真菌无毒基因克隆与抗性蛋白互作研究

无毒基因（avirulence genes, Avr）是病原物遗传因子,能诱发寄主植物产生抗病性。真菌Avr基因的克隆、编码产物结构和功能分析,以及与寄主抗性蛋白的互作机制等方面的研究对于深入了解植物的抗真菌病害分子机理具有重要意义。其编码AVR蛋白（又可以称为效应子,effector）通常富含半胱氨酸,效应子通过吸器或侵入丝被分泌到寄主细胞内促发抗性反应。在抗性反应过程中,效应子能直接或间接地被植物细胞相应的抗性蛋白识别,这种识别机制与效应子和抗性蛋白的区段相关。以几个真菌病害为例,综述了近年来有关无毒基因克隆、表达、以及与抗性蛋白的互作机制等方面的主要研究进展,以期为相关研究的深入提供参考。     

利用田间抗病基因近等混合系防治马铃薯晚疫病

马铃薯是世界上第四大粮食作物,是我国七大农作物之一。晚疫病是由致病疫霉菌（Phytophthora infestans）引起的毁灭性的病害,19世纪中叶曾造成举世闻名的“爱尔兰大饥荒”,至今仍然是马铃薯最严重的病害。提高抗晚疫病的水平是马铃薯育种的重要目标。马铃薯抗晚疫病育种是通过遗传操作来抵抗植物病害的最早的人类实践之一,但至今收效甚微,从野生种导入的抗病基因在田间很容易失效。马铃薯和致病疫霉菌的互作关系符合经典的“基因对基因”假说,只有马铃薯的抗病基因和致病疫霉菌的非毒力基因同时存在并表达时,抗病反应才能发生。致病疫霉菌是一种进化潜力非常高的病原,可以快速突变本身的非毒力基因,因而造成对应的马铃薯抗病基因的失效。要提高抗病基因的持久性,目前唯一的途径是同时释放多个抗病基因,人为造成田间抗病基因的多态性。马铃薯抗晚疫病基因的克隆取得了快速的发展,为通过转基因的手段创造田间抗晚疫病基因的多态性提供了基础。     

Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators

Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.     

The arms race between Magnaporthe oryzae and rice: Diversity and interaction of Avr and R genes

Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevolution of the host and pathogen, and provide the appropriate methods for preventing or controlling rice blast disease, researchers have focused on the evolution of virulence factors and resistance genes. Thus far, more than 30 rice blast resistance(R) genes and 12 avirulence(Avr) genes have been cloned. This review summarizes the cloned rice blast R genes, cloned Avr genes of M. oryzae and the interaction between them. This discussion also considers some of the major unanswered questions concerning this pathosystem and the opportunities for future investigations.     

植物三型信号分泌系统下的抗病基因防御机制

近年来随着对植物抗病机理及信号转导途径的深入研究和探索,以及通过分子生物学手段新的抗病基因和病原菌无毒基因不断被克隆,科研人员对于植物三型信号分泌系统中抗病(R)基因和无毒(Avr)基因的结构、功能,作用模式及作用机制具有更加深入的认识。深入研究病原菌—寄主之间的相互作用关系,为制定更为有效的植物病害防治措施提供了依据。该文通过对三型信号分泌系统中植物病原识别受体的组成部分,抗病基因的结构域及种类,抗病基因与无毒基因及相互作用的两种模式及具体机制的总结,从植物抗病基因角度探讨了三型信号分泌系统下植物的抗病机制并在此基础上进行了前景展望。     

Cladosporium Avr2 inhibits tomato Rcr3 protease required for Cf-2-dependent disease resistance

How plants recognize pathogens and activate defense is still mysterious. Direct interaction between pathogen avirulence (Avr) proteins and plant disease resistance proteins is the exception rather than the rule. During infection, Cladosporium fulvum secretes Avr2 protein into the apoplast of tomato leaves and, in the presence of the extracellular leucine-rich repeat receptor-like Cf-2 protein, triggers a hypersensitive response (HR) that also requires the extracellular tomato cysteine protease Rcr3. We show here that Avr2 binds and inhibits Rcr3 and propose that the Rcr3-Avr2 complex enables the Cf-2 protein to activate an HR.     

植物病害中的信号传导

植物由抗病基因介导的防卫过程存在一系列生理生化和分子生物学反应,这些反应从病原菌侵染点开始的超敏反应(HypersensitiveResponse,HR),并延伸到远处组织的系统抗性或获得性抗性(SystemicAcquiredResistance,SAR),受制于一种信号传导网络的调控。这个信号系统有抗病蛋白和病原菌非病毒性蛋白,在一种配体—受体的互作模式下激发,并由信号分子H2O2、NO和系统信号分子水杨酸(SA)、茉莉酸(JA)和乙烯(ET),通过关键调控基因传递和放大,最终诱导一系列防卫反应基因的表达和代谢的变化而产生抗性。植物防卫信号的产生类似于动物免疫系统因子的介导,并可由非寄主病原菌或诱导子诱发。这些信号途径所产生的广谱抗性为植物抗病基因工程的应用奠定了基础。     

The Trichoderma-plant interaction is mediated by avirulence proteins produced by this fungus

The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased development of root/aerial systems, in improved yields and in plant disease control. Other beneficial effects, such as the induction of plant systemic resistance, have also been described. To understand the mechanisms involved we are using different approaches, including the making…     

Cloning and functional analysis of the genes involved in signal transduction in tomato Cf-4-Avr4 pathosystem

Hypersensitive response (HR) is one of the most efficient and common resistance mechanisms in plants. Cloning signaling genes are very important to elucidate the resistance mechanisms. A gene in tomato homologous to several resistance proteins in plant was involved in HR and named as RGL (Resistance Gene Like). RGL protein was used as a bait to screen interacting protein(s) from tomato cDNA library through the yeast two-hybrid system. Two interacting proteins were found, which were called as RGLIP-1 and RGLIP-2 (RGL Interacting Protein), respectively. RGLIP-1 is a protein of 291 amino acids with significant homology with thylakoid lumen protein. RGLIP-2 is a protein of 248 amino acids with significant homology with transducin protein. Virus-Induced Gene Silencing (VIGS) of the two genes results in a partial and complete suppression of Avr4-induced HR, which indicates that both genes are involved in hypersensitive response. __________ Translated from Acta Horticulturae Sinica, 2006, 33(1): 52–57 [译自: 园艺学报]     

棉花黄萎病菌AD-cDNA文库的构建

以黄萎病菌(从邯208中分离到的致病性中等的和从中棉所8号中分离到的致病性弱的菌株)为材料,构建了AD-cDNA文库.该文库的转化效率为4×105cfu/μg,库容量为4.8×106克隆,插入片段的大小在750～1 500bp之间.筛选获得的候选蛋白测序结果表明含有TRRAP结构域(TRansformation/tRa...     

Activation of a phytopathogenic bacterial effector protein by a eukaryotic cyclophilin

Innate immunity in higher plants invokes a sophisticated surveillance system capable of recognizing bacterial effector proteins. In Arabidopsis, resistance to infection by strains of Pseudomonas syringae expressing the effector AvrRpt2 requires the plant resistance protein RPS2. AvrRpt2 was identified as a putative cysteine protease that results in the elimination of the Arabidopsis protein RIN4. RIN4 cleavage serves as a signal to activate RPS2-mediated resistance. AvrRpt2 is delivered into the plant cell, where it is activated by a eukaryotic factor that we identify as cyclophilin. This activation of AvrRpt2 is necessary for protease activity. Active AvrRpt2 can then directly cleave RIN4.     

Regulatory role of SGT1 in early R gene-mediated plant defenses

Animal SGT1 is a component of Skp1-Cullin-F-box protein (SCF) ubiquitin ligases that target regulatory proteins for degradation. Mutations in one (SGT1b) of two highly homologous Arabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss of SGT1b function in resistance is not compensated for by SGT1a. R genes differ in their requirements for SGT1b and a second resistance signaling gene, RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively to RPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.     

谷瘟病菌无毒基因型鉴定及分析

【目的】鉴定不同地区谷瘟病菌（Magnaporthe oryzae）所含无毒基因的类型,确定无毒基因在菌株中的分布及变异情况,为深入研究谷瘟病菌无毒基因变异机制提供参考。【方法】从中国北方谷子主产区不同区域内采集并分离76个谷瘟病菌的单孢菌株,提取其基因组DNA,根据目前已成功克隆的稻瘟病菌的7个无毒基因的核苷酸序列设计特异性引物,进行PCR扩增及电泳检测,并对部分菌株的无毒基因进行测序分析。【结果】在76个谷瘟病菌中,无毒基因ACE1、Avr-pita、Avr1-CO39和AvrPiz-t的扩增率为100％,无毒基因Avr-pik、Avr-pia和Avr-pii的扩增率分别为63.2%、42.1%和21.1%。在谷瘟病菌菌株P11和P34中,Avr1-CO39的扩增条带较预期片段大490 bp,测序结果发现菌株P11和P34中的Avr1-CO39基因序列完全一致,均在启动子区插入了490 bp核苷酸,该插入序列与non-LTR retrotransposon: Mg-SINE的相似度达99.16％。Avr-pita的测序结果发现,谷瘟病菌菌株中的Avr-pita基因序列变异较为丰富,其变异形式主要为单核苷酸的变异,包括单碱基的插入、缺失及多位点的SNP。Avr-pia的变异类型主要为整个无毒基因的缺失,经测序验证等位基因序列分为4种类型。Avr-pia-A与参考序列（AB498873.1）一致,包含10个菌株;Avr-pia-B包含20个菌株,在-116、-109和-16 bp处分别存在C/T、G/T和C/A变异,但与参考序列的CDS区序列相同;Avr-pia-C仅包含菌株P10,在+150 bp处存在T/G变异,但为同义突变;Avr-pia-D仅包含菌株P18,在+212 bp位点处存在C/T变异,导致该变异位点由编码苏氨酸突变为编码异亮氨酸。谷瘟病菌Avr-pii包含3种等位基因类型。Avr-pii-A型与参考序列（AB498874.1）一致,共包含14个菌株;Avr-pii-B型和Avr-pii-C型分别在+139和+64 bp处存在A/G变异,核苷酸的变异导致该位点由编码苏氨酸改为丙氨酸。Avr-pii-B型和Avr-pii-C型变异均为首次报道。单元型分析表明,AG2包含23个菌株,占供试菌株的30.2%,为优势单元型。【结论】明确了不同地区的谷瘟病菌中无毒基因ACE1、Avr-pita、Avr1-CO39和AvrPiz-t不存在地理来源的差异;而无毒基因Avr-pik、Avr-pia和Avr-pii在各地分布有差异。谷瘟病菌AG2单元型为优势单元型,其次是单元型AG1和AG5。     

Broad-spectrum mildew resistance in Arabidopsis thaliana mediated by RPW8

Plant disease resistance (R) genes control the recognition of specific pathogens and activate subsequent defense responses. We show that the Arabidopsis thaliana locus RESISTANCE TO POWDERY MILDEW8 (RPW8) contains two naturally polymorphic, dominant R genes, RPW8.1 and RPW8.2, which individually control resistance to a broad range of powdery mildew pathogens. Although the predicted RPW8.1 and RPW8.2 proteins are different from the previously characterized R proteins, they induce localized, salicylic acid-dependent defenses similar to those induced by R genes that control specific resistance. Apparently, broad-spectrum resistance mediated by RPW8 uses the same mechanisms as specific resistance.     

水稻抗瘟性遗传和抗性关联蛋白(英文)

对10个抗瘟性品种Tetep、砦糖、矮梅早3号、IR36、IR9782、IR58、水源300、红阳矮4号、矮脚白米仔和国际油占1号对4个中国小种ZB15、ZC3、ZC15和ZG1的抗性遗传进行了研究.结果表明这些品种的抗性分别由1个或2个显性重复抗性基因控制.共发现19个不同的抗性基因,其中一些是连锁的.经聚丙烯酰胺凝胶电泳和等电聚焦分析表明1至2个过氧化物酶同工酶带与抗性有关.在抗性系H7R中鉴定出5个抗性关联蛋白(RR-1至RR-5),等电点为4.2～5.5、分子量为17～54KD,可能由抗性基因编码.在接种后5天的感病系H7S中发现等电点为4.4～6.7,分子量为12～22KD的4个新蛋白质点(S-1至S-4).     

与马铃薯晚疫病菌无毒基因Avr1连锁的AFLP标记

 以具有和不具有无毒基因Avr1表现型的马铃薯晚疫病菌株80029 (AVR1) 和88133 (avr1)以及其杂交F1代50个菌株群体为试材，限制性内切酶PstI和HhaI为酶切组合，采用荧光AFLP技术和混合分组分析法(BSA)，通过256对引物筛选得到了5个与Avr1连锁的候选AFLP标记并进行了菌株个体验证，遗传分析表明5个标记中有2个标记与Avr1连锁且在F1代中共分离，无交换重组发生。