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.     

Cleavage of Arabidopsis PBS1 by a bacterial type III effector

Plant disease-resistance (R) proteins are thought to function as receptors for ligands produced directly or indirectly by pathogen avirulence (Avr) proteins. The biochemical functions of most Avr proteins are unknown, and the mechanisms by which they activate R proteins have not been determined. In Arabidopsis, resistance to Pseudomonas syringae strains expressing AvrPphB requires RPS5, a member of the class of R proteins that have a predicted nucleotide-binding site and leucine-rich repeats, and PBS1, a protein kinase. AvrPphB was found to proteolytically cleave PBS1, and this cleavage was required for RPS5-mediated resistance, which indicates that AvrPphB is detected indirectly via its enzymatic activity.     

Pre- and postinvasion defenses both contribute to nonhost resistance in Arabidopsis

Nonhost resistance describes the immunity of an entire plant species against nonadapted pathogen species. We report that Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi that in nature colonize grass and pea species. The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism. Postinvasion fungal growth is blocked by a separate resistance layer requiring the EDS1-PAD4-SAG101 signaling complex, which is known to function in basal and resistance (R) gene-triggered immunity. Concurrent impairment of pre- and postinvasion resistance renders Arabidopsis a host for both nonadapted fungi.     

Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease

Homologs of the Yersinia virulence effector YopJ are found in both plant and animal bacterial pathogens, as well as plant symbionts. These YopJ family members were shown to act as cysteine proteases. The catalytic triad of the protease was required for inhibition of the mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) signaling in animal cells and for induction of localized cell death in plants. The substrates for YopJ were shown to be highly conserved ubiquitin-like molecules, which are covalently added to numerous regulatory proteins. YopJ family members exert their pathogenic effect on cells by disrupting this posttranslational modification.     

烟草糖基转移酶基因SM-Ngt1在拟南芥中抗菌核病的初步研究

通过对转糖基转移酶基因SM-Ngt1的拟南芥T2代阳性植株接种油菜菌核病菌,鉴定其对菌核病的抗性,并应用实时荧光定量PCR(RT-PCR)确定植株中SM-Ngt1的表达水平。结果表明,拟南芥中SM-Ngt1基因对菌核病具有一定的抗性,且抗性的高低与体内SM-Ngt1的表达量具有明显的一致性。     

拟南芥在植物抗病性分子机制研究中的作用

植物对病原物的抗性是一个很复杂的生命现象。目前人们认识到植物的抗病过程有很多基因和一些相互关联的信号传导途径参与。人们对植物抗病的分子机制的了解,很多是来自以拟南芥作为材料所进行的大量研究。对植物抗病性的了解是一步步深入的,其进展与研究方法和技术的不断改革密不可分,其中实验材料的模式化使得研究更加集中、更具有通用性,所使用的资源更丰富,强有力地推进了研究的深入进行。在此,对拟南芥这个模式植物在植物抗病性的遗传和分子机制研究中的作用做了阐述。     

转NDR1基因烟草对赤星病和晚疫病的抗性增强

 NDR1(nonrace-specific disease resistance)基因在植物系统获得性抗性的信号传导途径中起着重要的作用,过量表达该基因的拟南芥对不同病原菌的抗性都有提高。利用PCR技术首次克隆了拟南芥Wassilewskija生态型的NDR1基因,与Columbia生态型相比,该基因共有7处碱基不同,引起编码氨基酸变化4处。构建了植物高效表达载体,利用农杆菌介导法转化烟草,经PCR和Southern鉴定,外源基因已整合到植物基因组中。随机挑选10个转基因株系进行抗病性分析,其中3个株系对赤星病和晚疫病的抗性明显增强。     

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.     

UV-B辐照下拟南芥突变体npq4、vtc2、ndr1叶绿素荧光特性研究

通过对野生型拟南芥及npq4（非光化学猝灭减弱）、vtc2（抗坏血酸生物合成途径受阻）、ndr1（NDR1基因突变导致系统获得性抗性上游信号途径中断）3种拟南芥突变体进行UV-B周期性处理,探究其在UV-B辐照下的叶绿素荧光特性并检测其叶绿素含量。结果表明：随着UV-B辐照辐射时间的延长,野生型和突变体的NPQ、Fv/Fm、ΦPSⅡ、qN、qP、叶绿素含量及叶绿素a/b均有所降低。npq4、vtc2两个突变体的NPQ、Fv/Fm、ΦPSⅡ、叶绿素a含量及叶绿素a/b值较野生型和ndr1下降幅度大而且差异显著,表现出更严重的伤害症状。据推测,这是因为非光化学猝灭途径及抗氧化物质在植物抵抗短期高强度UV-B辐照中起重要作用,而系统获得性抗性途径不能被短期的UV-B辐照所启动,因而在植物抵抗UV-B辐照中作用较弱。     

ATAF2正调控拟南芥对橡胶树白粉菌的抗病性

橡胶树白粉菌(Oidium heveae)侵染拟南芥野生型Col-0,激发拟南芥的抗病反应。该抗病反应依赖于EDS1（enhanced disease susceptibility 1）,EDS1在TIR-NB-LRR（Toll-Interleukin1 Receptor-nucleotide binding-leucine-rich repeat）类抗性基因介导的抗病信号通路中发挥非常重要的作用,但目前还不清楚具体的EDS1上、下游作用元件是什么。为了进一步研究橡胶树白粉菌在拟南芥上激发的抗疾信号通路,本实验室对接种橡胶白粉菌oidium heveae HN1106的拟南芥野生型Col-0进行了RNA-Seq数据分析,结果表明,拟南芥Col-0在接种橡胶树白粉菌4 d后,NAC家族 (ATAF1,2 and CUC2, NAM)转录因子ATAF2基因上调表达20倍,并且正调控拟南芥对橡胶树白粉菌的抗病性。另外,笔者通过体外蛋白质pull-down试验和体内原生质体免疫共沉淀试验,发现ATAF2可以直接与EDS1发生相互作用,这为将来进一步阐明EDS1抗病信号通路奠定了很好的基础。     

拟南芥不同ROP蛋白对病原细菌增殖的影响

ROP蛋白是植物特有的一类小G蛋白,在植物信号传导途径中起重要作用.拟南芥共编码11种ROP蛋白,为明确ROP蛋白在拟南芥抗病反应中的作用,将病原细菌Pseudomonas syringaepv.tomatoDC3000接种于各AtROP的激活和失活突变体后,观察其增殖情况.结果表明,AtROP2和AtROP11抑制病原菌的增殖,而AtROP10则促进病原菌的增殖,其他At-ROP对Pst.DC3000的增殖没有影响.     

Independently evolved virulence effectors converge onto hubs in a plant immune system network

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.     

玉米CCCH基因家族鉴定及分析

CCCH锌指蛋白与植物生物和非生物胁迫、生长发育及抗病性密切相关。以玉米为材料,应用在线工具与公共数据库,共鉴定出63个CCCH基因,命名为Zm CCCHs,进一步对玉米与水稻、拟南芥、小立碗藓的CCCH基因进行生物信息学分析。结果表明:玉米的CCCH家族基因在染色体上呈不均匀分布,蛋白质氨基酸序列长度在110～962 aa之间。Zm CCCHs基因具有相对保守的结构,即包含CCCH结构域、WD40结构域、C2H2结构域、C3HC4结构域,CCCH蛋白结构中含有2个α螺旋和4个β折叠结构域。进化树分析表明玉米CCCH蛋白与水稻CCCH蛋白序列具有较高的同源性。上述结果为深入研究该基因家族的功能与表达调控提供了参考信息。     

Transgenic Arabidopsis thaliana expressing a wheat oxalate oxidase exhibits hydrogen peroxide related defense response

Oxalic acid(OA) is considered as an important pathogenetic factor of some destructive diseases caused by some fungal pathogens such as Sclerotinia sclerotiorum. Oxalate degradation is important for plant health, and plants that contain oxalate oxidase(OXO) enzymes could breakdown oxalate into CO_2 and H_2O_2, which subsequently evokes defense responses. However, some species, such as Arabidopsis thaliana, have no oxalate oxidase activity identified to date. The present study aims to develop transgenic Arabidopsis expressing a wheat oxalate oxidase, to test for the response to OA exposure and fungal infection by S. sclerotiorum. The results showed that the transgenic Arabidopsis lines that expressed the wheat OXO exhibited enhanced resistance to OA exposure and S. sclerotiorum infection in the tolerance assays. In the same manner, it could convert OA to CO_2 and H_2O_2 to a higher extent than the wild-type. Intensive osmotic adjustments were also detected in the transgenic Arabidopsis lines. The higher level of produced H_2O_2 subsequently induced an elevated activity of antioxidant enzymes including superoxide dismutase(SOD) and peroxidase(POD) in the transgenic Arabidopsis plants. The present study indicated that the expression of a gene encoding wheat OXO could induce intensive osmotic adjustments and hydrogen peroxide related defense response, and subsequently increased tolerance to S. sclerotiorum in transgenic A. thaliana.     

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.     

转录因子AtWRKY28在拟南芥与甘蓝链格孢菌(Alternaria brassicicola)亲和性互作中的功能分析

WRKY是植物特有的一类转录因子,是一个超级大家族,与植物多种逆境胁迫应答密切相关,在植物防御病原菌的侵染中发挥十分重要的作用。拟南芥转录因子WRKY28属于WRKY家族的成员,包括一个WRKYGQK核心序列和一个Cys2His2锌指型结构。为了鉴定拟南芥WRKY28的功能,构建了反义抑制表达株系和转基因过表达株系并进行抗病性鉴定。研究表明,反义抑制表达株系降低了对腐生型真菌甘蓝链格孢菌(Alternaria brassicicola)的抗性,而转基因过表达株系提高了对甘蓝链格孢菌的抗性。由此可见,转录因子WRKY28参与了拟南芥对甘蓝链格孢菌的免疫应答,是其抗病反应中的一个正调控因子。     

过表达胡杨PeRIN4基因拟南芥提高质膜H+-ATPase活性和耐盐性

本文克隆了RIN4(RPM1-interacting protein 4)在胡杨中的同源基因PeRIN4,并在拟南芥中进行过表达,通过研究转基因株系的耐盐表型、质膜H+-ATPsae活性及H+ 、Na+、K+等的动态离子流,揭示了PeRIN4基因在植物响应和适应盐胁迫环境中的作用。利用定位载体pGreen0029-PeRIN4-GFP瞬时转化拟南芥叶肉细胞原生质体的方法,对胡杨PeRIN4蛋白进行亚细胞定位,发现该蛋白定位在细胞的胞质中。耐盐表型实验结果显示,在100 mmol/L NaCl处理下,拟南芥PeRIN4过表达株系(OE1和OE8)的生存率和根长均明显高于野生型(WT)和转空载体拟南芥(VC),说明PeRIN4基因能够提高拟南芥的耐盐性。与WT和VC相比,拟南芥PeRIN4过表达株系质膜H+-ATPsae的活性较高。动态离子流数据显示,在盐胁迫下,PeRIN4过表达株系外排H+和Na+ 离子的能力强于野生型和转空载体拟南芥,然而K+的外流却弱于WT和VC。因此,PeRIN4蛋白具有调节质膜H+-ATPsae活性的功能。拟南芥质膜H+-ATPsae活性的提高主要有两方面的作用:一是可以增强H+泵的质子动力势,驱动Na+/H+逆向转运蛋白,提高Na+外排的能力;二是抑制质膜的去极化,减少K+离子通过去极化激活的外向型K+通道(DA-KORCs)和非选择性阳离子通道(DA-NSCCs)外流,维持了K+/Na+平衡,从而提高PeRIN4转基因拟南芥的耐盐性。      

沙冬青热胁迫相关蛋白基因AmHsa32超表达提高大肠杆菌的抗热性

热胁迫相关蛋白32（Heat stress associated protein 32,Hsa32）与植物的抗逆性密切相关。本文将从沙冬青低温干旱EST库中获得的热胁迫相关蛋白基因AmHsa32进行了非生物胁迫下的表达分析与转化大肠杆菌研究。生物信息学分析表明,AmHsa32的编码区全长858 bp,编码286个氨基酸,预测分子量约32 kD,与拟南芥HSA32亲缘关系较近。qRT PCR分析显示,AmHsa32在沙冬青幼苗中受热胁迫诱导后表达量迅速提高,1 h后达到对照的25倍;对其他非生物胁迫如低温、干旱及盐均有响应,表达量有不同程度的增加,说明AmHsa32对沙冬青非生物胁迫抗性的提高可能发挥着重要作用。将AmHsa32基因转化大肠杆菌,同野生菌相比,热胁迫（50 ℃）条件下,转基因大肠杆菌存活率明显提高。本研究结果表明,AmHsa32可用于通过转基因技术提高热敏感植物抗热性的研究。      

人核糖体40S亚基RPS11蛋白的原核表达和纯化

RPS11是核糖体小亚基40S的组成部分,属于核糖体蛋白S17p家族,由RPS11基因所编码,主要存在于真核生物中。本研究通过PCR扩增RPS11基因,构建pET43a-RPS11和pGEX-4T-1-RPS11原核表达载体。将重组质粒转入E.coli DH5α,序列测定正确后,将其转入E.coli BL21(DE3),加入IPTG诱导表达。表达蛋白经SDS-PAGE分析后,利用亲和层析法纯化蛋白。结果成功克隆RPS11基因,构建了原核表达载体pET43a-RPS11和pGEX-4T-1-RPS11,转化宿主菌E.coli BL21(DE3)中进行了表达。SDS-PAGE分析证实表达目的蛋白正确。通过Ni-TNA和GSH-Sepharose层析柱获得纯化蛋白,为进一步研究该蛋白的生物学功能奠定了基础。