The phytopathogenic mollicute-insect vector interface: a closer look

ABSTRACT Spiroplasma citri, transmitted by phloem-feeding leafhoppers, moves from the gut lumen through the gut wall, hemolymph, and salivary glands and multiplies in insect tissues. Nontransmissible lines were deficient in their ability to cross these barriers. Molecular analysis revealed extensive chromosomal rearrangements between the transmissible and nontransmissible spiroplasma lines including a large chromosomal inversion and deletions of about 10 kb at each inversion border. One open reading frame of the deleted region, cloned from the transmissible strain BR3-3X, encodes an integral membrane protein of 58 kDa that shares limited sequence similarity with major adhesin proteins of two zoopathogenic mycoplasmas. Adhesion of spiroplasmas to cultured leafhopper cells was inhibited by proteases, suggesting that adherence to host cells is mediated by spiroplasma membrane protein(s). A hypothetical model for insect transmission of phytopathogenic mollicutes is presented.     

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.     

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.     

Detection of Spiroplasma citri in plants and insect hosts by ELISA

Enzyme-linked immunosorbent assay (ELISA) was shown to be a sensitive method for the detection of Spiroplasma citri in plants and insect hosts. S. citri was detected in Vinca rosea less than 1 week after infection by grafting, and reached a peak litre of up to 10⁹ spiroplasmas per gram of tissue in infected shoots and root tips. Multiplication of S. citri in the leafhopper Euscelidius variegatus was also monitored by ELISA. S. citri could be detected in a single insect, showing that this technique is suitable for screening insect populations in the field for potential vectors of Spiroplasma diseases. A method is described for raising a pathogen-specific antiserum from V. rosea infected with S. citri which reacted with cultured S. citri and also with S. citri in plant tissue.     

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.     

Interaction of Tomato Spotted Wilt Tospovirus (TSWV) Glycoproteins with a Thrips Midgut Protein, a Potential Cellular Receptor for TSWV

ABSTRACT Interactions between viral and cellular membrane fusion proteins mediate virus penetration of cells for many arthropod-borne viruses. Electron microscope observations and circumstantial evidence indicate insect acquisition of tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) is receptor mediated, and TSWV membrane glycoproteins (GP1 and GP2) serve as virus attachment proteins. The tospoviruses are plant-infecting members of the family Bunyaviridae and are transmitted by several thrips species, including Frankliniella occidentalis. Gel overlay assays and immunolabeling were used to investigate the putative role of TSWV GPs as viral attachment proteins and deter mine whether a corresponding cellular receptor may be present in F. occidentalis. A single band in the 50-kDa region was detected with murine monoclonal antibodies (MAbs) to the TSWV-GPs when isolated TSWV or TSWV-GPs were used to overlay separated thrips proteins. This band was not detected when blots were probed with antibody to the non-structural protein encoded by the small RNA of TSWV or the TSWV nucleocapsid protein, nor were proteins from nonvector insects labeled. Anti-idiotype antibodies prepared to murine MAbs against GP1 or GP2 specifically labeled a single band at 50 kDa in Western blots and the plasmalemma of larval thrips midguts. These results support the putative role of the TSWV GPs as viral attachment proteins and identified potential cellular receptor(s) in thrips.     

Genetic Variation in Spiroplasma citri

Spiroplasmas are members of the Class Mollicutes, wall-less prokaryotes having a high adenosine–thymidine content in their small genomes. Spiroplasma citri is a plant pathogen that inhabits phloem. Like other phytopathogenic spiroplasmas and the related phytoplasmas, it is transmitted from plant to plant by phloem-feeding leafhoppers that serve as alternate hosts for the spiroplasma as well as vectors. Genetic information in spiroplasmas is carried on a circular chromosome, on plasmids and/or in virus genomes. A picture emerging from recent research on the S. citri genome is one of frequent and often extensive variation, resulting from a number of different mechanisms. Expansion and contraction events must continually be occurring in about equal proportions so that the net genome size varies within defined boundaries. Particularly impressive are large changes in genome size that can occur in only a few generations. As with most organisms, genetic variation in S. citri results from variation in extrachromosomal DNA content, changes due to DNA replication and repair processes and changes due to recombination. The implied flux of genetic information into and out of the S. citri genome should be beneficial to the bacterium, allowing it, with its small genome size, to adapt to new environments.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

Partial characterization of the extracellular matrix released onto hydrophobic surfaces by conidia and conidial germlings ofColletotrichum graminicola

The extracellular matrix (ECM) released by conidia ofColletotrichum graminicolawhen incubated on artificial hydrophobic substrata was investigated. Time-course experiments revealed that proteins and carbohydrates were released in the ECM of both ungerminated and germinated conidia. Stains for protein and carbohydrate revealed that part of the ECM remained attached to the substratum after the fungus had been removed. The ECM isolated from substrata where ungerminated conidia had adhered and from substrata where germinated conidia had adhered exhibited the same profiles when analysed by SDS-PAGE for proteins and glycoproteins and by HPLC for carbohydrate analysis. The ECM was found to be composed of glycoproteins with a molecular weight of around 200 kDa. Deglycosylation of the isolated ECM material resulted in the release of a protein with a molecular weight of approx. 116 kDa, indicating that the original protein was extensively glycosylated. Carbohydrate analysis demonstrated that mannose was the predominant sugar. That the ECM remained attached to the surface of the hydrophobic substrata suggests that it has a role in adhesion.     

Some characteristics of the transmission of grapevine leafroll associated virus 3 by Planococcus citri Risso

Some characteristics of the acquisition and transmission of GLRaV-3 by Planococcus citri were determined by ELISA testing and transmission experiments. Groups of five insects were used, i.e. the advisable minimum group size suggested by the results of ELISA of insect groups of various sizes. The virus was transmitted to only 1/10 test plants each of which had been exposed to a group of insects fed on GLRaV-3 infected plants for at least three days, eventhough more than 80% of the insect groups were expected to contain viruliferous individuals under these conditions. Viruliferous mealybugs transferred to potato plants could retain the virus for up to 24 h, but lost the capacity for effective transmission to vines within 1 h after transfer. In newly infected vines, the virus remained latent or undetectable by ELISA for at least 13 months.     

大豆蚜Aghsp75基因对吡虫啉及温度胁迫的应激表达

热激蛋白在昆虫抵御温度胁迫和药剂胁迫反应中具有重要作用。本试验通过高通量测序获得一条大豆蚜热激蛋白基因的cDNA序列，该序列全长2982 bp，含1个长度为2052 bp的开放阅读框，编码683个氨基酸组成的多肽，多肽等电点约为6.30，分子质量约为77.8 kDa；推导的氨基酸序列与棉蚜Aphis gossypii HSP75同源性高达99.12%，属于hsp 75家族。我们把该基因定名为Aghsp75，已提交至GenBank（登录号为MN068810）。Aghsp75通过不同浓度吡虫啉药剂和不同温度胁迫成蚜后发现，经LC₅₀吡虫啉浓度胁迫3、6和24 h时以及在LC₃₀浓度吡虫啉胁迫12 h时该基因表达量显著升高；经6℃处理6 h时以及36℃处理3 h和6 h时该基因表达量显著升高。本研究表明该基因可能参与大豆蚜的抗逆过程，为利用分子生物技术手段防治大豆蚜提供理论保障。     

Characterisation of a 16SrII phytoplasma strain associated with bushy stunt of hawkweed oxtongue (Picris hieracioides) in south-eastern Serbia and the role of the leafhopper Neoaliturus fenestratus (Deltocephalinae) as a natural vector

A 2-year study of host association, molecular characterisation and vector transmission of a phytoplasma related to the 16SrII group in a vineyard of south-eastern Serbia was conducted. Grapevine, eight common weeds and 31 Auchenorrhyncha species were collected and analysed for phytoplasma presence. PCR-RFLP analyses of the 16S rRNA gene identified the presence of a new strain of phytoplasma related to the 16SrII group in P. hieracioides with symptoms of stunting or bushy stunting. Grapevine samples, all without symptoms, were negative for phytoplasma presence. Plants of Erigeron annuus, Cynodon dactylon, Daucus carota and P. hieracioides, either exhibiting symptoms of yellowing or without symptoms, were positive for the presence of stolbur phytoplasma. Among the tested cicada species, seven were infected with phytoplasmas from the aster yellows group, two with stolbur phytoplasma, two with 16SrII phytoplasma, and one with the 16SrV-C phytoplasma subgroup. The phytoplasma strain of the 16SrII group was recorded in approximately 50?% of the collected leafhopper species Neoaliturus fenestratus and in a few specimens of the planthopper Dictyophara europaea. The vector status of N. fenestratus was tested using the second generation of the planthopper in two separate transmission trials with P. hieracioides and periwinkle seedlings. In both tests, the leafhopper successfully transmitted 16SrII phytoplasma to exposed plants, proving its role as a natural vector of this phytoplasma in Europe. A finer molecular characterisation and phylogenetic relatedness of the 16SrII phytoplasma strain by sequence analyses of the 16S rRNA and ribosomal protein genes rpl22-rps3 indicated that it was most closely related to the 16SrII-E subgroup.     

苏云金芽胞杆菌GS8菌株的生物学特性及其cry基因型鉴定

采用桑叶浸渍法和饲料表面覆盖法测定发现,从土壤中分离的一株苏云金芽胞杆菌菌株GS8对家蚕Bombyx mori、甜菜夜蛾Spodoptera exigua和棉铃虫Helicoverpa armigera初孵幼虫均表现出较高的杀虫活性,经形态学和生理生化反应鉴定该菌株为东北亚种(Bacillus thuringiensis subsp.tohokuensis)。聚丙烯酰胺凝胶电泳(SDS-PAGE)分析结果表明,GS8菌株的杀虫蛋白晶体主要由分子质量为130、81和60 kDa的蛋白组成。聚合酶链反应-限制性片段长度多态性(PCR-RFLP)鉴定结果显示,GS8菌株含有 cry1Aa、cry1Ab、cry1Ib、cry2Ab和cry9Ba 等基因。     

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。     

Recovery of solubilized δ‐endotoxin from Bacillus thuringiensis subsp kurstaki fermentation broth

Insecticidal δ‐endotoxin proteins, degraded from parasporal crystals by protease, were recovered by a simple procedure using heat treatment, solubilization, and ultrafiltration of a fermentation broth of Bacillus thuringiensis subsp kurstaki HD‐1. A 68 kDa insecticidal protein was obtained and characterized by SDS‐PAGE. The procedure described gave a nearly quantitative recovery of toxicity. Furthermore, bioassay results on larvae of the diamondback moth (Plutella xylostella) showed that the 68 kDa δ‐endotoxin fraction (P1) was the principal insecticidal component to this target insect. A similar molecular mass polypeptide P2 (65 kDa) which was solubilized together with P1 from the parasporal crystals, gave relatively low mortality. © 2000 Society of Chemical Industry     

Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.     

暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析

cDNA文库免疫筛选到编码暗黑鳃金龟幼虫几丁质脱乙酰酶HpCDA5基因,序列分析表明HpCDA5含有1个几丁质脱乙酰酶结构域,属于Group V类CDA蛋白。构建重组杆状病毒表达载体pFastBac-HpCDA5,转染昆虫细胞sf9,Western blot分析表明HpCDA5在昆虫细胞sf9中成功表达42 kDa的蛋白。利用qRT-PCR方法分析HpCDA5基因组织表达,结果显示HpCDA5基因在中肠中表达最高,为中肠特异表达蛋白。几丁质结合活性表明HpCDA5蛋白只能被强洗脱剂洗脱,具有很强的几丁质结合活性。本研究通过对暗黑鳃金龟几丁质脱乙酰酶HpCDA5的生化特性研究,为进一步明确HpCDA5的生理功能提供理论依据,并为以HpCDA5蛋白为靶标的暗黑鳃金龟生物防治提供支撑。     

31种中草药提取物对柑橘全爪螨的杀螨活性

室内测定了31种植物的乙醇提取物对柑橘全爪螨的杀螨活性。结果表明:小茴香等4种植物的活性较高,24 h校正死亡率大于90%;藁本等7种植物的活性中等,校正死亡率为60%~90%;其余植物提取物的校正死亡率在60%以下。进一步测定了4种植物提取物对柑橘全爪螨的雌成螨和卵的触杀毒力,表明:小茴香对柑橘全爪螨的毒力最高,LC50值分别为0.065 8 g/L和144.180 5 g/L,可以作为下一步的研究对象。     

Identification and transmission of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) in Sri Lanka

Sri Lankan black pepper with symptoms of yellow mottle disease contained a mixture of viruses: Piper yellow mottle virus (PYMV) particles (30 × 130 nm), Cucumber mosaic virus (CMV, 30 nm diameter isometric particles), and unidentified, isometric virus-like particles (30 nm diameter). An effective purification procedure is described for PYMV. Immunosorbent and conventional electron microscopy successfully detected badnavirus particles only when at least partially purified extracts were used. PYMV was confirmed as the cause of the disease, with the other two viruses apparently playing no part in producing symptoms. PYMV was transmitted by grafting, by the insect vectors citrus mealy bug ( Planococcus citri ) and black pepper lace bug ( Diconocoris distanti ), but not by mechanical inoculation or through seeds. The CMV isolate was transmitted to indicator plants by mechanical inoculation and by the vector Aphis gossypii , but not by Myzus persicae ; but neither mechanical nor insect transmission of CMV to black pepper was successful. A sensitive polymerase chain reaction assay was developed to detect PYMV in black pepper.