The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

Genome-Wide Identification of Zn_(2)Cys_(6 ) Class Fungal-Specific Transcription Factors(ZnFTFs)and Functional Analysis of UvZnFTFI in Ustilaginoidea virens

Transcription factors(TFs)orchestrate the regulation of cellular gene expression and thereby determine cell functionality.In this study,we analyzed the distribution of TFs containing domains,which named as ZnFTFs,both in ascomycete and basidiomycete fungi.We found that ZnFTFs were widely distributed in these fungal species,but there was more expansion of the ZnFTF class in Ascomycota than Basidiomycota.We identified 40 ZnFTFs in Ustilaginoidea virens,and demonstrated the involvement of UvZnFTF1 in vegetative growth,conidiation,pigment biosynthesis and pathogenicity.RNA-Seq analysis suggested that UvZnFTF1 may regulate different nutrient metabolism pathways,the production of secondary metabolites,and the expression of pathogen-host interaction genes and secreted protein-encodi ng genes.Analysis of the distributi on of differe nt fungal TFs in U.virens further dem on strated that UvZnFTFs make up a large TF family and may play essential biological roles in U.virens.

Site-Specifc Gene Targeting Using Transcription Activator-Like Effector(TALE)-Based Nuclease in Brassica oleracea

Site-specific recognition modules with DNA nuclease have tremendous potential as molecular tools for genome targeting. The type III transcription activator-like effectors （TALEs） contain a DNA binding domain consisting of tandem repeats that can be engineered to bind user-defined specific DNA sequences. We demonstrated that customized TALE-based nucleases （TALENs）, constructed using a method called ＂unit assembly＂, specifically target the endogenous FRIGIDA gene in Brassica oleracea L. var. capitata L. The results indicate that the TALENs bound to the target site and cleaved double-strand DNA in vitro and in vivo, whereas the effector binding elements have a 23 bp spacer. The T7 endonuclease I assay and sequencing data show that TALENs made double-strand breaks, which were repaired by a non- homologous end-joining pathway within the target sequence. These data show the feasibility of applying customized TALENs to target and modify the genome with deletions in those organisms that are still in lacking gene target methods to provide germplasms in breeding improvement.

Generation of knockout rabbits using transcription activator-like effector nucleases

Zinc-finger nucleases and transcription activator-like effector nucleases are novel gene-editing platformscontributing to redefine the boundaries of modern biological research. They are composed of a non-specificcleavage domain and a tailor made DNA-binding module, which enables a broad range of genetic modifications byinducing efficient DNA double-strand breaks at desired loci. Among other remarkable uses, these nucleases havebeen employed to produce gene knockouts in mid-size and large animals, such as rabbits and pigs, respectively.This approach is cost effective, relatively quick, and can produce invaluable models for human disease studies,biotechnology or agricultural purposes. Here we describe a protocol for the efficient generation of knockout rabbitsusing transcription activator-like effector nucleases, and a perspective of the field.

The root-knot nematode effectorMi2G02 hijacks a host plant trihelix transcription factor to promote nematode parasitism

Root-knot nematodes(RKNs)cause huge agricultural losses every year.They secrete a repertoire of effectors to facilitate parasitism through the induction of plant-derived giant feeding cells,which serve as their sole source of nutrients.However,the mode of action of these effectors and their targeted host pro-teins remain largely unknown.In this study,we investigated the role of the effector Mi2G02 in Meloidogyne incognita parasitism.Host-derived Mi2G02 RNA interference in Arabidopsis thaliana affected giant cell development,whereas ectopic expression of Mi2G02 promoted root growth and increased plant sus-ceptibility to M.incognita.We used various combinations of approaches to study the specific interactions between Mi2G02 and A.thaliana GT-3a,a trihelix transcription factor.GT-3a knockout in A.thaliana affected feeding-site development,resulting in production of fewer egg masses,whereas GT-3a overex-pression in A.thaliana increased susceptibility to M.incognita and also root growth.Moreover,we demon-strated that Mi2G02 plays a role in maintaining GT-3a protein stabilization by inhibiting the 26S proteasome-dependent pathway,leading to suppression of TOZ and RAD23C expression and thus promoting nematode parasitism.This work enhances our understanding of how a pathogen effector manipulates the role and regulation of a transcription factor by interfering with a proteolysis pathway to reprogram gene expression for development of nematode feeding cells.

rAL Effectors Drive Transcription Bidirectionally in Plants

TAL effectors delivered by phytopathogenic Xanthomonas species are DNA-sequence-specific transcrip- tional activators of host susceptibility genes and sometimes resistance genes. The modularity of DNA recognition by TAL effectors makes them important also as tools for gene targeting and genome editing. Effector binding elements （EBEs） recognized by native TAL effectors in plants have been identified only on the forward strand of target promoters. Here, we demonstrate that TAL effectors can drive plant tran- scription from EBEs on either strand and in both directions. Furthermore, we show that a native TAL effector from Xanthomonas oryzae pv. oryzicola drives expression of a target with an EBE on each strand of its promoter. By inserting that promoter and derivatives between two reporter genes oriented head to head, we show that the TAL effector drives expression from either EBE in the respective orientations, and that activity at the reverse-strand EBE also potentiates forward transcription driven by activity at the forward-strand EBE. Our results reveal new modes of action for TAL effectors, suggesting the possibility of yet unrecognized targets important in plant disease, expanding the search space for off-targets of custom TAL effectors, and highlighting the potential of TAL effectors for probing fundamental aspects of plant transcription.

Stripe Rust Effector PstGSRE1 Disrupts Nuclear Localization of ROS-Promoting Transcription Factor TaLOL2 to Defeat ROS-Induced Defense in Wheat

Puccinia striiformis f.sp.tritici(Pst),a biotrophic plant pathogen,secretes numerous effectors to modulate host defense systems.Understanding the molecular mechanisms by which Pst effectors regulate wheat immunity is of great importance for the development of novel strategies for durable control of stripe rust.In this study,we identified a glycine-serine-rich effector gene,PstGSRE1,which is highly induced dur-ing early infection.Transgenic expression of PstGSRE1 RNAi constructs in wheat significantly reduced virulence of Pst and increased H2O2 accumulation in wheat.PstGSRE1 was shown to target the reactive ox-ygen species(ROS)-associated transcription factor TaLOL2,a positive regulator of wheat immunity.PstGSRE1 disrupted nuclear localization of TaLOL2 and suppressed ROS-mediated cell death induced by TaLOL2,thus compromising host immunity.This work reveals a previously unrecognized strategy whereby rust fungi exploit the PstGSRE1 effector to defeat ROS-associated plant defense by modulating the subcellular compartment of a host immune regulator and facilitate pathogen infection.

基因编辑技术在免疫缺陷动物模型研究中的应用进展

免疫缺陷动物模型在临床前研究中发挥重要作用,是现代生物医学研究领域的重要实验工具,广泛应用于免疫学、遗传学、肿瘤学与微生物学等研究领域。基因编辑是针对生物体基因组进行靶向修饰的技术,从出现到应用,极大推动了生物医学研究领域的发展。基因编辑技术主要包括HEs、ZFNs、TALENs与CRISPR/Cas9系统。目前,研究者已利用该技术建立了多种类型的免疫缺陷动物模型,各有其优势和局限性。近年来,大量研究证实人源化免疫缺陷动物模型能够精准模拟癌细胞、药物以及免疫系统在人体内的作用,可以更好地模拟人类疾病,广泛用于研究人类免疫生物学及人类复杂疾病的潜在机制。本文对利用基因编辑技术构建免疫缺陷动物模型的研究应用进展进行综述,对基因编辑技术在免疫缺陷动物模型制备中存在的问题及优化策略深入探讨,并对其未来发展前景进行了展望,以期为研究者选用和建立免疫缺陷动物模型提供参考。

类转录激活因子效应物核酸酶(TALEN)介导的基因组定点修饰技术

人工构建的序列特异性核酸内切酶能够识别并切割特定的DNA靶序列,造成双链断裂,从而引起基因组结构的定点改变。人工核酸内切酶技术使得研究人员有可能对任意物种的基因组进行定点修饰,开启了反向遗传学研究的新天地。类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)自2010年底开始成功应用于基因打靶,很快成为一种比锌指核酸酶(Zinc-finger nuclease,ZFN)更容易设计、特异性更高和毒性更低的人工核酸内切酶。文章综述了TALEN技术的研究进展及应用前景,重点介绍TALEN的结构、作用机制与构建方法和利用TALEN进行基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,特别是在斑马鱼中的应用,为开展这一领域的研究工作提供参考。

应用TALEN技术对兔CCR5基因进行靶向修饰

缺少合适的能够被人免疫缺陷病毒1型（Human immunodeficiency virus 1,HIV-1）感染的动物模型是获得性免疫缺陷综合征/艾滋病（Acquired immunedeficiency syndrome,AIDS）疫苗和药物研发的瓶颈.HIV-1能在野生型兔子中形成持续感染,在共表达人CD4和CCR5的兔细胞系中,HIV-1能高效复制,并形成合胞体.若在家兔中高表达人CD4和CCR5,就可能获得研究AIDS的理想动物模型.文章采用高效基因打靶技术—类转录激活因子效应物核酸酶（Transcription activator-like effector nuclease,TALEN）,探讨在家兔CCR5基因位点定点敲入人CD4和CCR5,获得能够感染HIV-1家兔模型的可能性.针对家兔CCR5基因,设计了两对TALENs和一个同源打靶载体,TALEN mRNAs和DNA同源片段显微注射到家兔受精卵中,体外培养3～5 d后,收集24枚胚胎,对胚胎的基因突变情况进行PCR和测序分析.结果显示,在家兔CCR5位点,24枚胚胎均发生了基因敲除,5枚胚胎还发生了人CD4和CCR5基因敲入.该结果为建立艾滋病研究新动物模型奠定了基础.

TALEN介导的MYH9基因沉默及对细胞周期与凋亡的影响

目的利用TALEN技术敲除人胃癌细胞系MGC803细胞株MYH9基因,观察MYH9基因沉默后细胞周期及凋亡改变。方法根据斯丹赛Fast TALETMTALEN试剂盒说明书,设计并构建靶向MYH9基因的TALEN质粒对。通过质粒转染、DNA测序、RT-PCR和Western blot等检测质粒活性,成功挑取MYH9基因敲低单克隆株,并对构建好的细胞株进行周期和凋亡检测。结果成功挑选的MGC803单克隆细胞株未检测到MYH9基因完全敲除;MYH9基因敲低后,MGC803细胞周期受阻于G2/M期(P<0.05),早期凋亡增加(P<0.05)。结论利用TALEN技术成功构建MGC803细胞MYH9基因敲低单克隆株,该模型有助于后期深入探讨胃癌MYH9基因功能。

大豆疫霉PsMPK1沉默突变体的基因表达谱分析

[目的]促有丝分裂原蛋白激酶(MAPK)是真核生物细胞信号转导途径中的关键蛋白激酶,对植物病原菌的生长发育与侵染致病具有重要作用。我们前期发现1个MAPK基因Ps MPK1参与调控大豆疫霉的细胞壁完整性、菌丝生长、游动孢子形成,以及对大豆的致病性等生物学性状。本文旨在进一步探究Ps MPK1如何调控大豆疫霉的上述性状。[方法]利用数字基因表达谱技术对大豆疫霉Ps MPK1沉默突变体在游动孢子囊阶段的基因表达情况进行分析。[结果]样品间基因表达水平的整体相关性分析和实时定量PCR结果表明:数据具有较高的可靠性。与野生型菌株相比,Ps MPK1沉默突变体中分别有1 451个显著下调和981个显著上调的基因,占所有被检测基因的23%。与之相比,被检测的细胞壁相关基因和分泌蛋白编码基因中,显著差异表达的基因比例更高(分别占37%和28%),且大部分基因为下调表达(分别占87%和78%)。显著下调表达的分泌蛋白编码基因中,37个基因可能与侵染过程相关,主要编码Rx LR、NLP和CRN等家族的效应分子,CBEL蛋白,富含半胱氨酸蛋白,氧化还原相关蛋白以及水解酶等。[结论]首次通过转录组学方法揭示了特定的功能基因Ps MPK1沉默后大豆疫霉基因组的转录变化,鉴定了可能与Ps MPK1信号途径相关的基因和基因家族,为进一步研究大豆疫霉中MAPK的调控网络奠定了基础。

TALEN构建与斑马鱼基因组定点突变的实验方法与流程

类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)是最近发展起来的一类新型的人工核酸内切酶,它由特异性的TALE DNA结合结构域和非特异性的FokⅠ核酸内切酶切割结构域组成。TALEN能够根据用户需要切割特定的核苷酸靶序列,造成DNA双链断裂,从而诱导该靶序列产生indel突变,目前已成功地应用于多个物种或体外培养细胞的基因组定点突变。文章介绍TALEN靶点的选择与确认,采用"单元组装法"构建人工TALEN的原理与步骤,以及通过显微注射TALEN mRNA诱导并筛选斑马鱼突变体的实验流程与经验。这些方法理论上也适用于对其他物种进行基因打靶。

组蛋白甲基化修饰效应分子的研究进展

作为一种重要的表观遗传学调控机制,组蛋白甲基化修饰在多种生命过程中发挥了重要的作用。细胞内有多种组蛋白甲基化酶和去甲基化酶共同调节组蛋白的修饰状态,在组蛋白甲基化状态确定后,多种效应分子特异的读取修饰信息,从而参与基因转录调控过程。文章从组蛋白甲基化效应分子的作用机制方面综述了这一领域的研究进展。

水稻与白叶枯病菌互作机制研究进展

水稻白叶枯病由Xanthomonas oryzaepv.Oryzae(Xoo)致病菌引起,为水稻三大病害之一,对世界水稻生产造成了严重危害。水稻与Xoo互作符合"基因对基因"假说,是研究植物与细菌互作的典型模式系统。水稻基因组以及Xoo基因组测序的完成,极大地推动了水稻-Xoo互作分子机理的研究。就有关水稻与Xoo互作机制的最新研究进展作一概述。

SD大鼠超排及TALEN质粒显微注射构建基因敲除大鼠

目的 应用TALEN技术构建基因敲除大鼠,并探讨SD大鼠超数排卵、显微注射浓度、胚胎移植方式对构建大鼠出生率、阳性率的影响.方法 将SD雌鼠超数排卵后,取受精卵显微注射由TALEN质粒体外转录成的mRNA,注射后存活的受精卵输卵管移植,观察大鼠的出生率,并测序判定出生大鼠的基因敲除阳性率.结果 4～5周和5～6周的雌鼠超数排卵见栓率[（74±9）％vs（77±6％）％]、取卵数量[（25±2）枚vs （23±2）枚]以及卵的受精率[（85±2）％vs（93±2）％]差异均无统计学意义（P＞0.05）;结扎公鼠和受体雌鼠在8～10周时见栓率最高[（23.02±5.26％）％],使用12周后见栓率明显下降[（11.66±2.40）％];单侧和双侧输卵管移植大鼠出生率差异无统计学意义（37.88％ vs 36.49％）,但单侧移植大鼠存活率高于双侧移植（37.88％ vs 20.27％,P＜0.05）,且单细胞移植大鼠出生率显著高于双细胞移植.质粒注射浓度在20 ng/μl组大鼠基因敲除阳性率最高（7.5％）.结论 应用TALEN技术成功构建基因敲除大鼠,显微注射质粒浓度20 ng/μl时大鼠出生率不受影响且基因敲除阳性率最高.

TALEN技术研究进展

类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)是近年来新兴的分子生物学工具。该技术可高效地介导DNA编辑修饰,如基因敲除、基因敲入、碱基替换、点突变等。近年来,随着人们对TALEN系统研究的不断深入和改造,TALEN已成功地应用于人类细胞、鼠、斑马鱼等物种中,成为一项具有广阔应用前景的基因编辑技术。对TALEN的来源、构建及应用情况进行阐述,并对其未来发展进行展望。

TALENs技术在基因功能研究中的应用

基因敲除技术广泛应用于研究各类细胞和生物的基因功能。转录激活子样效应因子核酸酶技术(TALENs)是近年发展起来的一种新型基因敲除方法,其利用TALEs蛋白核酸结合域氨基酸序列与其靶位点核酸序列之间存在着恒定对应关系,从而组装出能特异性结合任意DNA序列的模块化蛋白。TALENs技术以其快速、高效的优势对生物基因研究领域产生深远影响,为遗传疾病的治疗提供了新的策略。

TALENs编辑绵羊成纤维细胞FGF 5基因

类转录激活因子效应物(Transcription activator-like effector,TALE)已经成为基因组编辑和基因转录调控的有效工具,在多个物种的基因组中实现特定位点的删除、插入和突变。针对绵羊成纤维细胞生长因子5(Fibroblast growth factor 5,FGF5)基因起始密码子ATG位点,设计构建TALENs(Transcription activator-like effector nucleases,TALENs)。通过比较分析正常培养和基因组编辑处理的绵羊成纤维细胞,电转TALENs组合,Surveyor突变检测,筛选获得1对有效的TALENs。有限稀释细胞传代培养,PCR扩增FGF5基因片段,经PAGE检测筛选发生突变的细胞,测序确认FGF5基因ATG位点产生缺失突变细胞。获得具有编辑活性的TALENs,为该基因的定点编辑奠定基础。Surveyor检测和测序结果表明,在绵羊FGF5基因ATG起始密码子上游104碱基位点,存在1个G/C单核苷酸多态。

基因编辑技术原理及其在动植物研究中的应用

自人类基因组计划开展以来,越来越多生物的基因组序列得到了测定,基因的功能逐步得到鉴定.人们期望通过对基因表达的改变,来治疗人类疾病或提高生物的产量和品质.早期突变技术对基因的改变是不定向的,近年来,锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALEN)和CRISPR/Cas9等技术可对某个已知基因进行编辑.特别是CRISPR/Cas9技术,由于具有操作方便、效率高等优点,因此成为对基因进行定向操作的强有力工具.本文对几种基因编辑技术的原理和应用进行简要介绍和展望.