利用杨树原生质体瞬时表达系统筛选杨生褐盘二孢菌效应因子蛋白

效应因子蛋白在病原真菌侵染宿主植物过程中发挥着重要作用,效应因子的筛选和鉴定是目前研究的难点。笔者以杨生褐盘二孢菌全基因组测序结果为基础,根据真菌效应因子蛋白结构特征,预测出106个候选效应因子,并成功克隆其中25个。利用杨树原生质体瞬时表达系统对这25个候选效应因子蛋白进行细胞内功能筛选,鉴定出4个效应因子蛋白可以抑制杨树转录因子WRKY29启动子的活性,表明这4个效应因子蛋白在干扰植物免疫系统的信号传导途径中发挥重要作用。     

水稻幼苗叶片应答稻瘟病侵染的差异蛋白谱分析

为了探讨水稻感病基因型种质响应稻瘟病侵染的蛋白质表达谱的变化规律和作用途径,以水稻感稻瘟病种质日本晴为材料,采用接种稻瘟病菌分生抱子悬浮液,24,48和72 h后提取叶片蛋白质,采用i TRAQ蛋白质组学技术研究稻瘟病胁迫下水稻叶片蛋白质组的变化.结果表明,稻瘟病侵染诱导了水稻幼苗叶片内涉及氧化还原平衡、防御、信号传导、糖和能量代谢、氨基酸代谢、光合作用,以及蛋白质代谢等代谢途径相关的53个蛋白质的表达量发生了改变.GO分析表明稻瘟病主要调控了植株体内细胞内平衡、代谢过程和蛋白质代谢等生物学过程.稻瘟病侵染激活了活性氧代谢、防御,以及热休克蛋白等相关的途径,而抑制了蛋白质生物合成过程.结合这些差异表达蛋白的丰度变化结合它们可能的功能,描绘了水稻应答稻瘟病侵染的蛋白质代谢网络,有助于在蛋白质水平上了解其应答过程.     

植物多酚氧化酶的诱导

多酚氧化酶(Polyphenol oxidase,PPO)是植物体内众多防御化合物中的重要种类之一,属于植物抗营养型防御物质.其分布广泛,作用方式多样,并可被多种外界因素所诱导.本文将依据国内外文献,就引起植物体内PPO活性提高的诱导因子诱导机制两个方面进行一系统综述.     

用抑制差减杂交技术分离烯丙异噻唑诱导水稻特异表达的基因

烯丙异噻唑(PBZ)处理水稻根能使其产生对稻瘟病的系统获得性抗性,因此在东南亚稻区被广泛用于防治稻瘟病,然而关于其作用的分子机理还知之甚少．运用抑制差减杂交技术,试图通过分离鉴定受PBZ诱导调控的关键基因,探索其作用的分子机理．以PBZ处理后的水稻叶片cDNA为目标群体(tester),以未处理水稻叶片cDNA为对照群体(driver),用经过对照cDNA差减的、烯丙异噻唑处理的cDNA群体构建了一个含260个重组子的差减文库．通过差示筛选鉴定出了26个。PBZ诱导水稻特异表达和增强表达的候选克隆．对26个cDNA克隆进行了双向测序和同源性比较,发现其中3个克隆：rJAB1,rTAB2和蛋白磷酸酯酶2Aδ调节亚基同型物基因,位于抗病相关信号转导途径上,它们与哺乳动物和人类免疫途径上的信号因子有明显相似之处,因此推断可能与诱导抗性有关．另外8个克隆与已知基因同源性为70％～99％．经Northern杂交分析,其中rJAB1(编码c-jun激活区结合蛋白1)受烯丙异噻唑和稻瘟菌诱导表达;膜糖蛋白同源基因及肌动蛋白(actin)α1受烯丙异噻唑诱导表达,部分克隆为低丰度转录本．     

稻瘟病菌中己糖载体蛋白的生物信息学分析

己糖载体蛋白是一种重要的糖载体蛋白,主要用于己糖的摄取和运输。本文通过生物信息学的方法,研究发现稻瘟病菌有67个可能的己糖载体蛋白。其中,MGG 06203、MGG 03620、MGG 15700、MGG 00040、MGG 13651、MGG 05946的氨基酸序列分别与Neurosporacrassa、Aspergillusnidulans和Colletrotrichumgramini-cola中已鉴定出的己糖载体蛋白高度同源。此外,稻瘟病菌不同的己糖载体蛋白基因在附着胞形成的各个阶段表达量存在很大的差异,尤其是其中有25个基因在稻瘟病菌侵染水稻48 h后有明显上调表达,表明它们可能参与了稻瘟病菌的致病过程。     

稻瘟病菌中己糖载体蛋白的生物信息学分析

己糖载体蛋白是一种重要的糖载体蛋白,主要用于己糖的摄取和运输。本文通过生物信息学的方法,研究发现稻瘟病菌有67个可能的己糖载体蛋白。其中,MGG_06203、MGG_03620、MGG_15700、MGG_00040、MGG_13651、MGG_05946的氨基酸序列分别与Neurospora crassa、Aspergillus nidulans和Colletrotrichum graminicola中已鉴定出的己糖载体蛋白高度同源。此外,稻瘟病菌不同的己糖载体蛋白基因在附着胞形成的各个阶段表达量存在很大的差异,尤其是其中有25个基因在稻瘟病菌侵染水稻48 h后有明显上调表达,表明它们可能参与了稻瘟病菌的致病过程。     

内生芽孢杆菌防治植物病害的应用及作用机制研究进展

植物内生细菌由于其独特的生境,成为植物病害防治中的重要生防因子.芽孢杆菌在植物体内分布具有广泛性和多样性,其在防治病害和促进植物生长发育方面的作用被广泛研究和应用.芽孢杆菌控制植物病害的作用机制主要包括竞争、拮抗、促进植物生长和诱导植物抗病性四个方面.植物诱导抗性作为植物内生细菌防病的主要机制之一,具有非特异性、广谱性和系统性,在植物病害的生物防治中可被广泛应用.     

饥饿状态下HNF4α对MTP的表达调控

肝细胞核因子4α(HNF4α)是肝脏中一种重要的转录因子,其参与多个代谢途径的调控.微粒体甘油三酯转运蛋白(MTP)是极低密度脂蛋白装配和分泌过程的限速酶.C57BL/6J小鼠中研究发现,饥饿可以诱导肝细胞MTP基因的表达.在HepG2细胞中进一步证实饥饿可以诱导MTP表达,同时HNF4α表达也显著上升.实验证实MTP是HNF4α的靶基因之一,用腺病毒介导的HNF4α过表达和HNF4α的激动剂均显著提高MTP的表达;HNF4α特异性siRNA抑制HNF4α的表达,MTP的mRNA和蛋白水平相应下调.结果证明,在饥饿状态下可以诱导MTP和HNF4α的表达,MTP的表达量上升是受到HNF4α的转录调控.     

The genome sequence of the rice blast fungus Magnaporthe grisea

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.     

The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi

Pathogens have evolved different strategies to overcome the various barriers that they encounter during infection of their hosts. The rice blast fungus Magnaporthe grisea causes one of the most damaging diseases of cultivated rice and has emerged as a paradigm system for investigation of foliar pathogenicity. This fungus undergoes a series of well-defined developmental steps during leaf infection, including the formation of elaborate penetration structures (appressoria). This process has been studied in great detail, and over thirty M. grisea genes that condition leaf infection have been identified. Here we show a new facet of the M. grisea life cycle: this fungus can undergo a different (and previously uncharacterized) set of programmed developmental events that are typical of root-infecting pathogens. We also show that root colonization can lead to systemic invasion and the development of classical disease symptoms on the aerial parts of the plant. Gene-for-gene type specific disease resistance that is effective against rice blast in leaves also operates in roots. These findings have significant implications for fungal development, epidemiology, plant breeding and disease control.     

Obtaining transgenic rice resistant to rice fungal blast disease by controlled cell death strategy

The strategy of the two-component system,composed of Barnase and Barstar which encode RNase and a specific inhibitor to the RNase respectively, is adopted to obtain transgenic rice resistant to rice fungal blast disease. In this study, two chimeric promoters, induced by rice blast fungus pathogen (Magnaporthe grisea), are fused with Barnase respectively to construct two plant expression vectors, pWBNBS and pPBNBS together with the Barstar driven by CaMV 35S promoter. The resistance of the transgenic rice lines to rice blast fungus disease and rice blight disease are evaluated. The results show that (1) the expression of Barnase is induced in rice leaves when inoculated with the spores of Magnaporthe grisea; (2) the induced expression level of Barnase surpasses the level of Barstar, which elicits a similar hypersensitive response (HR) in the leaves, and the transgenic plant shows high resistance to the rice fungal blast disease; and (3) transgenic rice plants also show obvious resistance to rice bacterial blight disease. Taken together, these results suggest that the transgenic rice plants harboring this two-component system acquire relatively broad spectrum resistance against pathogens, especially high resistance to rice fungal pathogen.     

A P-type ATPase required for rice blast disease and induction of host resistance

To cause diseases in plants, pathogenic microorganisms have evolved mechanisms to deliver proteins directly into plant cells, where they suppress plant defences and facilitate tissue invasion. How plant pathogenic fungi, which cause many of the world's most serious plant diseases, deliver proteins during plant infection is currently unknown. Here we report the characterization of a P-type ATPase-encoding gene, MgAPT2, in the economically important rice blast pathogen Magnaporthe grisea, which is required for exocytosis during plant infection. Targeted gene replacement showed that MgAPT2 is required for both foliar and root infection by the fungus, and for the rapid induction of host defence responses in an incompatible reaction. DeltaMgapt2 mutants are impaired in the secretion of a range of extracellular enzymes and accumulate abnormal Golgi-like cisternae. However, the loss of MgAPT2 does not significantly affect hyphal growth or sporulation, indicating that the establishment of rice blast disease involves the use of MgApt2-dependent exocytotic processes that operate during plant infection.     

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.     

基于全基因组序列的尖孢镰刀菌分泌蛋白预测及其特征分析

尖孢镰刀菌是一种以土壤习居为主要特征的植物病原菌,广泛分布于世界各地,且寄主广泛,能引起豆科、茄科、葫芦科等科100多种植物根腐、茎腐、茎基腐等病害,严重时造成植株萎蔫死亡,严重影响着植物的产量和品质.同时,目前生产上对于该菌的防治多使用甲霜灵·锰锌、多菌灵、百菌清等化学药剂,然而,防治效果不佳,急需开发针对该菌新的作用靶标的药剂.前人已经明确植物病原真菌分泌蛋白在侵染、操控植物等过程中发挥着重要功能,然而,学术界尚未见有关该菌中分泌蛋白的报道.本研究以尖孢镰刀菌菌株(Fusarium oxysporumf.sp.lycopersici 4287)的蛋白序列为基础数据,以分泌蛋白所具有的N-端含有信号肽、不含有跨膜结构域、没有GPI锚定位点、将蛋白分泌在胞外4大特征为依据,利用生物信息学在线分析程序明确该菌中含有778个分泌蛋白,并对上述蛋白开展特征分析,明确上述蛋白的氨基酸长度主要集中在101～400aa、氨基酸组成中以G,T,S,A较多,信号肽长度主要集中在16～21aa、信号肽氨基酸组成中以P,S,A,T,G较多,信号肽切割位点为T-X-T类型.为今后进一步开展尖孢镰刀菌的分泌蛋白功能解析和开发新型药剂靶标提供了重要的理论基础.     

Phytophthora elicitor PB90 induced apoptosis in suspension cultures of tobacco

The protein elicitor PB90 secreted by Phytophthora boehmeriae is an efficient elicitor inducing the hypersensitive response and systemic acquired resistance in tobacco plants. Here, we observed cell death in suspension-cultured cells of Nicotiana tabacum BY-2 with PB90 treatment using Trypan blue staining method. And this cell death could be suppressed by cycloheximide, an inhibitor of proteins synthesis, which implies that PB90-induced celldeath was an active cell death process requiring new protein synthesis. DAPI staining revealed that PB90 induce rapid chromatin condensation, margination, apoptotic bodies‘ formation and DNA laddering, further TUNEL assay also observed the specific breakage of 3 ‘-OH ends. All of the above common morphological characteristics indicated that PB90 induced apoptosis in suspension cultures of tobacco, suggesting that hypersensitive response induced by PB90 is an apoptotic process.