Role of the α subunit of heterotrimeric G-protein in probenazole-inducing defense signaling in rice

 Certain cellular responses to stresses and stimuli are regulated by a G-protein-mediated signaling pathway. A rice dwarf mutant that is defective in the α subunit of the heterotrimeric G-protein was found to be fully protected from blast fungus by the plant activator probenazole (PBZ) despite the 1-day delay in induction of the PR-10 gene PBZ1 by PBZ. These results suggest that the signaling pathway for protection by PBZ is not via the G-protein, although G-protein is involved in the induction of PBZ1 by PBZ. Received: March 27, 2002 / Accepted: August 20, 2002     

不同抗逆诱导剂对水稻细胞PBZ 1 基因表达的影响

以水稻品种中花8号的悬浮细胞为试材,分别研究了真菌激发子和盐激发子对用稻瘟灵(IPT)和脱落酸(ABA)预处理24 h的水稻悬浮细胞中PBZ 1 基因表达的影响。结果发现:单独用IPT、ABA处理可以诱导细胞中PBZ 1 基因表达;只用真菌激发子和盐激发子处理的细胞中也可以检测到PBZ 1 基因的表达,但两种激发子对PBZ 1 基因表达的影响不同,在真菌激发子处理后的0~3 h之间PBZ 1 基因表达逐渐增强,而在盐激发子处理后的0~3 h之间则呈现逐渐减弱的趋势,到第3 h就检测不到了;用IPT预处理24 h后,再分别用真菌激发子和盐激发子诱导,水稻细胞中PBZ 1 基因表达强度都显著高于单独用IPT处理的细胞,而且在处理后的第0.5~3 h期间一直保持较强的表达;ABA＋盐激发子处理使细胞中PBZ 1 基因表达推迟且短暂,只在处理后第1~2 h之间可检测到,ABA＋真菌激发子处理使细胞中PBZ 1 基因在0.5~3 h期间一直保持较强的表达,明显强于单独用ABA处理的细胞。可见,IPT和ABA都可以诱导抗病基因PBZ 1 的表达,PBZ 1 基因也可由盐激发子诱导表达,说明PBZ 1 基因对除病害以外的非生物逆境也有积极的作用。     

水稻纹枯病发病过程PR1和PBZ1的表达动态

 采用从福建省稻田分离纯化的纹枯病菌(Rhizoctonia solani)菌株FJ-15接种籼稻9311,分析了水稻在纹枯病菌侵染致病过程中,编码病程相关蛋白的基因表达动态,并观察了症状的变化。Northern blot分析表明:PR1在接种12 h后开始表达,在之后的4个时间段其表达量逐步增强;而PBZ1也在12 h开始表达,在48 h表达量激剧增强几乎与72 h表达量相当。组织和症状观察表明,接种12 h后叶鞘表面菌丝纵横分枝,接种36 h后出现零星病斑,接种48 h后表现典型的受害症状,接种72 h后病斑继续扩大,并可蔓延到非接种叶鞘。结果表明,PR1和PBZ1的表达与水稻和纹枯病菌亲和互作的过程存在对应关系。     

Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance.     

Expression Analysis of Rice Defense-Related Genes in Turfgrass in Response to Magnaporthe grisea

ABSTRACT Magnaporthe grisea (anamorph = Pyricularia grisea) causes blast on rice (Oryza sativa) and gray leaf spot on turfgrass. Gray leaf spot is a serious disease on St. Augustinegrass (Stenotaphrum secundatum), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea). Virulence assays performed in this study revealed that M. grisea collected from rice could also cause disease on St. Augustinegrass and tall fescue. One rice isolate, Che86061, caused similar disease reactions on susceptible cultivars of rice and St. Augustinegrass and an incompatible interaction on resistant cultivars of both species. To explore whether similar defense-related genes are expressed in rice and St. Augustinegrass, a rice cDNA library was screened using pooled cDNAs derived from M. grisea-infected St. Augustinegrass. Thirty rice EST (expressed sequence tag) clones showing differential expression in St. Augustinegrass following M. grisea inoculation were identified and classified into six putative functional groups. Northern blot analyses of seven EST clones that collectively represented each putative functional group confirmed that the expression of five out of seven EST clones was similar in both rice and St. Augustinegrass. This study represents one of the first attempts to use a broad-scale genomic approach and resources of a model monocot system to study defense gene expression in St. Augustinegrass following M. grisea infection.     

Behavior of <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with bioluminescence genes on rice plants

Erwinia ananas, the causal agent of bacterial palea browning of rice, was transformed with bioluminescence genes to clarify their behavior on rice plants. Transformant CTB009T2 was used to inoculate rice plants, and the subsequent bioluminescence of CTB009T2 was observed using a two-dimensional luminometer. Luminous spots frequently appeared on anthers after flowering and on dead tissues such as leaf tips, lower leaf sheaths, and leaf blades. In spikelets that developed the disease symptom on the palea, luminous spots appeared 48h after flowering on stigmas, basal parts of ovaries, and lodicules. These results indicate that postflowering anthers and dead tissues on rice plants are important sites for a rapid increase in the pathogen population, and that the multiplication of the pathogen on internal tissues of spikelets after flowering is associated with the appearance of browning.     

水稻抗病性反应的cDNA微阵列分析及一个新基因OsBTB的发现

 利用含1106条水稻独立基因的cDNA微阵列,检测水稻近等基因系H7R/H7S受稻瘟病菌诱导8h的表达谱差异。结果显示:基因表达谱变化明显,在980组有效数据中,共检测到170条基因的表达丰度发生显著变化,其中上调表达基因106条,下调表达基因64条。通过与NCBI水稻数据库进行BLASTn比对分析,发现这些差异表达基因包含β-1,3-葡聚糖酶、富甘氨酸蛋白在内的已知基因33条,未知基因137条。对10个未知基因进行生物信息学分析后,其中获得一个含全长基因并具有BTB/POZ结构域的克隆T007F02(http://www.estarray.org),该基因命名为OsBTB。该克隆的cDNA序列具有完整的ORF区,OsBTB上游具有典型的启动子区。结果经测序得到证实。高通量的RNA斑点杂交证实:在抗性供试水稻中,该基因在水稻接种稻瘟病菌早期(8~12h)受诱导高表达;在感病品种中,该基因表达无明显改变。结果提示OsBTB基因在功能上可能与水稻抗瘟性过程密切相关。     

表达的hrmA蛋白质具有诱导水稻细胞产生防卫反应的活性

 将丁香假单胞菌hrmA基因构建到原核表达载体pET 29中,得到重组载体pET hrmA。将重组载体转化大肠杆菌BL21(DE3),经IPTG诱导,获得了以包含体形式存在的融合蛋白。复性的融合蛋白能诱导水稻悬浮细胞的活性氧迸发,处理20min后达到峰值。Northern杂交结果表明hrmA蛋白能显著地诱导PBZ1基因在水稻悬浮细胞中表达,在所观察的时间内PBZ1基因表达丰度随处理时间延长而增加,用hrmA处理水稻植株,诱导了PAL基因的表达。实验结果表明hrmA融合蛋白具有激活水稻细胞防卫反应的活性。     

小麦条锈菌cDNA文库构建和表达序列标签(ESTs)分析

 小麦条锈菌(Puccinia striiformis f. sp. tritici)是全世界范围内小麦生产上的重要病原真菌, 但是对小麦条锈菌的基因组和基因功能却了解甚少。为了促进小麦条锈菌基因组学的发展和大规模基因发现, 我们以噬菌体λTrip1Ex2为载体, 采用SMART技术构建了小麦条锈菌萌发夏孢子的cDNA文库。原始文库的滴度为1.1×10⁶pfu/mL, 平均插入片段长度为750 bp。从文库中随机挑取279个cDNA克隆测序, 分析发现这些ESTs的平均GC含量为45.08%。通过聚类分析, 279个ESTs拼接成31个contigs和80 singletons。BLASTx分析表明, 47%的ESTs与GenBank中报道的功能已知或未知蛋白具有相似性。tBLASTx分析表明12个uniseqs与EST数据库中的序列具有相似性, 其中9个是来自担子菌的cDNA文库。几个EST与已知的真菌致病相关基因具有高度相似性。RT-PCR分析了几个基因在小麦条锈菌侵染过程中的表达水平。这些结果为小麦条锈菌夏孢子萌发以及侵染寄主过程中的基因表达研究奠定了很好的分子生物学基础。     

Differentially expressed genes in dicamba‐resistant and dicamba‐susceptible biotypes

In an ongoing effort to investigate the mechanism of auxinic herbicide resistance in Kochia scoparia (kochia), polymerase chain reaction‐based cDNA suppression subtractive hybridization was used to identify genes that are differentially expressed between dicamba‐resistant (HRd) and dicamba‐susceptible (S1) kochia biotypes in response to herbicide treatment. Both the HRd and S1 adaptor‐ligated cDNAs were used in separate hybridizations in order to generate biotype‐specific clones. A total of 710 cDNAs, representing putative differentially expressed mRNAs, were isolated and subjected to further screening. The false‐positive cDNAs were removed by conducting two colony hybridizations and at least one Northern hybridization. Differential or biotype‐specific expression was confirmed for six clones each from the HRd and S1 plants. The S1‐related genes were constitutively expressed at higher levels than in the HRd plants, but none had significant sequence similarity to known genes. Among the HRd‐related genes, HRd‐88 had 42% amino acid sequence identity to a conserved domain within thiol peptidases, which might be involved in auxin‐regulated gene expression. The constitutively expressed and inducible (by the dicamba treatment) HRd‐39 had 40% identity and 60% similarity to a domain from the Fe(II)/α‐ketoglutarate‐dependent hydroxylase superfamily. The HRd‐39 gene product had the characteristics of an enzyme that is able to detoxify dicamba via oxidative hydroxylation and thus its overexpression might confer the dicamba resistance phenotype.     

Ratio of rice reflectance for estimating leaf blast severity with a multispectral radiometer

 Rice reflectance was measured to determine the spectral regions most sensitive to leaf blast infection with a multispectral radiometer. As disease severity increased, reflectance also increased in the 400–500 nm (blue), 570–700 nm (red), and 900–2000 nm regions but decreased in the 500–570 nm and 700–900 nm regions. The increased reflectance in the blue and red regions may be attributed to decreased chlorophyll and carotenoid contents in response to the blast infection. The maximum and minimum reflectance differences occurred at 680 nm and 760 nm for the nondiseased and diseased rice, respectively. The spectral location of maximum sensitivity was 675 nm regardless of disease severity. Rice reflectance ratios were evaluated as indicators of leaf blast severity. Two ratios, R550/R675 (reflectance at 550 nm divided by reflectance at 675 nm), and R570/R675 quantified the significant disease severity. These wavelengths were selected based on the sensitivity minima and maxima. The ratios of nondiseased rice plants varied depending on growth stage. The variation in ratios must be considered when they are used to estimate leaf blast severity. Received: April 2, 2002 / Accepted: August 12, 2002     

Possible connection between imidacloprid-induced changes in rice gene transcription profiles and susceptibility to the brown plant hopper Nilaparvatalugens Stål (Hemiptera: Delphacidae)

The chemical pesticide, imidacloprid (IMI) has long-lasting effectiveness against Hemiptera. IMI is commonly used to control the brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae). Some chemical pesticides, however, can induce the susceptibility of rice to BPH, which has indirectly led to the resurgence of BPH. The mechanism of the chemical induction of the susceptibility of rice to BPH was not previously understood. Here, a 44 K Agilent Rice Expression Microarray was used to identify changes in gene expression that accompany IMI-induced rice susceptibility to BPH. The results showed that 225 genes were differentially expressed, of which 117 were upregulated, and 108 were downregulated. Gene ontology annotation and pathway analysis revealed that differentially expressed genes were mainly classified into the eight functional groups: oxidation reduction, regulation of cellular process, response to stress, electron carrier activity, metabolic process, transport, signal transducer, and organismal development. The genes encoding plant lipid transfer protein, lignin peroxidase, and flavonol-3-O-methyltransferenase may be important responses to the IMI-induced susceptibility of rice to BPH. The reliability of the microarray data was verified by performing quantitative real-time PCR and the data provide valuable information for further study of the molecular mechanism of IMI-induced susceptibility of rice.     

Silicon content in rice seedlings to protect rice blast fungus at the nursery stage

To control rice blast effectively at the nursery stage, the absolute SiO₂ content necessary for rice plants to resist blast disease was investigated using various rice cultivars and soils. Nine rice cultivars with different complete resistance genes and different degrees of partial resistance were grown on nursery soils amended with silica gel at different rates to change the SiO₂ content of rice plant. The rice seedlings were then inoculated 28 days after sowing with Pyricularia grisea to estimate their blast resistance. In all rice cultivars, the number of lesions was significantly reduced when SiO₂ content increased in the rice seedling; lesions were reduced to 5%–20% of the number on the seedlings grown in soil without silica gel when the seedling SiO₂ content reached 5%. Additionally, the susceptibility to blast disease of rice seedlings grown on eight soils collected from different districts, with varying amounts of silica gel, was compared. The number of lesions decreased significantly when the SiO₂ content in the seedlings reached 5%. These results suggest that SiO₂ content of at least 5% in the rice plant can control this disease at the nursery stage under any conditions.     

Culex pipiens pallens: identification of genes differentially expressed in deltamethrin-resistant and -susceptible strains

Insecticide-resistance is a major obstacle to controlling insect vectors of microorganisms that cause human diseases. Identification of genes associated with resistance to insecticides has been a valuable tool for understanding mechanisms underlying resistance to commonly used insecticides such as deltamethrin. To identify such genes, we used suppression subtractive hybridization to obtain 809 differentially expressed clones in deltamethrin resistant versus susceptible laboratory strains of Culex pipiens pallens. Using cDNA microarrays and reverse Northern blots, a subset of 16 clones was confirmed to have greater than 3-fold difference in expression levels. Within this subset, we identified 2 clones uniquely expressed in the deltamethrin-resistant strain, eight clones exhibiting higher expression in the resistant strain and six in the susceptible strain. Of these 16 clones, 13 clones have sequence homology to known genes, such as ribosomal RNA, ribosome proteins, trypsin, and chymotrypsin-like proteins. Our data suggests resistance to deltamethrin may be a polygenic phenotype.     

水稻磷酸核酮糖激酶基因OsPRK克隆、亚细胞定位与诱导表达分析

为探究水稻磷酸核酮糖激酶基因OsPRK在水稻诱导抗虫反应中的功能,以水稻秀水110为材料克隆OsPRK基因的全长,通过生物信息学软件分析其序列特征,并应用实时荧光定量PCR技术分析OsPRK基因在水稻不同组织中的分布情况以及在虫害诱导、激素和机械损伤处理水稻中的表达特征。结果显示,水稻OsPRK基因序列全长为1 212 bp,编码403个氨基酸,分子量为44.86 kD,具有1个磷酸核酮糖激酶保守结构域。OsPRK蛋白亚细胞定位结果显示其定位于叶绿体。OsPRK基因在水稻中的表达具有组织特异性,其在内叶、外叶、内叶鞘、外叶鞘和根系这5个组织中相对于内参基因ACTIN的表达量分别为35.83、20.53、6.25、3.21和0.03。与对照相比,二化螟Chilo suppressalis为害能够强烈抑制水稻茎秆中OsPRK基因的表达;褐飞虱Nilaparvata lugens怀卵雌成虫为害1.5、24 h、白背飞虱Sogatella furcifera怀卵雌成虫为害1、8、24 h以及机械损伤处理3、6、24 h均能显著诱导水稻茎秆中OsPRK基因的表达;而OsPRK基因的表达量在茉莉酸处理6、12 h时以及水杨酸处理0.5、1.5 h时被显著抑制,在茉莉酸处理48 h和水杨酸处理24 h时被显著诱导。表明OsPRK基因可能参与了水稻对害虫的诱导防御反应。     

水稻草矮病特异蛋白抗血清的制备及应用

 采用不同pH值沉淀法及超速离心获得提纯的水稻草矮病特异蛋白。提纯的S-蛋白具有典型的蛋白紫外吸收曲线,提纯产量为28 mg/100 g病叶。从SDS-PAGE精提纯的S-蛋白免疫兔子上获得抗血清,经微量沉淀反应、琼脂双扩散测定,其效价分别为1:1024和1:256。获得的S-蛋白抗血清与提纯的S-蛋白及感病植株,在琼脂双扩散反应中,均有明显的沉淀线出现,对照健株及提纯的水稻草矮病毒则无反应。利用制备的S-蛋白抗血清经A-蛋白酶联免疫吸附法检测,结果表明:S-蛋白抗血清与提纯S-蛋白及感病植株有专化性反应,且不同品种、不同部位、接种后不同发病时间的水稻病株中S-蛋白的积累量有差异,这就为RGSV的检测提供了一种快速、简便的方法。     

Resistance to anthracnose is decreased by tissue culture but increased with longer acclimation in the resistant strawberry cultivar

Inoculation of tissue-cultured plants of strawberry cultivar Ichigochukanbohon-Nou2gou (Nou-2) with the anthracnose pathogen (Glomerella cingulata) results in wilting and plant death, whereas inoculation of strawberry runners grown in greenhouses results in leaf spots, not wilting or death. When tissue-cultured Nou-2 plants were acclimated for 3, 9 or 15 days, plant resistance to anthracnose increased as the acclimation period increased, suggesting that the resistance of Nou-2 may be induced by external factors. To clarify the mechanisms of resistance, we used cDNA microarray analyses to compare gene expressions among tissue-cultured and acclimated plants of the resistant cultivar Nou-2 and the susceptible cultivar Tochiotome. As a result, we identified 18 cDNA clones that were upregulated during acclimation in Nou-2 but not in Tochiotome. In a real-time RT-PCR of the 18 clones, the expression levels of three were significantly higher in acclimated plants of Nou-2. Two of the three clones showed close homology to enzymes related to flavonoid biosynthesis, i.e., leucoanthocyanidin dioxygenase (LDOX, ANS) and UDP-glucosyltransferase, putative (3-GT). The clones spotted onto the cDNA microarrays were rechecked, and 23 nonredundant cDNA clones of 13 enzymes were estimated to be flavonoid biosynthetic enzymes. Most of these clones were upregulated by acclimation in Nou-2.     

西瓜与枯萎病菌非亲和互作相关基因的分离及表达分析

 从整体水平阐明西瓜对西瓜枯萎病菌1号生理小种的抗病分子机制和抗病相关基因的表达特征,以高抗枯萎病菌1号生理小种的西瓜品种“卡红(Calhoun Gray)”为试材,接种西瓜枯萎病菌和蒸馏水的根尖组织作为测验方(Tester)和驱动方(Driver),构建西瓜枯萎病菌胁迫的SSH-cDNA正向文库。利用反向 Northern 斑点杂交技术对文库中克隆进行杂交筛选。随机测序300个阳性克隆,序列比对分析,利用RT-PCR技术分析抗病相关基因的表达特性。259条EST成功测序,167条与已知基因具有较高的同源性,占全部ESTs的65.5%,其中与抗病和防卫相关的有64条23种,占24.7%;卡红对枯萎病菌1号生理小种的抗性相关基因主要涉及抗病信号传导、抗病防卫、转录因子、次生代谢合成和细胞保护等方面;Aquaporin和Peroxidase基因在接种后表达量均增加。Calhoun Gray对枯萎病菌侵染作出的反应是全方位多方面的,抗病相关基因主要集中在系统获得性抗性反应中,获得了一些Calhoun Gray与野生西瓜PI296341在与枯萎病生理小种1互作中差异表达的基因,为深入研究西瓜与枯萎病菌互作的分子机制以及关键基因的功能分析奠定基础。     

乙烯信号传导途径因子OsEIL 6调控水稻抗稻瘟病反应

稻瘟病(rice blast)是水稻生产上最严重的病害之一。抗病相关基因的挖掘对稻瘟病的防治具有重要意义。研究表明植物EIN3/EIL家族基因在抗病过程中发挥着重要作用。本研究采用RNAi技术探究OsEIL6参与的水稻抗稻瘟病反应。稻瘟菌侵染时基因表达谱检测结果表明,OsEIL6在水稻和稻瘟菌非亲和组合中受到诱导表达。稻瘟菌接种结果显示,水稻OsEIL6沉默株系和野生型植株‘TG394’相比抗性下降;实时荧光定量RT-PCR结果分析表明,OsEIL6的表达量下降导致乙烯合成途径中OsACO1和乙烯信号传导途径的OsERF063和OsERF073的转录水平下降。亚细胞定位研究发现该基因定位于水稻细胞质。OsEIL6沉默株系中ROS合成途径标记基因OsrbohA和OsrbohB的表达量均明显下调,表明该基因可能通过影响ROS的合成调控水稻抗稻瘟病反应。本研究结果将有助于进一步揭示OsEIL6参与的乙烯信号传导途径介导的水稻抗稻瘟病反应机制。