Putative protein digestion in a sap-sucking homopteran plant pest (rice brown plant hopper; Nilaparvata lugens: Delphacidae)--identification of trypsin-like and cathepsin B-like proteases

Sap-sucking phytophagous insect species of the order Hemiptera have been assumed not to carry out digestive proteolysis, but instead to rely on free amino acids in the phloem and xylem saps for their nutritional requirements. Extracts prepared from isolated guts of rice brown planthopper (Nilaparvata lugens), a homopteran crop pest, were shown to contain protease activity, with hydrolysis of both protein and synthetic peptide substrates being observed. Assays with specific inhibitors suggested that a trypsin-like serine protease was responsible for most of hydrolytic activity against synthetic substrates. A cDNA library was prepared from RNA extracted from N. lugens gut tissue, and screened for protease-encoding sequences. cDNAs for a cathepsin B-like protease and a trypsin-like protease were isolated and fully characterised; the latter exhibits a novel C-terminal region and an unusual activation mechanism, and represents a small gene family. Soya bean Kunitz trypsin inhibitor (SKTI) is an effective inhibitor of protein hydrolysis by N. lugens gut extracts in vitro, explaining why transgenic rice plants expressing this protein are partially resistant to the insect (Mol. Breed. 5 (1999) 1). It is suggested that digestive proteolysis may be widespread in sap-sucking homoptera, and can make a significant contribution to nutrition.     

稻飞虱种群发生的调查与取样技术

稻飞虱是中国和许多国家水稻生产上的主要害虫,属r对策暴发危害类型。其中,褐飞虱和白背飞虱是典型的远距离迁飞性害虫,具有大区域同期"突增"和"突减"的灾变特性;灰飞虱则主要以本地越冬为主,远距离迁飞对种群结构的影响较小,但其传播的病毒病对水稻生产的影响更甚。针对3种稻飞虱的生物学特性,本文发展了稻飞虱田间种群发生与危害等的调查与取样技术,为其种群发生及其监测预警提供技术支撑。     

Expression of snowdrop lectin (GNA) in transgenic rice plants confers resistance to rice brown planthopper

Snowdrop lectin ( Galanthus nivalis agglutinin; GNA) has been shown previously to be toxic towards rice brown planthopper ( Nilaparvata lugens ; BPH) when administered in artificial diet. BPH feeds by phloem abstraction, and causes ‘hopper burn’, as well as being an important virus vector. To evaluate the potential of the gna gene to confer resistance towards BPH, transgenic rice ( Oryza sativa L.) plants were produced, containing the gna gene in constructs where its expression was driven by a phloem-specific promoter (from the rice sucrose synthase RSs1 gene) and by a constitutive promoter (from the maize ubiquitin ubi1 gene). PCR and Southern analyses on DNA from these plants confirmed their transgenic status, and that the transgenes were transmitted to progeny after self-fertilization. Western blot analyses revealed expression of GNA at levels of up to 2.0% of total protein in some of the transgenic plants. GNA expression driven by the RSs1 promoter was tissue-specific, as shown by immunohistochemical localization of the protein in the non-lignified vascular tissue of transgenic plants. Insect bioassays and feeding studies showed that GNA expressed in the transgenic rice plants decreased survival and overall fecundity (production of offspring) of the insects, retarded insect development, and had a deterrent effect on BPH feeding. gna is the first transgene to exhibit insecticidal activity towards sap-sucking insects in an important cereal crop plant.     

Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA)

Transgenic rice plants expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA) were screened for resistance to green leafhopper (Nephotettix virescens; GLH), a major homopteran pest of rice. Survival was reduced by 29% and 53% (P<0.05) respectively, on plants where GNA expression was tissue-specific (phloem and epidermal layer) or constitutive. Similar levels of resistance in GNA-expressing transgenic rice were previously reported for rice brown planthopper (Nilaparvata lugens; BPH). GNA binding to glycoproteins in gut tissues showed that BPH contained more "receptors" than GLH, and that the binding affinity was stronger, particularly in the midgut. Subsequent toxicity of GNA is thus unlikely to be directly related to the amount of lectin bound. GNA was not detected in the honeydew of either insect species when they were fed on GNA-expressing plants, in contrast to results from artificial diet studies. This result suggests that GNA is not being delivered to the insect efficiently. When offered a free choice vs control plants, BPH nymphs tended to avoid plants expressing GNA; avoidance was less pronounced and took longer to develop on plants where GNA expression was tissue-specific, In contrast to BPH, GLH nymphs were attracted to plants expressing GNA, whether constitutively or in a tissue-specific manner.     

Pyramided rice lines harbouring Allium sativum (asal) and Galanthus nivalis (gna) lectin genes impart enhanced resistance against major sap-sucking pests

We have developed transgene pyramided rice lines, endowed with enhanced resistance to major sap-sucking insects, through sexual crosses made between two stable transgenic rice lines containing Allium sativum (asal) and Galanthus nivalis (gna) lectin genes. Presence and expression of asal and gna genes in pyramided lines were confirmed by PCR and western blot analyses. Segregation analysis of F₂ progenies disclosed digenic (9:3:3:1) inheritance of the transgenes. Homozygous F₃ plants carrying asal and gna genes were identified employing genetic and molecular methods besides insect bioassays. Pyramided lines, infested with brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH), proved more effective in reducing insect survival, fecundity, feeding ability besides delayed development of insects as compared to the parental transgenics. Under infested conditions, pyramided lines were found superior to the parental transgenics in their seed yield potential. This study represents first report on pyramiding of two lectin genes into rice exhibiting enhanced resistance against major sucking pests. The pyramided lines appear promising and might serve as a novel genetic resource in rice breeding aimed at durable and broad based resistance against hoppers.     

Transgenic indica rice resistant to sap-sucking insects

Agrobacterium-mediated genetic transformation has been optimized in indica rice susceptible to sap-sucking insects, viz., brown planthopper (BPH) and green leafhopper (GLH). Snowdrop lectin gene (gna) from Galanthus nivalis, driven by phloem-specific rice-sucrose-synthase promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was employed for genetic transformation. Embryogenic calli--after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-gna--were selected on the medium containing phosphinothricin. PCR and Southern blot analyses confirmed the stable integration of both the genes into genomes of transgenic (T0) rice plants. Northern and Western blot analyses revealed the expression of gna in the transgenic plants. In the T1 and T2 generations, the gna and bar transgenes showed co-segregation at a ratio of 3 : 1. Plant progenies expressing gna, in T1 and T2, exhibited substantial resistance against BPH and GLH pests. This is the first report dealing with transgenic indica rice exhibiting high resistance to both insects.     

褐飞虱中肠内膜结合短肽P2S的筛选

筛选与褐飞虱Nilaparvata lugens中肠内膜相结合的短肽, 探索能阻断褐飞虱传播水稻病毒的新方法。【方法】通过改良的膜饲喂法和噬菌体短肽文库展示技术, 模拟褐飞虱正常取食的行为, 筛选能够与其中肠内膜结合的短肽。将筛选到的短肽与加强绿色荧光蛋白进行融合表达, 经纯化后, 将融合蛋白饲喂给褐飞虱若虫, 通过荧光显微镜观察饲喂后若虫的中肠。【结果】使用改良的膜饲喂法, 16 h后褐飞虱若虫的取食率达到91%, 24 h后达到95%。经形态学验证, 确定了一种短肽与褐飞虱中肠内膜具有结合活性, 并将其命名为P2S。【结论】改良了针对褐飞虱的膜饲喂法, 筛选并验证了能够与褐飞虱中肠内膜结合的活性短肽P2S。研究结果为阻断相关的水稻病毒与褐飞虱中肠内膜间的相互作用研究提供了科学依据, 也为褐飞虱新型膜穿孔毒素蛋白的研究提供了理论基础。     

Food assimilated by two sympatric populations of the brown planthopper Nilaparvata lugens (Delphacidae) feeding on different host plants contaminates insect DNA detected by RAPD-PCR analysis

Contamination of insect DNA for RAPD-PCR analysis can be a problem because many primers are non-specific and DNA from parasites or gut contents may be simultaneously extracted along with that of the insect. We measured the quantity of food ingested and assimilated by two sympatric populations of brown planthopper (BPH), Nilaparvata lugens, one from rice and the other from Leersia hexandra (Poaceae), a wetland forage grass, and we also investigated whether host plant DNA contaminates that of herbivore insects in extractions of whole insects. Ingestion and assimilation of food were reduced significantly when individuals derived from one host plant were caged on the other species. The bands, OPA3 (1.25), OPD3 (1.10), OPD3 (0.80), OPD3 (0.60), pUC/M13F (0.35), pUC/M13F (0.20), BOXAIR (0.50), peh#3 (0.50), and peh#3 (0.17) were found in both rice-infesting populations of brown planthopper and its host plant (rice). Similarly, the bands, OPA4 (1.00), OPB10 (0.70), OPD3 (0.90), OPD3 (0.80), OPD3 (0.60), pUC/ M13F (0.35), pUC/M13F (0.20), and BOXAIR (0.50) were found in both Leersia-infesting populations of brown planthopper and the host plant. So, it is clear that the DNA bands amplified in the host plants were also found in the extracts from the insects feeding on them.     

重庆市秀山县稻飞虱发生特点及其原因分析

重庆市秀山地区稻飞虱的发生主害种类为白背飞虱Sogatellafurcifera和褐飞虱Nilaparvatalugens,白背飞虱的危害重于褐飞虱 ;白背飞虱 1年发生 5～ 6代 ,以 4 ( 3)代为主害代 ,田间发生 2～ 3次若虫高峰 ,以第 2次为主害代峰、虫量最大 ;褐飞虱 1年发生 4～ 5代 ,以 5 ( 4 )代为主害代 ,田间发生 3～ 4次若虫高峰 ,以第 3次为主害代峰、虫量最大 ;( 3)稻飞虱重发年比例高 ,达 85 %以上 ;( 4 )白背飞虱呈逐年加重和主害期提前、褐飞虱呈逐年减轻和主害期推迟的趋势。其主要原因是 :特殊的槽谷地形、迁入虫量大、品种及栽培技术的变化、防治技术的提高和气候条件有利等。     

水稻黑条矮缩病传毒昆虫的防治实践与研究

水稻黑条矮缩病 (RBSDV)是由传毒媒介灰飞虱Laodelphaxstriatellus (Fall n)传播所致 ,治虫防病是目前防治水稻黑条矮缩病的重要手段。生产实践证明 ,防治该病应以控制灰飞虱种群数量增长为首选目标 ,把带毒灰飞虱尽可能地扑灭在迁移到水稻秧苗进行传毒侵染之前。因此掌握防治适期 ,选用高效低毒农药 ,切断初次侵染来源 ,才能达到控制该病发生的目的。     

Antimetabolic effects of plant lectins and plant and fungal enzymes on the nymphal stages of two important rice pests,Nilaparvata lugens andNephotettix cinciteps

Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.     

Transgenic rice expressing Allium sativum leaf lectin with enhanced resistance against sap-sucking insect pests

Mannose binding Allium sativum leaf agglutinin (ASAL) has been shown to be antifeedant and insecticidal against sap-sucking insects. In the present investigation, ASAL coding sequence was expressed under the control of CaMV35S promoter in a chimeric gene cassette containing plant selection marker, hpt and gusA reporter gene of pCAMBIA1301 binary vector in an elite indica rice cv. IR64. Many fertile transgenic plants were generated using scutellar calli as initial explants through Agrobacterium-mediated transformation technology. GUS activity was observed in selected calli and in mature plants. Transformation frequency was calculated to be ~12.1%±0.351 (mean ± SE). Southern blot analyses revealed the integration of ASAL gene into rice genome with a predominant single copy insertion. Transgene localization was detected on chromosomes of transformed plants using PRINS and C-PRINS techniques. Northern and western blot analyses determined the expression of transgene in transformed lines. ELISA analyses estimated ASAL expression up to 0.72 and 0.67% of total soluble protein in T₀ and T₁ plants, respectively. Survival and fecundity of brown planthopper and green leafhopper were reduced to 36% (P<0.01), 32% (P<0.05) and 40.5, 29.5% (P<0.001), respectively, when tested on selected plants in comparison to control plants. Specific binding of expressed ASAL to receptor proteins of insect gut was analysed. Analysis of T₁ progenies confirmed the inheritance of the transgenes. Thus, ASAL promises to be a potential component in insect resistance rice breeding programme.     

褐飞虱和白背飞虱的取食为害对水稻营养生长的影响

对塑料钵栽培的水稻进行罩宠试验,研究了褐飞虱和白背飞虱在不同若虫密度下取食为害对水稻营养生长的影响．结果表明,两种飞虱的成虫干重、水稻叶面积和其地上部干重因若虫密度的增加而下降．叶片干重占地上部干重的比例和稻株分配给叶片干物质量随为害程度的加重而增大;褐飞虱和白背飞虱总干重（X）与稻株地上部损失量（Y）之间存在着极显著的线性关系．两种飞虱干重每增加1mg,水稻地上部干重则分别损失26.01mg和21．90mg．讨论了稻飞虱取食为害对水稻致害的可能机制．     

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.     

Role of hydroperoxide lyase in white-backed planthopper (Sogatella furcifera Horváth)-induced resistance to bacterial blight in rice, Oryza sativa L.

A pre-infestation of the white-backed planthopper (WBPH), Sogatella furcifera Horváth, conferred resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae ( Xoo ) in rice ( Oryza sativa  L.) under both laboratory and field conditions. The infestation of another planthopper species, the brown planthopper (BPH) Nilaparvata lugens Stål, did not significantly reduce the incidence of bacterial blight symptoms. A large-scale screening using a rice DNA microarray and quantitative RT-PCR revealed that WBPH infestation caused the upregulation of more defence-related genes than did BPH infestation. Hydroperoxide lyase 2 ( OsHPL2 ), an enzyme for producing C₆ volatiles, was upregulated by WBPH infestation, but not by BPH infestation. One C₆ volatile, ( E )-2-hexenal, accumulated in rice after WBPH infestation, but not after BPH infestation. A direct application of ( E )-2-hexenal to a liquid culture of Xoo inhibited the growth of the bacterium. Furthermore, a vapour treatment of rice plants with ( E )-2-hexenal induced resistance to bacterial blight. OsHPL2 -overexpressing transgenic rice plants exhibited increased resistance to bacterial blight. Based on these data, we conclude that OsHPL2 and its derived ( E )-2-hexenal play some role in WBPH-induced resistance in rice.     

水稻生育期对褐飞虱和白背飞虱卵巢发育及起飞行为的影响

研究了取食分蘖初期、拔节期和孕穗抽穗期稻株上的褐飞虱和白背飞虱成虫的卵巢发育和起飞情况。其中,水稻生育期对褐飞虱的影响相对较小,不同生育期稻株上试虫的卵巢发育级别羽化后的4d内均无显著差异,羽化后第5天的褐飞虱在孕穗抽穗期卵巢发育最快,分蘖初期最慢,拔节期居中。褐飞虱的起飞率在各个生育期均无显著差异。白背飞虱卵巢发育、起飞率均受水稻生育期的显著影响,在水稻分蘖初期,卵巢发育最快,孕穗抽穗期则卵巢发育最慢,拔节期居中,水稻分蘖初期,起飞率最低,孕穗抽穗期起飞率最高,拔节期居中。结果表明,水稻生育期对两种飞虱卵巢发育和起飞行为的影响明显不同。认为水稻生育期对两种飞虱卵巢发育与起飞行为的不同影响,应是水稻田间白背飞虱发生较早、褐飞虱发生较晚的一个重要原因。