beneath intact basal lamina. These observations are consistent with spiroplasma entry into the insect cells by receptor-mediated endocytosis. Cytopathological effects of spiroplasma infection in salivary cells included loss of membrane and basal lamina integrity, presence in some cells of irregular inclusion-like structures containing dense matrices of filamentous material that labeled with anti S. citri antibodies, and apparent disorganization of the endoplasmic reticulum. Compared to the tightly aligned fiber bundles in healthy muscle cells, bundles in spiroplasma-containing muscle cells appeared fragmented and loosely arranged. Such symptoms could contribute to the reduction in longevity and fecundity that has been previously reported for S. citri-infected C. tenellus.     

Detection of corn stunt spiroplasma in vivo by ELISA using antisera to extracts from infected corn plants (Zea mays)

Antisera were raised in rabbits to extracts from healthy corn plants and from plants infected with corn stunt spiroplasma (CSS); they were prepared by partially purifying sap or tissue homogenates from whole stems by differential centrifugation and filtration. The titres of CSS-specific antibody in the antisera to diseased plant extracts (DPE) were monitored by spiroplasma deformation tests. After cross-absorption against healthy plant extracts, the DPE antisera detected cultured CSS at concentrations exceeding 10⁶ cells/ml, compared with less than 10⁵ cells/ml for homologous antiserum, and reacted more strongly against extracts from diseased than from healthy plants. A comparison of techniques for extracting diseased plant tissues showed that the reaction of stems and midribs against cultured CSS antiserum was two to three times stronger if the samples were lyophilized before extraction. When extracts from lyophilized tissues were used as test antigens the DPE antisera discriminated between diseased and healthy corn stems, midribs and roots as effectively as antiserum to cultured CSS. Preliminary attempts to detect CSS in its vector, Dalbulus maidis , were complicated by non-specific reactions of insect tissue extracts but concentrations equivalent to at least one CSS-infected insect/ml of sample were detected by antiserum to cultured CSS.     

Spiroplasma citri Surface Protein P89 Implicated in Adhesion to Cells of the Vector Circulifer tenellus

ABSTRACT Two microtiter plate assays were developed to study the adherence of the plant-pathogenic mollicute Spiroplasma citri to a monolayer of cultured cells of its leafhopper vector, Circulifer tenellus. Adherence was significantly reduced by prior treatment of the spiroplasmas with proteinase K or pronase. Electrophoresis and western blotting of spiroplasma membrane proteins, before and after exposure of intact spiroplasmas to proteases, revealed the concomitant reduction in intensity of a major membrane protein (P89) and a new polypeptide of approximately 46 kDa in protease-treated preparations (P46). Triton X-114 phase partitioning demonstrated that P89 and P46 are amphiphilic, and labeling of the new polypeptide P46 with anti-P89 serum suggested that this molecule may be a breakdown product of P89. Regeneration of P89 after proteinase K treatment of spiroplasmas was directly associated with restoration of the pathogen's attachment capability. Treatment of spiroplasmas with any of several carbohydrates and glycoconjugates or with tetramethyl-urea, a compound that interferes with hydrophobic associations, had a negligible effect on attachment. These results suggest that a spiroplasma surface protein, P89, has a role in S. citri adherence to C. tenellus cells.     

Properties of Tribolium gut chitin synthetase

An insect chitin synthetase (CS) is readily assayed using the microsomal fraction (～0.5 mg protein) from an homogenate of Tribolium castaneum larvae. This enzyme preparation is incubated at 22°C with uridine 5′-diphospho-N-acetyl[³H]glucosamine in 355 μl of 25 mM Tris-HCl buffer containing 10 mM MgCl₂, 17 mM N-acetylglucosamine, and 1 mM dithiothreitol. Other divalent cations and amino sugars are less effective activators or are inhibitory. T. castaneum CS is strongly inhibited by polyoxin D and uridine 5′-diphosphate. These activation and inhibition properties of Tribolium castaneum gut CS are similar to those of fungal CS. The polymerization product formed by the Tribolium enzyme is stable in alkali but hydrolyzed by chitinase. Enzymes of Tribolium confusum, Tribolium brevicornis, Tenebrio molitor, and Galleria mellonella are also active under the same conditions. These enzymes are from the gut and probably from the peritrophic membrane. Integumental CS activity is not detected under the indicated assay conditions.     

Bacillus thuringiensis vegetative insecticidal protein family Vip3A and mode of action against pest Lepidoptera

Vip3A proteins are widely used for controlling pest Lepidoptera. Different binding sites with different receptors in the insect midgut membrane and lack of cross‐resistance with crystal (Cry) proteins enhance their applicability, as both single proteins and proteins pyramided with Cry proteins in transgenic Bt crops. Vip3A proteins are effective but there is relatively little information about their structure, function, activation, specificity, and mode of action. In addition, the mechanism of insect resistance to these proteins is unknown. Phylogenetic analysis and multiple sequence alignment showed that Vip3A proteins are genetically distant from Cry proteins. The mode of action and insecticidal activity of Vip3A proteins are discussed in this review. This review also provides detailed information about the Vip3A protein family that may aid in the design of more efficient pest management strategies in response to insect resistance to insecticidal proteins. © 2020 Society of Chemical Industry     

Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes

BACKGROUND: Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt) were first commercialised in 1996. The risk that pests have the potential to evolve resistance to Bt toxins is one of the most serious challenges to this technology. Gene stacking, pyramiding two Bt genes into one variety, is considered to be an effective insect resistance management (IRM) strategy. In this study, insect‐resistant rice expressing two Bt genes was developed by sexual crossing, and then characterised. RESULTS: Homozygous rice lines of two pyramided Bt genes were obtained in the F₃ generation. Quantification of Bt toxin showed that protein concentrations of Cry1Ab, Cry1Ac and Cry2A in the two‐gene lines were comparable with their single‐gene parents, while the expression of cry1C gene decreased after gene stacking. Four two‐gene lines showed higher activity to striped stem borer (Chilo suppressalis Walker) than parental lines in the laboratory bioassay. All pyramided lines and their hybrids exhibited excellent efficacy against stemborers and leaffolders in field evaluation, while most pyramided lines had no significant differences from original variety in yield under spraying of insecticide. CONCLUSION: These results demonstrate that the two‐gene lines have commercial potential and could serve as a valuable IRM strategy. Copyright © 2011 Society of Chemical Industry     

Can the mammalian organoid technology be applied to the insect gut?

Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self‐organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance. © 2020 Society of Chemical Industry     

Gut bacteria associated with the Pacific Coast wireworm,<Emphasis Type="Italic">Limonius canus</Emphasis>, inferred from 16s rDNA sequences and their implications for control

A multitude of bacteria have been isolated from the guts of several insect species. Some of these have been modified to interfere with the development of the host insect or with the development and transmission of plant and animal pathogens transmitted by the host insect. We surveyed the gut flora of the Pacific Coast wireworm,Limonius canus LeConte, a serious pest of potato, at two sites in Oregon and Washington. Isolates were obtained from surface-sterilized triturated larvae by dilution plating on standard media. A rich diversity of species was found in 86 isolates, including spore-formers, non-spore-formers and aerobic and facultatively anaerobic species collected on four sampling dates at each location. Twenty-one of the isolates were identified to species based on rDNA sequence (nine distinct species). An additional 34 isolates were identified to genus from the sequence data while six isolates could be assigned only to family based on sequence comparisons. Twenty-seven additional isolates were identified to species (9), genus (17) or family (1) based on side-by-side morphological comparisons with isolates identified from rDNA sequence. The most frequently isolated bacterium wasBacillus megaterium, followed byRahnella aquatilis. A naturally occurring bacterium found in the gut and/or environment of a targeted insect that is modified to express toxins or other detrimental substances could provide certain advantages (such as persistence and recycling) over inundatively applied microbial control agents, particularly within soil habitats. The hypothesis that these species or others from the survey represent candidates for genetic modification to provide control options forL. canus is discussed.     

Inhibition of Tribolium gut chitin synthetase

The chitin synthetase (CS) of Tribolium castaneum gut is inhibited 50% by 0.02 μM nikkomycin and 4 μM polyoxin D, two pyrimidine nucleoside fungicides, in in vitro assays with 10-min preincubation of enzyme and inhibitor prior to substrate addition. Tribolium CS is also sensitive to inhibition by the pyrimidine nucleotides uridine and cytidine di- and triphosphates. Captan, a known inhibitor of insect chitin synthesis, and the related fungicides captafol and dichlofluanid are highly potent inhibitors of Tribolium CS. Moderately active CS inhibitors are the acaricide oxythioquinox and the herbicide barban. One phenylcarbamate insect growth reatardant, H-24108, is weakly active in inhibiting Tribolium gut CS, as are three of its analogs but not 26 others. Many triazines are not inhibitory including several herbicides and an azido derivative, CGA 19255, which is active in blocking insect growth and chitin synthesis. Although the benzoylphenyl urea insecticides diflubenzuron and SIR 8514 are potent in vivo inhibitors of the polymerization step in insect chitin synthesis, they do not affect T. castaneum gut CS activity in vitro and greatly stimulate Tribolium brevicornis gut CS activity in vivo. These studies and preliminary findings on an integumental enzyme indicate that CS of these tissues is not sensitive to the direct action of benzoylphenyl ureas. This leads to speculation that the benzoylphenyl ureas act either as CS inhibitors via active metabolites formed in the integument or as blocking agents by direct binding to non-CS sites important in chitin polymerization and fibrillogenesis.     

Insect resistance to Nilaparvata lugens and Cnaphalocrocis medinalis in transgenic indica rice and the inheritance of gna+sbti transgenes

Molecular genetic analysis and insect bioassay of transgenic indica rice 'Zhuxian B' plants carrying snowdrop lectin gene (gna) and soybean trypsin inhibitor gene (sbti) were investigated in detail. PCR, 'dot' blot and PCR-Southern blot analysis showed that both transgenes had been incorporated into the rice genome and transmitted up to R3 progeny in most lines tested. Some transgenic lines exhibited Mendelian segregation, but the other showed either 1:1 (positive: negative for the transgenes) or other aberrant segregation patterns. The segregation patterns of gna gene crossed between R2 and R3 progeny. In half of transgenic R3 lines, gna and sbti transgenes co-segregated. Two independent homozygous lines expressing double transgenes were identified in R3 progeny. Southern blot analysis demonstrated that the copy numbers of integrated gna and sbti transgenes varied from one to ten in different lines. Insect bioassay data showed that most transgenic plants had better resistance to both Nilaparvata lugens (Stahl) and Cnaphalocrocis medinalis (Guenee) than wild-type plants. The insect resistance of transgenic lines increased with the increase in transgene positive ratio in most of the transgenic lines. In all, we obtained nine lines of R3 transgenic plants, including one pure line, which had better resistance to both N lugens and C medinalis than wild-type plants.     

基于酵母双杂交系统筛选灰飞虱体内与大麦黄条点花叶病毒核衣壳蛋白互作的蛋白质

 为了获取影响大麦黄条点花叶病毒(BYSMV)在灰飞虱体内增殖、积累和传播的相关介体因子,本研究利用分离泛素酵母双杂交膜系统,以BYSMV核衣壳蛋白(N)为诱饵对灰飞虱cDNA文库进行了筛选。 将BYSMV N基因构建到诱饵载体pDHB1上进行表达检测和功能验证,结果表明重组载体pDHB1-N能在酵母内正常表达并行使功能。利用诱饵载体筛选pPR3-N空文库对文库筛选条件进行优化,确定3-氨基-1,2,4-三唑(3-AT)浓度为12 mmol·L^-1的QDO平板为筛选文库培养基条件,去除可能存在的轻微筛库背景。在此筛选条件下以诱饵载体从灰飞虱cDNA文库中筛选得到57个阳性克隆,序列比对结果表明这些阳性克隆编码17种候选蛋白,包括表皮蛋白、泛素B、核糖体膜相关蛋白、细胞色素b5以及海藻糖转运蛋白等。 经酵母双杂交共转验证和β-半乳糖苷酶检测进一步确认了这17个候选蛋白与BYSMV N发生互作。本研究成功从灰飞虱分离泛素酵母双杂交膜系统cDNA文库筛选到与BYSMV N互作的蛋白质,为进一步探索弹状病毒与介体昆虫的分子互作机制奠定了基础。     

泡桐丛枝植原体Sec分泌蛋白转运系统3个亚基基因的克隆及蛋白特性分析

采用PCR方法扩增Sec分泌蛋白转运系统3个亚基基因,并进行了生物信息学分析。通过SWISS MODEL同源建模与3D PSSM折叠子识别法构建蛋白质的三维结构,并通过PROCHECK程序验证构建的三维结构的可靠性。首次从泡桐丛枝(PaWB)植原体基因组中分离出Sec分泌蛋白转运系统3个亚基基因,各基因全长依次为2 508、1 242 bp和411 bp,分别编码835、204个及136个氨基酸的蛋白。SecA、SecY和SecE均为脂溶性稳定蛋白,SecA无明显跨膜区,SecY和SecE分别含有10个和3个明显的疏水跨膜区。二级结构主要为螺旋,其次为折叠和无规则卷曲,没有转角。构建的三维结构符合立体化学原则。泡桐丛枝(PaWB)植原体中存在的Sec分泌蛋白转运系统作为最主要的运输途径,可能直接转运菌体蛋白如毒素等直接到寄主细胞质中或介体昆虫细胞中,引起寄主病症。研究植原体的Sec蛋白转运系统对于了解植原体的致病机理及预防这类病害的发生提供了理论依据。     

A recombinant form of chagasin from Trypanosoma cruzi: inhibitory activity on insect cysteine proteinases

BACKGROUND: The activity of the major digestive cysteine proteinase detected in the intestinal tract of larvae of the bean weevil, Acanthoscelides obtectus (Say), was efficiently inhibited by the well-characterized cysteine proteinase synthetic inhibitor E-64 and also by a recombinant form of chagasin (r-chagasin), a tight-binding cysteine proteinase inhibitor protein from Trypanosoma cruzi. RESULTS: Incorporation of r-chagasin into an artificial diet system at 0.1 g kg(-1) retarded growth rate, decreased larval survival and led to complete mortality of A. obtectus at the end of the trial. The observed differences in growth rates occurred particularly in the first and second development stages. Artificial seeds containing high levels of r-chagasin (0.5-30 g kg(-1)) completely inhibited larval penetration. CONCLUSION: Together, the results reported in this paper support the hypothesis that the inhibitory activity of r-chagasin towards the major insect gut cysteine proteinase in vitro and in vivo is an accurate prediction of its insecticidal effects. The selectivity of this inhibitor against insect digestive proteinases supports the key role in parasite virulence by affecting the endogenous proteinase activity in its natural host.     

小菜蛾中肠氨肽酶N2在昆虫细胞中的表达

利用昆虫细胞-杆状病毒表达系统(Bac-to-Bac baculovirus expression system)表达获得小菜蛾 Plutella xylostella(L.)中肠氨肽酶N2(aminopeptidase N,APN2)蛋白,成功建立了该蛋白在昆虫细胞中的表达体系。将对Cry1Ac毒素敏感和已产生抗性的小菜蛾中肠APN2基因插入表达载体pFAST Bac HTB中,并在草地夜蛾 Spodoptera frugiperda 细胞系(Sf9)中进行了重组表达。聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白免疫印迹技术(Western-blotting)结果显示,在107 kDa处有1条特异性蛋白条带。研究表明,小菜蛾中肠APN2基因可在Sf9细胞中成功表达,这为继续研究其功能及抗性的关系奠定了基础。     

利用酵母双杂交膜系统筛选与小麦蓝矮植原体免疫膜蛋白互作的异沙叶蝉蛋白

正小麦蓝矮病(Wheat blue dwarf,WBD)是我国西北麦区一种重要植原体病害,在我国西部地区危害严重。该病害由异沙叶蝉(Psammotettix alienus L.)专化性传播,介体传毒成为病害流行的重要中心环节[1]。本实验室前期通过免疫荧光标记研究发现WBD植原体免疫膜蛋白(immunodominant membrane protein, IMP)与介体异沙叶蝉肌动蛋白互作,说明IMP在植原体传播和致病过程中起关键作用。     

利用酵母双杂交膜系统筛选与小麦蓝矮植原体免疫膜蛋白互作的异沙叶蝉蛋白

In order to investigate interactive proteins in leafhopper (Psammotettix alienus L.) with WBD phytoplasma, the protein interaction analysis was performed by using WBD phytoplasma IMP (immunodominant membrane protein) as bait protein to screen a cDNA library of leafhopper using a split-ubiquitin yeast membrane system. Through the screening test, 30 clones were obtained from the cDNA library and 8 proteins were identified by searching against NCBI database, such as tubulin, peptidyl-prolyl cis-trans isomerase, Cdc42 protein, ribosomal proteins and ATP-F0 subunit protein, etc. The interactions between IMP and 8 putative proteins were further confirmed by co-transformation and β- Galactosidase assays. This study could be useful for understanding the molecular interaction mechanism between WBD phytoplasma and insect vector and the specificity of transmission.     

Isolation and Characterization of Derivative Lines of the Onion Yellows Phytoplasma that Do Not Cause Stunting or Phloem Hyperplasia

ABSTRACT Two lines of onion yellows phytoplasma producing milder symptoms were isolated from the original line (OY-W). One has an additional characteristic, non-insect-transmissibility (OY-NIM), compared with the other (OY-M). OY-M was established after maintaining OY-W for 11 years on a plant host (Chrysanthemum coronarium) with an insect vector (Macrosteles striifrons), and OY-NIM was isolated after subsequent maintenance of OY-M in plants by periodic grafting. Polymerase chain analysis suggested that OY-NIM cannot traverse the gut or survive in the hemolymph of the leafhopper. OY-W results in witches'-broom formation and stunted growth in the host plant. In contrast, OY-M and OY-NIM do not cause stunting in the host plant, although they result in witches'-broom. Histopathological analysis of these lines revealed that the hyperplastic phloem tissue and severe phloem necrosis seen in OY-W did not exist in OY-M and OY-NIM. This was attributed to a reduction in the population of phytoplasma in tissues in both OY-M- and OY-NIM-infected plants. The results suggest that the cause of stunting and phloem hyperplasia may be genetically different from the cause of witches'-broom. Pulsed field gel electrophoresis analysis showed that OY-M had a smaller genome size ( approximately 870 kbp) than OY-W ( approximately 1,000 kbp). Thus, some of the OY-W genes responsible for pathogenicity may not be present in OY-M.     

Insect midgut structures and molecules as targets of plant‐derived protease inhibitors and lectins

The midgut of insects is involved in digestion, osmoregulation and immunity. Although several defensive strategies are present in this organ, its organization and function may be disturbed by some insecticidal agents, including bioactive proteins like lectins and protease inhibitors (PIs) from plants. PIs interfere with digestion, leading to poor nutrient absorption and decreasing amino acid bioavailability. Intake of PIs can delay development, cause deformities and reduce fertility. Ingestion of PIs may lead to changes in the set of proteases secreted in the insect gut, but this response is often insufficient and results in aggravation of the malnutrition status. Lectins are proteins that are able to interact with glycoconjugates, including those linked to cell surfaces. Their effects on the midgut include disruption of the peritrophic matrix, brush border and secretory cell layer; induction of apoptosis and oxidative stress; interference with nutrient absorption and transport proteins; and damaging effects on symbionts. In addition, lectins can cross the intestinal barrier and reach the hemolymph. The establishment of resistant insect populations due to selective pressure resulting from massive use of a bioactive protein is an actual possibility, but this can be minimized by the multiple mode‐of‐action of these proteins, mainly the lectins. © 2018 Society of Chemical Industry     

Marker‐free,tissue‐specific expression of Cry1Ab as a safe transgenic strategy for insect resistance in rice plants

BACKGROUND: Rice is the major food resource for nearly half of the global population; however, insect infestation could severely affect the production of this staple food. To improve rice insect resistance and reduce the levels of Bt toxin released into the environment, the Cry1Ab gene was conjugated to the rice rbcS promoter to express Bt toxin in specific tissues of transgenic plants. RESULTS: Eight marker‐free, T₂ lines were separated from the T₀ cotransformants. Using RT‐PCR, high levels of Cry1Ab expression were detected in the leaf but not in the seed. The Cry1Ab protein level ranged from 1.66 to 3.31 µg g^?1 in the leaves of four transgenic lines, but was barely detectable in their seeds by ELISA. Bioassays showed that the mortality rate of silkworm larvae feeding on mulberry leaves dipped in transgenic rice flour and pollen was less than that of the positive control (KMD), and that their average weight was higher than that of KMD, suggesting that the Cry1Ab protein was not expressed in the seed and pollen. CONCLUSION: The transgene conferred a high level of resistance to insects and biosafety to the rice plants, which could be directly used in rice breeding. Copyright © 2012 Society of Chemical Industry