imple Bioassay for PAMP-Triggered Immunity in Rice Seedlings Based on Lateral Root Growth Inhibition

Pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)is an essential layer of plant disease resistance.Robust bioassays for PTI are pre-required to dissect its molecular mechanism.In this study,we established that lateral root growth inhibition as a simple and robust measurement of PTI in rice seedlings.Specifically,flg22,a well-characterized PAMP from bacterial flagellin,was used to induce PTI in rice seedlings.While flg22 treatment induced PR gene expression and mitogen-activated protein kinase activation in the roots of rice seedlings to support the PTI triggered,this treatment substantially repressed lateral root growth,but it did not alter primary root growth.Moreover,treatments with chitin(i.e.,a fungal PAMP)and oligogalacturonides(i.e.,classical damage-associated molecular pattern)clearly inhibited the lateral root growth,although a priming step involving ulvan was required for the chitin treatment.The bioassay developed was applicable to various rice cultivars and wild species.Thus,lateral root growth inhibition represents a simple and reliable assay for studying PTI in rice plants.

Tomato SlPti5 plays a regulative role in the plant immune response against Botrytis cinerea through modulation of ROS system and hormone pathways

While SlPti5 has been shown to play a crucial role in the regulation of antagonistic genes in Solanum lycopersicum and Arabidopsis against pathogen infection,there have been no comprehensive studies on the effects of SlPti5 on the regulatory response mechanism of reactive oxygen species(ROS) system and hormone pathways during growth and disease resistance of tomato plants.Here,we investigated the function of SlPti5 in the defense response of tomato against Botrytis cinerea utilizing a virus-induced gene silencing(VIGS)-based system.Expression profile analysis showed that SlPti5 was significantly induced upon B.cinerea infection,with high expression levels in the leaves and fruit of tomato.VIGS-based silencing of SlPti5 inhibited early vegetative growth,increased the plant’s susceptibility to infection,promoted the development of ROS,affected the expression of genes involved in the ROS scavenging system,and attenuated the expression of genes associated with pathogenesis and the ethylene/jasmonic acid signaling pathways.In sum,our data demonstrated that SlPti5 stimulates the immune response of tomato plant to Botrytis cinerea infection by involving the ethylene(ET)-and jasmonic acid(JA)-mediated pathways and modulating the expression of some key pathogenesis-related(PR) genes.

植物PTI天然免疫信号转导研究进展

病害是危害农业生产的重要因素之一,植物对于病原菌的抗性依赖于植物的天然免疫(plant innate immunity)系统。因此,对于植物天然免疫的研究将为农作物抗病育种提供重要理论基础。植物天然免疫系统包含彼此相互关联的两个层面,即PTI(PAMP-triggered immunity)和ETI(effector-triggered immunity)。近年来,国内外对于PTI的研究取得了一系列重要进展。PTI是由病原物相关分子模式PAMP(pathogen-associated molecular pattern)所诱发的植物免疫反应。PAMP被位于植物细胞表面的受体识别后,将免疫信号通过胞质类受体激酶BIK1(Botrytis-induced kinase 1)、MAPK级联、CDPK(calcium-dependent protein kinase)等向下游传递,诱导活性氧的爆发、气孔的关闭、免疫基因的表达等,从而抑制病原微生物的生长。免疫信号在传递过程中会在多个层次上被精细调控,以保证合适的反应强度和持续时间。本文从植物免疫受体FLS2及其他免疫受体的发现、免疫信号转导组分的发现以及其生物学功能、参与天然免疫的转录因子、植物免疫反应的负调控及植物天然免疫在抗病育种中的应用等方面综述了近年来植物PTI天然免疫的分子机理和信号转导方面的研究进展。并对该领域的发展提出展望,我们认为农作物天然免疫信号转导和作物与致病真菌互作系统的研究将会是未来研究的重点和主要方向,天然免疫的重要理论与基因编辑技术的结合,必将使作物抗病育种迎来新时代。

Signaling Mechanisms in Pattern-Triggered Immunity （PTI）

In nature, plants constantly have to face pathogen attacks. However, plant disease rarely occurs due to efficient immune systems possessed by the host plants. Pathogens are perceived by two different recognition systems that initiate the so-called pattern-triggered immunity （PTI） and effector-triggered immunity （ETI）, both of which are accompanied by a set of induced defenses that usually repel pathogen attacks. Here we discuss the complex network of signaling pathways occurring during PTI, focusing on the involvement of mitogen-activated protein kinases.

CPR5 positively regulates pattern-triggered immunity via a mediator protein

Plant cells possess a two-layered immune system consisting of pattern-triggered immunity(PTI)and effector-triggered immunity(ETI), mediated by cell surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors(NLRs), respectively. The CONSTITUTIVE EXPRESSION OF PR GENES 5(CPR5) nuclear pore complex protein negatively regulates ETI, including ETI-associated hypersensitive response. Here, we show that CPR5 is essential for the activation of various PTI responses in Arabidopsis, such as resistance to the non-adapted bacterium Pseudomonas syringae pv. tomato DC3000 hrc C-. In a forward-genetic screen for suppressors of cpr5, we identified the mediator protein MED4. Mutation of MED4 in cpr5 greatly restored the defective PTI of cpr5. Our findings reveal that CPR5 plays opposite roles in regulating PTI and ETI, and genetically regulates PTI via MED4.

Arabidopsis RNA polymerase Ⅱ C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase(MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1(C-terminal domain phosphatase-like 1)as a negative regulator of microbe-associated molecular pattern(MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide fg22. Furthermore, fg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with fg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefy luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.

水稻抗稻瘟病天然免疫机制及抗病育种新策略

稻瘟病是水稻最严重的病害之一,由子囊菌(Magnaporthe oryzae)引起。利用抗病品种是防治稻瘟病最经济、最有效的措施。近年来,稻瘟病已发展为研究植物与病原真菌分子互作机制的模式系统,在水稻与稻瘟菌互作和寄主抗性分子生物学、基因组学和蛋白组学等领域取得了一系列重要的研究成果。文章综述了近年来水稻抗稻瘟病两种天然免疫机制,即病原菌相关分子模式诱导和效应蛋白诱导的抗病机制研究的最新进展,讨论了GWAS、TALLEN、CRISPR和HIGS等基因组研究新方法和新技术在水稻抗病育种中的应用,并对目前稻瘟病抗性机制研究和抗病育种中的问题和挑战进行了探讨和展望。

植物天然免疫系统研究进展

很多植物病原菌严重地损害植物的生长和繁殖。植物与病原体协同进化过程中,也逐渐形成了一系列复杂高效的保护机制来抵御病原物的侵染。植物中抵抗外界微生物刺激所形成的系统被称为植物天然免疫系统,可分为两个层次。第1个层次是植物模式识别受体(PRRs)识别病原相关分子模式(PAMPs),触发病原相关分子模式触发的免疫反应(PTI),激活植物体中促丝裂原活化蛋白激酶(MAPK)信号通路使植物产生早期应答反应。PTI适应性较广,可识别和响应包括非致病菌的许多类微生物。第2个层次是病原菌产生效应因子抑制基础免疫响应PTI,而植物产生针对性更强的抗性蛋白(R蛋白)识别效应因子,并通过效应因子触发型免疫(ETI)来重建植物的抗性。笔者综述了近年来植物天然免疫系统的研究进展,认为随着对植物天然免疫系统研究的深入,应重视PTI和ETI的结合利用,有效扩大植物抗菌谱,改良植物ETI抗性。

大豆胞囊线虫效应蛋白Hg16B09对植物免疫反应的抑制作用

为明确大豆胞囊线虫效应蛋白Hg16B09是否对植物免疫反应具有调控作用,本研究借助本氏烟草叶片瞬时表达Hg16B09,利用免疫激发子触发PTI和ETI反应,考察激发子诱导烟草叶片细胞过敏性坏死反应发生、活性氧爆发以及防御相关基因的表达变化情况。结果表明:Hg16B09在烟草细胞中瞬时表达能够显著抑制鞭毛蛋白flg22诱导H;O;的产生及防御基因PTI5、WRKY22-A、WRKY22-B和ACRE31的表达,其表达量相比对照分别降低了45%、61%、67%和61%。Hg16B09也能抑制由丁香假单胞杆菌Pst DC3000和线虫无毒效应子Rbp-1触发的HR发生,并减少了细胞坏死面积;qRT-PCR分析显示,Hg16B09对Pst DC3000激活的SA途径防御基因PR1a、PR2、WRKY51及PI1上调表达具有显著抑制作用,与对照相比表达量分别降低了67%、71%、48%和81%。以上结果表明Hg16B09对植物的PTI和ETI免疫反应具有双重抑制作用,说明调控寄主免疫防御是Hg16B09促进SCN寄生的一个关键毒性功能。

小麦条锈菌效应蛋白Hasp58抑制植物免疫的功能分析

为明确小麦条锈菌吸器特异表达效应蛋白Hasp58抑制植物免疫的功能,利用PCR获得Hasp58的全长cDNA序列;借助生物信息学软件预测Hasp58的信号肽;将Hasp58构建到pGR106载体用于农杆菌(Agrobactrium tumefacien)介导的烟草瞬时表达系统,明确Hasp58抑制细胞坏死的毒性功能;将Hasp58构建到pCambia1302载体用于烟草细胞定位,明确Hasp58定位情况;将Hasp58构建到pEDV6载体用于荧光假单胞杆菌(Pseudomonas fluorescens)介导的小麦瞬时表达系统,对小麦胼胝质、过敏性坏死面积、活性氧、菌丝面积、菌丝长度进行统计分析,明确Hasp58抑制寄主植物PAMPs引发的免疫反应(PTI)和效应蛋白诱导的免疫反应(ETI)功能。结果表明,条锈菌Hasp58的cDNA全长为1 026 bp,编码342个氨基酸,N端含26_aa的信号肽;通过农杆菌侵染,在烟草中瞬时表达Hasp58,发现其能够抑制由BAX诱导的细胞坏死,为条锈菌的候选效应蛋白;烟草细胞定位发现,含有信号肽和缺失信号肽的Hasp58与GFP融合表达均定位在细胞质,推测该效应蛋白在胞质作用;利用细菌的三型分泌系统在小麦中瞬时表达Hasp58,发现其能够抑制荧光假单胞杆菌引起的胼胝质积累,导致由无毒性条锈菌小种CYR23引起的小麦过敏性坏死面积和活性氧减少,使菌丝面积和长度增加。推测小麦条锈菌效应蛋白Hasp58可抑制寄主植物的PAMPs引发的免疫反应(PTI)和效应蛋白诱导的免疫反应(ETI),并促进自身侵染。

Recent Progress in Understanding PAMP- and Effector-Triggered Immunity against the Rice Blast Fungus Magnaporthe oryzae

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is one of the most destructive diseases of rice worldwide. The rice-M, oryzae pathosystem has become a model in the study of plant-fungal interactions because of its scientific advancement and economic importance. Recent studies have identified a number of new pathogen- associated molecular patterns （PAMPs） and effectors from the blast fungus that trigger rice immune responses upon perception. Interaction analyses between avirulence effectors and their cognate resistance proteins have provided new insights into the molecular basis of plant-fungal interactions. In this review, we summarize the recent research on the characterization of those genes in both M. oryzae and rice that are important for the PAMP- and effector-triggered immunity recognition and signaling processes. We also discuss future directions for research that will further our understanding of this pathosystem.

Phytocytokines function as immunological modulators of plant immunity

Plant plasma membrane-resident immune receptors regulate plant immunity by recognizing microbe-associated molecular patterns(MAMPs),damage-associated molecular patterns(DAMPs),and phytocytokines.Phytocytokines are plant endogenous peptides,which are usually produced in the cytosol and released into the apoplast when plant encounters pathogen infections.Phytocytokines regulate plant immunity through activating an overlapping signaling pathway with MAMPs/DAMPs with some unique features.Here,we highlight the current understanding of phytocytokine production,perception and functions in plant immunity,and discuss how plants and pathogens manipulate phytocytokine signaling for their own benefits during the plant-pathogen warfare.

PBS3 Protects EDS1 from Proteasome-Mediated Degradation in Plant Immunity

Plant immunity is controlled by both positive regulators such as PBS3 and EDS1 and negative regulators such as NPR3 and NPR4.However,the relationships among these important immune regulators remain elusive.In this study,we found that PBS3 interacts with EDS1 in both the cytoplasm and the nucleus,and is required for EDS1 protein accumulation?NPR3 and NPR4,which function as salicylic acid receptors and adaptors of Cullin3-based E3 ligase,interact with and mediate the degradation of EDS1 via the 26S proteasome.We further discovered that PBS3 inhibits the polyubiquitination and subsequent degradation of EDS1 by reducing the association of EDS1 with the Cullin3 adaptors NPR3 and NPR4.Furthermore,we showed that PBS3 and EDS1 also contribute to PAMP-triggered immunity in addition to effector-triggered immunity.Collectively,our study reveals a novel mechanism by which plants fine-tune defense resporises by inhibiting the degradation of a positive player in plant immunity.

TOPLESS promotes plant immunity by repressing auxin signaling and is targeted by the fungal effector Naked1

In plants,the antagonism between growth and defense is hardwired by hormonal signaling.The perception of pathogen-associatedmolecularpatterns(PAMPs)frominvadingmicroorganismsinhibits auxin signalingand plant growth.Conversely,pathogens manipulate auxin signaling to promote disease,but how this hormone inhibits immunity is not fully understood.Ustilago maydis is a maize pathogen that induces auxin signaling in its host.We characterized a U.maydis effector protein,Naked1(Nkd1),that is translocated into the host nucleus.Through its native ethylene-responsive element binding factor-associated amphiphilic repression(EAR)motif,Nkd1 binds to the transcriptional co-repressors TOPLESS/TOPLESS-related(TPL/TPRs)and prevents the recruitment of a transcriptional repressor involved in hormonal signaling,leading to the derepression of auxin and jasmonate signaling and thereby promoting susceptibility to(hemi)biotrophic pathogens.A moderate upregulation of auxin signaling inhibits the PAMP-triggered reactive oxygen species(ROS)burst,an early defense response.Thus,our findings establish a clear mechanism for auxin-induced pathogen susceptibility.Engineered Nkd1 variants with increased expression or increased EAR-mediated TPL/TPR binding trigger typical salicylic-acid-mediated defense reactions,leading to pathogen resistance.This implies that moderate binding of Nkd1 to TPL is a result of a balancing evolutionary selection process to enable TPL manipulation while avoiding host recognition.

水稻-病原菌互作途径研究进展

水稻稻瘟病和白叶枯病分别由真菌病原菌Magnaporthe oryzae(M.oryzae)和细菌病原菌Xanthomonas oryzae pv.oryzae(Xoo)引起,是造成世界范围内水稻减产的主要病因,水稻-稻瘟病菌及水稻-白叶枯病原菌互作已成为研究植物-病原菌互作的模式系统。本研究归纳了目前已克隆的抗稻瘟病及白叶枯病基因与其分子结构和功能,概括了近年来鉴定的一些病原菌相关分子(Pathogen-associated molecular patterns,PAMPs)及稻瘟病菌和白叶枯菌分泌的效应蛋白,并总结了针对稻瘟病菌和白叶枯菌介导的病原物分子诱导的抗病反应(PAMP-triggered immunity,PTI)和效应蛋白诱导的抗病性(Effectortriggered immunity,ETI)及其信号传导途径的研究成果,指出效应蛋白-抗病蛋白间互作将为探索植物-病原菌间互作提供新的分子基础,并为水稻抗病育种实践提供借鉴与指导。

Coordinated regulation of plant immunity by poly(ADP-ribosyl)ation and K63-linked ubiquitination

Poly(ADP-ribosyl)ation(PARylation)is a posttranslational modification reversibly catalyzed by poly(ADP-ribose)polymerases(PARPs)and poly(ADP-ribose)glycohydrolases(PARGs)and plays a key role in multi-ple cellular processes.The molecular mechanisms by which PARylation regulates innate immunity remain largely unknown in eukaryotes.Here we show that Arabidopsis UBC13A and UBC13B,the major drivers of lysine 63(K63)-linked polyubiquitination,directly interact with PARPs/PARGs.Activation of pathogen-associated molecular pattern(PAMP)-triggered immunity promotes these interactions and enhances PARylation of UBC13.Both parp1 parp2 and ubc13a ubc13b mutants are compromised in immune responses with increased accumulation of total pathogenesis-related(PR)proteins but decreased accu-mulation of secreted PR proteins.Protein disulfide-isomerases(PDIs),essential components of endo-plasmic reticulum quality control(ERQC)that ensure proper folding and maturation of proteins destined for secretion,complex with PARPs/PARGs and are PARylated upon PAMP perception.Significantly,PARylation of UBC13 regulates K63-linked ubiquitination of PDIs,which may further promote their disulfide isomerase activities for correct protein folding and subsequent secretion.Taken together,these results indicate that plant immunity is coordinately regulated by PARylation and K63-linked ubiquitination.

植物免疫激酶AtCERK1的重组表达与结晶研究

植物免疫激酶中的CERK1(chitin elicitor receptor kinase 1)家族是能够感知细菌及真菌侵染的模式识别受体。此类受体蛋白可以通过识别多糖类的病原微生物相关分子,引起病原微生物相关分子模式触发的免疫反应,是植物产生先天性免疫应答以应对微生物感染的重要元件,同时也是诱导植物-微生物共生的关键蛋白质。为推进CERK1激酶区真实蛋白质三维空间构象的解析,本研究针对拟南芥源的CERK1蛋白构建了野生型及突变型AtCERK1的E.coli-pRSF原核表达系统,经原核表达得到带有6个His标签的融合蛋白,经镍柱亲和层析、离子交换层析和凝胶过滤层析等一系列纯化过程得到高纯度蛋白质样品后对其进行结晶条件筛选。最终,AtCERK1^(F460V)突变型成功在0.2 mol/L氟化铵和20%PEG3350溶液环境中析出蛋白质晶体。经上海光源BL17U1线站采集,蛋白质晶体X射线衍射数据的分辨率为3.2A。综上所述,本研究成功获得了高纯度的AtCERK1蛋白样品,且突变型蛋白质成功析出晶体;同时,利用X射线收取了一套中等分辨率的衍射数据,为AtCERK1介导的植物免疫的结构机理进行了实验探索。

Suppression of ETI by PTI priming to balance plant growth and defense through an MPK3/MPK6-WRKYs-PP2Cs module

Pattern-triggered immunity(PTI)and effector-triggered immunity(ETI)are required for host defense against pathogens.Although PTI and ETI are intimately connected,the underlying molecular mechanisms remain elusive.In this study,we demonstrate that flg22 priming attenuates Pseudomonas syringae pv.tomato DC3000(Pst)AvrRpt2-induced hypersensitive cell death,resistance,and biomass reduction in Arabidopsis.Mitogen-activated protein kinases(MAPKs)are key signaling regulators of PTI and ETI.The absence of MPK3 and MPK6 significantly reduces pre-PTI-mediated ETI suppression(PES).We found that MPK3/MPK6 interact with and phosphorylate the downstream transcription factor WRKY18,which regulates the expression of AP2C1 and PP2C5,two genes encoding protein phosphatases.Furthermore,we observed that the PTI-suppressed ETI-triggered cell death,MAPK activation,and growth retardation are significantly attenuated in wrky18/40/60 and ap2c1 pp2c5 mutants.Taken together,our results suggest that the MPK3/MPK6-WRKYs-PP2Cs module underlies PES and is essential for the maintenance of plant fitness during ETI.

对植物病害相关受体-PRR的研究

先前大量的研究表明植物与病原体博弈进化的过程中,病原体配备了一系列的进攻之“矛”,而同时植物也配备了一套复杂有效的防御之“盾”,植物的免疫系统可大致分为PTI和ETI两个层面。即PAMPs或DAMPs诱导的免疫反应PTI是植物抵御病原菌的第1层反应,由病原体释放的效应子(effectors)引发的免疫反应ETI是植物的第2层防卫反应。植物所配置的各种类型的受体在植物免疫及生长发育等过程中启着重要作用。而模式识别受体(Pattern Recognition Receptor,PRR)在PTI反应中发挥重要作用,研究就PRR的结构分类,对病原的识别,信号传导,转录表达以及调控下游免疫的方面展开讨论。

西瓜食酸菌Ⅲ型效应蛋白AopAI的鉴定及其功能初步分析

【背景】西瓜食酸菌(Acidovorax citrulli,Ac)引起的细菌性果斑病是葫芦科植物重要的病害之一,通过Ⅲ型分泌系统(typeⅢsecretedsystem,T3SS)分泌至植物体内的Ⅲ型效应蛋白(typeⅢeffector,T3E)是该菌重要的致病因子,目前对Ac T3E的认识仍然非常有限。【目的】鉴定西瓜食酸菌候选的T3E Acidovorax outer protein AI(AopAI),分析其对Ac致病力的影响和干扰植物免疫的方式。【方法】利用生物信息学方法分析AopAI序列特征、AvrBs1无毒报告系统验证蛋白转运功能;通过荧光定量PCR技术分析aopAI基因表达的调控及其对植物病原相关分子模式(pathogen-associated molecular pattern,PAMP)激发的免疫反应(PAMP-triggered immunity,PTI)信号通路标记基因表达的影响;利用基因插入突变和基因功能互补方法,检测菌的致病力、植物组织过氧化氢和胼胝质积累量的变化;运用瞬时表达技术分析AopAI亚细胞定位和其抑制激发子诱导细胞死亡的能力。【结果】AopAI蛋白序列中不含跨膜螺旋区和信号肽,含有二磷酸腺苷(adenosine diphosphate,ADP)核糖基转移酶保守结构域;在T3SS核心基因hrpG和hrpX突变体中aopAI基因表达量显著降低;表达AopAI及AvrBs1功能区(59-445 aa)的avrBs1突变体可诱导ECW-10R辣椒叶发生过敏性坏死反应,表明AopAI具有转运功能;aopAI基因突变体在黄瓜子叶上的致病力减弱,但与其互作的黄瓜子叶组织中过氧化氢和胼胝质的含量均显著增加;AopAI在本氏烟叶瞬时表达后,显示其定位于细胞膜和细胞核,还表现抑制激发子NIP诱导的叶细胞死亡,导致叶细胞的PTI信号通路标记基因GRAS2和ACRE31的表达量显著降低。【结论】在西瓜食酸菌中具有一个定位于细胞核和细胞膜、有ADP核糖基转移酶结构域的T3E蛋白AopAI,该T3E是能够抑制NIP诱导的细胞死亡的毒性蛋白,通过抑制ACRE31调节的免疫途径降低植物过氧化氢和胼胝质的积累,以抑制植物PTI防御反应机制。