Identif ication of a hypersensitive response core peptide of HrpZ and its role in increasing grape downy mildew resistance

Harpins play a key role in inducing disease resistance in crops,and identifying their core functional regions and establishing a system for their efficient expression would be very valuable.In this study,large amounts of soluble fusion proteins of harpin HrpZ and its subpeptides were obtained via the optimized induction conditions(28℃ with 0.5 mmol·L^(-1) IPTG for 6 h)in Escherichia coli BL21(DE3).Hypersensitive response(HR)assays demonstrated that the C-terminal 66 aa of HrpZ(HrpZ_C_2_2)elicited a strong HR in tobacco(Nicotiana benthamiana)and grape(Flame Seedless)leaves.Additionally,treatment with HrpZ,and particularly HrpZ_C_2_2,significantly reduced the disease incidence and severity index of field vine leaves and those inoculated with downy mildew.The determination of the physiological parameters indicated that HrpZ,and especially HrpZ_C_2_2,improved the photosynthesis-and chlorophyll fluorescence-related parameters,enhanced the activity of defense-related enzymes,including SOD,POD,CAT and PAL,and increased the H_(2)O_(2) level.Collectively,we efficiently expressed a core peptide of HrpZ and elucidated its strong ability to elicit a HR and resistance to downy mildew.This research provides insight into understanding the structure and function of HrpZ and will advance the application of HrpZ_C_2_2 to increase the resistance of grapevine to downy mildew.

A Selective‑Response Hypersensitive Bio‑Inspired Strain Sensor Enabled by Hysteresis Effect and Parallel Through‑Slits Structures

Flexible strain sensors are promising in sensing minuscule mechanical signals,and thereby widely used in various advanced fields.However,the effective integration of hypersensitivity and highly selective response into one flexible strain sensor remains a huge challenge.Herein,inspired by the hysteresis strategy of the scorpion slit receptor,a bio-inspired flexible strain sensor(BFSS)with parallel through-slit arrays is designed and fabricated.Specifically,BFSS consists of conductive monolayer graphene and viscoelastic styrene–isoprene–styrene block copolymer.Under the synergistic effect of the bio-inspired slit structures and flexible viscoelastic materials,BFSS can achieve both hypersensitivity and highly selective frequency response.Remarkably,the BFSS exhibits a high gage factor of 657.36,and a precise identification of vibration frequencies at a resolution of 0.2 Hz through undergoing different morphological changes to high-frequency vibration and low-frequency vibration.Moreover,the BFSS possesses a wide frequency detection range(103 Hz)and stable durability(1000 cycles).It can sense and recognize vibration signals with different characteristics,including the frequency,amplitude,and waveform.This work,which turns the hysteresis effect into a"treasure,"can provide new design ideas for sensors for potential applications including human–computer interaction and health monitoring of mechanical equipment.

Exchangeability of Two hrp Gene Fragments from Xanthomonas oryzae pv.oryzae and pv.oryzicola for Hypersensitive Response on Tobacco and Pathogenicity on Rice

hrp mutants were produced from strain JXOIII of Xanthomonas oryzae pv. oryzae (Xoo) and strain RS105 of X.o. pv. oryzicola (Xooc), respectively, by using diethyl sulfate (DES) as a mutagenic che mical. All the hrp mutants lost their pathogenicity on a susceptible host plant, rice (Shanyou63), and elicitation of the hypersensitive response (HR) on a nonhost plant, tobacco (NC89). Extracellular enzyme (amy lase, pectate lyase, proteinase, cellulase and lipase) activities of all the hrp mutants were similar to those of the corresponding wild type strains. The response of tobacco to cell sonicated integrations of the wild type strains and the hrp mutants demonstrated that there existed an HR eliciting substance which was heat stable and sensitive to protease. No HR appeared on tobacco after infiltration of the lipopolysaccharide (LPS) of both the wild strains and hrp mutants into tobacco leaves. The ability of the Xooc hrp mutants to induce HR on tobacco and cause streak disease on rice was restored by complementation with pUHRX245 from JXOIII genomic DNA library and by pUHRS138 from RS105 genomic DNA library, respectively. Subcloning of a 38.6 kb hrp fragment insert in pUHRX245 and a 39.3 kb insert in pUHRS138 revealed that a 3.3 kb Sac Ⅰ fragment from pUHRX245 and a 4.5 kb Bam HⅠ Kpn Ⅰ fragment from pUHRS138 were the minimal functional portions required for restoration of the ability of Xooc hrp mutants to induce HR on tobacco and cause disease on rice. The disease symptom caused by the conjugant (M1005 plus 3.3 kb) on rice was similar to that caused by the wild type of Xooc. It suggests that the two fragments contain the same hrp gene(s) and are responsible reciprocally for HR induction on tobacco and pathogenicity on rice.

Alpha-picolinic acid,a fungal toxin and mammal apoptosis-inducing agent,elicits hypersensitive-like response and enhances disease resistance in rice

Alpha-picolinic acid (PA),a metabolite of tryptophan and an inducer of apoptosis in the animal cell,has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here,we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS),rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride,indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence,it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice,which also provides a simple eliciting tool for studying apoptosis in the plant cell.

Induction of Hypersensitive Response and Nonhost Resistance by a Cladosporium fulvum Elicitor CfHNNI1 is Dose-Dependent and Negatively Regulated by Salicylic Acid

Nonhost resistance is a phenomenon that enables plants to protect themselves against the majority of potential pathogens, and thus has a great potential for application in plant protection. We recently found that CfHNNI1 （for Cladosporium fulvum host and nonhost plant necrosis inducer 1） is an inducer of plant hypersensitive response （HR） and nonhost resistance. In this study, its functional mechanism was analyzed. CfHNN11 was a single copy gene in C. fulvum genome. The functional ORF of the CfHNN11 cDNA was ATG3-TAG780, which showed homology with genes encoding bZIP transcription factors. The functional ORF included in frame an inner one ATG273-TAG780, which was sufficient to induce HR in plants. CfIINN11 induced plant HR in a dose-dependent manner. CfHNNIl-induced necrosis in NahG transgenic tomato plants was significantly stronger than that in their wild type controls. However, the necrosis in Nr and defl tomato mutants was similar to that in their corresponding wild type plants. These data demonstrate that induction of HR and nonhost resistance by CfHNNI1 is negatively regulated by salicylic acid signalling pathway but independent of ethylene and jasmonic acid signalling pathways.

OsbZIP53 Negatively Regulates Immunity Response by Involving in Reactive Oxygen Species and Salicylic Acid Metabolism in Rice

The basic region/leucine zipper(bZIP)transcription factors play important roles in plant development and responses to abiotic and biotic stresses.OsbZIP53 regulates resistance to Magnaporthe oryzae in rice by analyzing APIP5-RNAi transgenic plants.To further investigate the biological functions of OsbZIP53,we generated osbzip53 mutants using CRISPR/Cas9 editing and also constructed OsbZIP53 over-expression transgenic plants.Comprehensive analysis of phenotypical,physiological,and transcriptional data showed that knocking-out OsbZIP53 not only improved disease resistance by inducing a hypersensitivity response in plants,but also regulated the immune response through the salicylic acid pathway.Specifically,disrupting OsbZIP53 increased H2O2 accumulation by promoting reactive oxygen species generation through up-regulation of several respiratory burst oxidase homologs(Osrboh genes)and weakened H2O2 degradation by directly targeting OsMYBS1.In addition,the growth of osbzip53 mutants was seriously impaired,while OsbZIP53 over-expression lines displayed a similar phenotype to the wild type,suggesting that OsbZIP53 has a balancing effect on rice immune response and growth.

小麦与叶锈菌互作过程中的H_2O_2与HR

以小麦品种洛夫林10为材料,与叶锈菌生理小种165和260分别组成亲和组合和不亲和组合,以荧光探针DCFHDA标记H2O2的变化,并借助荧光显微镜原位地直观显现了叶锈菌侵染过程中小麦叶片中H2O2的动态变化。结果表明,亲和组合中H2O2爆发表现为单峰曲线,峰值出现在接种后12h;不亲和组合中表现为双峰曲线,峰值分别出现在接种后12h和20h,第二个峰比第一个高约一倍,且不亲和组合中H2O2爆发的强度远远大于亲和组合。另外,通过药理学试验,采用活体注射法对小麦叶片在接种叶锈菌小种260之前分别预注射抗氧化药物AsA、DTT以及NADPH氧化酶抑制剂DPI和咪唑,观察这些药物对不亲和组合中寄主叶片产生的H2O2和HR的影响。结果表明,抗氧化药物和NADPH氧化酶抑制剂均使寄主叶片中H2O2爆发的强度降低,表现在两个H2O2峰均受到抑制,其中第二个峰受抑制的程度更明显,同时寄主细胞发生HR的面积减小。

Whirly转录因子对非寄主菌诱导水稻HR反应的负调控作用

用RNA干扰技术研究了Whirly转录因子对水稻非寄主抗性反应HR的调控作用。PCR扩增编码Whirly转录因子的Oswhirly基因片段,分别以正向、反向顺序连接到载体pTCK303的两个多克隆位点中,构建Oswhirly基因沉默的RNA干扰载体pTCK303-Oswhy,经农杆菌EHA105菌株转化水稻细胞,结果发现转基因水稻细胞受非寄主菌诱导后,HR反应明显,细胞死亡率显著高于野生型及空载体转化水稻细胞。Oswhirly基因沉默导致HR反应增强,初步揭示Whirly转录因子可能是调控水稻HR反应的负调控因子。

植物病原细菌的hrp基因

hrp基因存在于4类革兰氏阴性植物病原细菌中,决定病原细菌对寄主植物致病性和诱导非寄主及抗病植物过敏性反应。从hrp基因簇,hrp基因avr基因的关系,hrp基因的编码产物harpin蛋白,hrp基因调控与功能以及hrp基因参与植物-细菌互作等几个方面,较为详细地阐述了当前植物病原细菌hrp基因的研究状况,并分析了hrp基因未来研究的趋势。

与植物超敏反应(HR)相关的细胞编程性死亡

在植物抗病的超敏反应(hypersensitiveresponse,HR)中发生的细胞死亡被证明为一种细胞编程性死亡(programmedcelldeath,PCD),与HR相关的PCD发生一般由R基因 /avr基因的不相容互作引起。离子流(Ca2+ )、活性氧(ROS)、水杨酸(SA)、茉莉酸(JA)等均是PCD发生过程中的重要信号分子;类半胱氨酸蛋白水解酶(caspases likeproteases,CLPs)、线粒体参与PCD过程;液泡在植物PCD中占重要的地位。

Ⅲ型分泌装置蛋白HrcJ决定条斑病菌在植物上的过敏反应和致病性的研究

【目的】水稻条斑病菌(Xanthomonas oryzae pv.oryzicola,Xooc)的hrp(hypersensitive response and pathogenicity)基因编码形成的Ⅲ型分泌系统(typeⅢsecretion system,T3SS),将致病性效应分子注入水稻细胞中,但Xooc的hrcJ基因在致病性中的作用以及其如何参与形成T3SS分泌装置,并不明确。【方法】本研究根据标记交换原理对Xooc的hrcJ基因进行了敲除。【结果】发现hrcJ突变体丧失在水稻上的致病性和在烟草上的HR激发能力。酵母双杂交显示,HrcJ蛋白N端脂蛋白结构域可与HrcC互作,HrcJ蛋白C端跨膜结构域可与HrcV互作,提示HrcJ蛋白联接于T3SS装置的内膜和外膜之间。功能互补结果显示,缺失脂蛋白结构域和跨膜结构域的hrcJ基因均不能恢复hrcJ突变体在烟草上的HR激发能力和在水稻上的致病性。RT-PCR结果显示,hrcJ的表达受hrpX基因调控,hrcJ基因突变后不影响效应分子hpa1的表达。【结论】hrcJ基因是水稻条斑病菌致病性和非寄主上激发HR的关键因子,其基因产物参与了T3SS装置的形成。

水稻白叶枯病菌hrp调节基因hrpXoo的克隆与序列分析

用化学方法诱变水稻白叶枯病菌PXO99A 菌株 ,获得 6株hrp-突变体 ,此突变体除丧失在非寄主烟草上激发过敏反应和在感病寄主水稻上的致病能力外 ,有些缺乏激发烟草产生HR的信号物质 ,有些在胞内存在此信号物质 ,而不能泌至胞外。来自Xanthomonasoryzaepv .oryzaeJXOIII粘粒基因文库的hrp基因克隆pUHRX2 4 5 ,所携hrp基因片段大小为 36 .8kb。系列亚克隆 36 .8kbhrp基因片段及各亚克隆对hrp-突变体功能互补作用的结果显示 ,3.3kbSacI片段为最小酶切功能片段。序列测定和分析结果表明 ,3.3kbhrp片段含hrpX oo基因和含与热激蛋白 90家族有关的 2个开放阅读框hspORF1和hspORF2。HspORF1在蛋白质数据库中未发现序列蛋白。HspORF2与Hsp90 Xo的同源性达 99%。hrpXoo与黄单胞菌中已报道的hrpX基因有很高的同源性(90 %以上 )。在黄单胞菌中高度保守的编码α 螺旋 转 α 螺旋结构的 6 0 bp核苷酸序列 ,在交叉功能互补时是必需的。不含此结构的hrpXoo (1.1kb)片段 ,可使JXOIII的hrp-突变体在水稻上具致病性和在非寄主烟草上激发产生HR ,但不能使来自PXO99A 和RS10 5的hrp-突变体恢复在烟草上激发产生HR的功能。黄单胞菌中已知HrpX的同列比较显示 ,X .oryzae和X .campestris种间在 88、196和 2 4

Ecc36菌株hrpN基因的克隆、表达及HrpN_(EccPR)蛋白的生物学活性

为了揭示hrp基因表达的Harpins蛋白能够广泛地诱导植物对植物病虫害防卫反应的分子机制,通过琼脂固体平板法,发现胡萝卜软腐欧文氏菌Ecc36菌株具有很高的胞外酶分泌活性,该菌接种非寄主植物烟草,能够引起过敏反应(HR),结果表明,Ecc36携带hrp基因。用地高辛标记的hrp N基因作探针,通过Southern杂交证明了Ecc36菌株中含有hrp N基因,命名为hrp NEcc PR基因;核苷酸序列分析表明,hrp NEcc PR基因的开放阅读框ORF为1 254 bp,编码的Harpin蛋白(命名为HrpN_(EccPR)蛋白)由417个氨基酸残基组成,其分子量为45.27 ku,等电点为5.73,与其他几种软腐欧文氏菌Harpin蛋白有较高的同源性。此外,将hrp NEcc PR基因克隆到表达载体p ET28a(+)上,将构建的hrp NEcc PR基因表达载体(p ET30a-Ecc PR)转化到大肠杆菌BL21(DE3)中,经IPTG(异丙基硫代-β-D半乳糖苷)诱导HrpN_(EccPR)蛋白表达,纯化后的HrpN_(EccPR)能诱导烟草发生过敏反应,表明HrpN_(EccPR)蛋白具有激发植物免疫反应的生物活性,可为进一步研究HrpN_(EccPR)蛋白诱导植物防卫反应的机制奠定基础。

胡萝卜软腐欧文氏菌CSSY002菌株hrpN基因的克隆、鉴定及其表达产物Harpin蛋白的活性分析

从感染软腐病的胡萝b组织中分离出胡萝卜软腐欧文氏菌胡萝b亚种（ Erwinia carotovora subsp, carotovora）CSSY002菌株．用该菌株接种非寄主植物烟草不能引起过敏反应．构建CSSY002菌株的基因组文库，应用α-P^32标记的hrpN基因作探针，通过菌落原位杂交技术从该文库中筛选到1．5～2．0 kb的hrpN-like阳性克隆，进一步将该片段亚克隆到pBluescript—II SK（＋）载体中，进行核苷酸序列测定．结果表明，hrpN-like基因（即hrpNCSSY002基因）的开放阅读框（ORF）为1071bp，其核苷酸序列已在Genbank注册，登录号为bankit AY999002．hrpN-like基因的编码产物Harpin—like蛋白（即HarpinCSSY002蛋白）由356个氨基酸残基组成，分子量为36．64×10^3，等电点pI5．9．将hrpN—like基因构建到表达质粒pET-28a（＋）的17强启动子下，并转化到大肠杆菌工程菌JM109（DE3）细胞中，经WTG（异丙基硫代-β-D半乳糖苷）诱导获得Harpin-like蛋白的高效表达，并通过接种烟草叶片的过敏反应检测Harpin—like蛋白的生物学活性．

hrcQ基因决定水稻条斑病菌在非寄主烟草上的过敏性反应和在寄主水稻上的致病性

水稻条斑病菌(Xanthomonas oryzaepv.oryzicola)hrcQ基因在动植物病原细菌中高度保守。不同植物病原细菌的HrcQ蛋白因在N端有变化,推测可能与分泌的效应分子特异性有关。但水稻条斑病菌HrcQ蛋白对Ⅲ型分泌系统(typeⅢsecretion system,T3SS)的形成以及对效应分子的分泌性影响还不清楚。为弄清HrcQ蛋白在此方面的作用,利用同源重组方式获得了水稻条斑病菌hrcQ基因的敲除突变体,该突变体丧失了在烟草上激发过敏性反应的能力和在水稻上的致病性。hrcQ基因与水稻细胞互作时受诱导表达。蛋白质-蛋白质互作结果显示,HrcQ可分别与Hpa1、HrcN、HrpB5和HrpB2互作。分泌性检测发现,HrcQ不通过T3SS分泌至胞外。hrcQ基因突变,影响Hpa1和HrpB2蛋白分泌。这些结果表明,HrcQ蛋白是形成Ⅲ型分泌系统的基本组分,并通过帮助Hpa1和HrpB2等T3SS效应因子的分泌,从而影响病菌在非寄主上的过敏性反应和在水稻上的致病性。这为进一步分析水稻黄单胞菌T3SS的形成和分泌机制奠定了基础。

EcbCSL101菌株hrpN基因的克隆、表达及HarpinEcbCSL101蛋白的生物学活性

胡萝卜软腐欧文氏菌甜菜亚种(Erwinia carotovora subsp.betavasculorum)EcbCSL101菌株具有很强胞外酶分泌活性,接种非寄主植物烟草引起过敏反应。Southern blotting结果表明EcbCSL101菌株中含有hrpN基因。PCR扩增含EcbCSL101完整开放阅读框的DNA片段并克隆到表达载体pET28a(+)中。核苷酸序列分析表明,EcbCSL101菌株的hrpN基因的ORF为1113bp,编码36.65 kD HarpinEcbCSL101蛋白(GenBank,DQ355519),与其它几种软腐欧文氏菌Harpin蛋白有较高的同源性。将含有hrpNEcbCSL101基因的重组质粒转化到大肠杆菌JM109(DE3)中进行表达,纯化后的HarpinEcbCSL101能诱导烟草发生过敏反应。

hrpZ基因在大肠杆菌中的高效表达与活性检测

PCR扩增hrpZ基因片段，克隆到pGEM-T载体中，经EmR Ⅰ／Xho Ⅰ酶切、连接将hrpZ基因插入大肠杆菌表达载体pET32b(+)硫氧还蛋白下游，构建重组表达质粒pET-hrpZ，再将其转化至E．coli BL21(DE3)中，经筛选得到阳性克隆子，IPTG诱导表达HrpZ蛋白。SDS-PAGE显示，目的蛋白在重组菌株中得到了可溶性高效表达。该重组蛋白分子量为55．8 kD，与理论值大小相符。采用叶片穿刺法，融合蛋白具有诱导烟草过敏反应的生物功能。

丁香假单胞大豆致病变种不同HrpZ蛋白差异区域功能研究

[目的]丁香假单胞大豆致病变种(Pseudomonas syringae pv.glycinea)A1和S1菌株编码产生的Hrp Z蛋白差异序列主要集中在C端的3个区域,并且这2个蛋白诱导非寄主HR的能力也存在差异。本研究将探究Hrp Z蛋白这3个差异区域在烟草上诱导HR(hypersensitive response)和抑制烟草花叶病毒(TMV)中所起的作用。[方法]采用常规PCR及重叠PCR方法将这2个hrp Z基因的3个差异区域分别进行互换,构建了相应的重组表达载体,表达并纯化得到了6个重组蛋白,相对分子质量均在41×103左右。并检测了其诱导HR活性、诱导植物抗病性。[结果]检测结果表明:重组蛋白Hrp ZS(S2→A2)和Hrp ZA(A3→S3)诱导HR的能力相比亲本都有所增强,6个重组蛋白诱导烟草抗TMV的活性都明显强于亲本,其中,Hrp ZA(A3→S3)和Hrp ZS(S3→A3)的活性最强。[结论]此试验表明Hrp Z蛋白的这些差异区域(第7、8、9个α-螺旋)是过敏反应能力的主要调控区域,同时C-端第8、9个α-螺旋区域与诱导植物抗病性也显著相关。本研究为改造Hrp Z类harpin蛋白激发子,提高其诱导抗性及功能域研究提供了理论依据。

水稻白叶枯病菌hrcU基因缺失突变体构建及功能研究

通过基因敲除方式,构建了水稻白叶枯病菌hrcU基因的缺失突变体。突变体丧失了在水稻上的致病性和在非寄主植物上的过敏反应。携带hrcU基因的黏粒完全互补了hrcU突变菌株的表型,互补菌在水稻组织中的生长能力也与野生型菌株相当。免疫杂交结果显示,hrcU基因突变后,水稻白叶枯病菌的Harpin蛋白Hpa1不能从细胞内分泌到胞外。这些结果表明,水稻白叶枯病菌hrcU基因的主要功能是形成Ⅲ型分泌系统,将效应分子分泌到病原细菌胞外,从而决定了在非寄主上的过敏反应和在水稻上的致病性。

Xoryp_08180 of Xanthomonas oryzae pv.oryzicola,Encoding a Hypothetical Protein,is Regulated by HrpG and HrpX and Required for Full Virulence in Rice

Xanthomonas oryzae pv.oryzicola（Xoc） causes a destructive bacterial leaf streak disease in rice.Some of the gene products annotated as hypothetical proteins in the genome of Xoc may contribute to its virulence in rice.A mutant,Mxoc1679,screened from our previous Tn5-tagged mutant library for Xoc strain RS105,showed reduced virulence in rice.In this mutant,a gene named as Xoryp_08180 was disrupted by Tn5 insertion.Xoryp_08180 encodes a 1 306-aa hypothetical protein which is highly conserved in Xanthomonas spp.Non-polar mutation of Xoryp_08180 in RS105 strain led to a significant reduction in bacterial virulence and growth in rice,a delayed hypersensitive response（HR） in non-host tobacco,and a decrease in extracellular protease activity.The deficiencies above were restored to wild-type level in the complementary strain by expressing Xoryp_08180 in trans.In addition,the expression of Xoryp_08180 was repressed in hrpG and hrpX mutants in planta but not in a nutrient-rich condition.These results suggested that Xoryp_08180 is a virulence factor required for extracellular protease production,HR induction and full virulence of Xoc.