Summary

VIGS介导的正向遗传学筛选参与非寄主抗性基因鉴定

Published: August 23, 2013
doi:

Summary

病毒诱导的基因沉默是一个有用的工具,用于识别植物非寄主抗性基因参与。我们演示了如何使用中表达GFPuv的识别基因沉默植株易受非寄主病原体的细菌病原体。这种做法是方便,快捷,有利于大规模筛选和类似协议可以应用到其他各种植物与微生物相互作用研究。

Abstract

非寄主植物对细菌病原体的抗病能力控制复杂的防御途径。了解这一机制是非常重要开发耐用的抗病植物对病原体的广泛。病毒诱导的基因沉默(VIGS)基于正向遗传学筛选是用于识别赋予非寄主抗性植物防卫基因的一个有用的方法。 烟草脆裂病毒 (TRV)的沉默载体是最有效的VIGS载体和有效地使用已内源性靶基因沉默在烟草

在这篇稿件中,我们展示了一个正向遗传学筛选方法沉默单个克隆cDNA文库在N本生和评估对非寄主病原体损害非寄主抗性基因沉默植株的响应, 野火病菌番茄T1,P.丁香甘油 PV。成人教育学院,和X油菜叶斑病病菌。这些细菌病原体设计到快递GFPuv蛋白质和绿色荧光的菌落肉眼可以看出,如果在非寄主植物病原体接种在紫外光下沉默目标基因参与传授非寄主抗性。这有利于可靠和更快的识别基因沉默植株易受非寄主病原体。此外,有希望的候选基因的信息可以被称为TRV载体序列的植物基因插入。在这里,我们展示了VIGS介导的正向遗传学的高吞吐能力,以确定基因参与非寄主抗性。大约100个​​cDNA可以在约2至3周,对一些非寄主细菌病原体的非寄主抗性的相关个别沉默可以研究在其后的一个星期。在这篇稿件中,我们列举参与筛查的具体步骤。 VIGS介导的正向遗传学甄选NG方法不仅可以延长参与非寄主抗性基因鉴定,但也研究基因传授各种植物物种的一些生物和非生物胁迫公差。

Introduction

非寄主抗性是对一个特定的病原体1,2种族的所有植物物种的阻力。这赋予广谱和持久抗病植物中2,3。然而,其作用机制,尤其是对细菌病原体,没有得到很好的理解妥协非寄主抗性,高通量转录分析识别差异表达的基因在非寄主抗性5-9有两种主要方法,以前用于解剖细菌非寄主抗性的突变体或沉默植株的筛选。因为非寄主抗性是由一个复杂的机制(S)4与许多基因的参与,高通量功能基因组学方法进行基因识别控制,是为更好地理解非寄主抗性机制(S)的关键。

病毒诱导的基因沉默(VIGS)已成功地用于植物内源沉默基因在许多植物物种10,11。 烟草(Nicotiana benthamiana)是最适合的植物病毒诱导的基因沉默10,12和它的基因组序列草图,现已13。 烟草脆裂病毒 (TRV)基于VIGS技术已被广泛用作反向遗传学工具表征基因参与非寄主抗性2,4,14。这VIGS载体及衍生产品现已通过拟南芥生物资源中心(ABRC, http://www.arabidopsis.org/abrc/catalog/individ_cloned_gene_1.html )。 VIGS也被用来确定参与植物免疫力15-17,特别是6,18非寄主抗性基因作为一个正向遗传学工具。评估过敏性反应(HR)介导的细胞死亡由一个特定的非寄主植物对病原体引起和评估疾病诱导的细胞死亡,是两个主要检测主要用于identifyin的克易感基因沉默植株。然而,HR细胞死亡不仅对II型非寄主病原体引起的,而不是针对I型非寄主病原体2。因此,人力资源分析不能被普遍用来识别非寄主抗性被植物所利用的策略,特别是针对广泛的I型非寄主病原体。此外,在基因沉默的植物非寄主抗性的部分损失并不总是导致疾病的症状,因此不能用来确定损害非寄主抗性的植物疾病的得分。相反,评估非寄主病原体的基因沉默植株的增长是一个更好的方法研究非寄主抗性基因沉默植株的损失。

以往的生长测定6,19,更快速的方法,以评估非寄 ​​主细菌生长的基因沉默的植物相比,可以缩短在正向遗传学筛选所需的时间。我们此前报道的方法观察细菌升病原体生长在紫外线(UV)光叶肉眼使用细菌表达绿色荧光蛋白(GFP)19。在这篇稿件中,我们表现出的实用性表达GFPuv的非寄主细菌病原体受到损害非寄主抗性基因沉默植株易于识别。这种方法是准确识别敏感的植物,适合高通量筛选。

Protocol

1。植物生长与靶基因沉默植物生长的条件: 母猪N.本生种子无土盆栽混合物,地铁混合350和发芽的种子在生长室。任何其它也可用于土壤或土壤的介质,而不是对新城混合。 移植三周龄的苗成单个盆种植在温室中保持在21±2℃以及与其他的生长条件,详细的在以前的文献12。移栽后的两到三天,植物可以用来为TRV接种。 成长TRV2克隆: TRV是?…

Representative Results

这项研究的主要目的是要证明的方法,方便,准确识别基因沉默N. benthamiana植物非寄主抗性受到损害。这种方法有四个主要步骤。第一个步骤是个别地大量使用TRV-VIGS基因沉默。我们已经沉默了大约5000个基因6,18过了一段约1.5年的使用协议, 如图1所示。部分的基因沉默的植物表现出各种表型的改变,包括生长发育迟缓,泛黄和光漂白的表型( 图2)。 <p c…

Discussion

植物免疫力限制非寄主病原体的生长,因此,很少或根本没有发出绿色荧光矢量控制植物的叶子接种非寄主病原体在长波长紫外光灯( 图3D)。然而,当在非寄主抗性有关的基因沉默,基因沉默的植物有利于生长的非寄主的病原体和绿色荧光出现( 图3E)。这是这个手稿中所描述的方法中所涉及的基本原理。这种方法有两个主要的优点,在人力资源为基础的检测。 在?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这个项目是由塞缪尔·罗伯茨诺贝尔基金会。作者感谢MSS。珍妮Gallaway和科琳埃尔斯优良的植物护理和凯蒂·布朗女士艺术品。我们也想感谢MSS。天合光能科特雷尔,普加Uppalapati,萨哈Moumita,Swetha Vinukonda,此协议在建立技术帮助的艾萨克Greenhut先生。

Materials

Name of the reagent/equipment Company Catalogue number Comments (optional)
96-well U-bottom plates Becton Dickinson Labware (Franklin Lakes, NJ, USA) 35-3077  
96-pin replicator stainless steel Nalge Nunc International (Naperville, IL, USA) 250520  
High Intensity UV Inspection Lamps, Model B-100ap Thomas scientific (Swedesboro, NJ, USA) 6283K50 Manufacturer ID 95-0127-01
Stuart SC6 colony counter Bibby Scientific Limited, Staffordshire, UK SC6PLUS  
Soil-less potting mixture, Metro-Mix 350 SUNGRO Horticulture Distribution, Inc., (Bellevue, WA, USA)    
Primers:
attB1 (GGGGACAAGTTTGTACAAAAAAGCAGGCT)
attB2 (GGGGACCACTTTGTACAAGAAAGCTGGGT)
Integrated DNA Technologies, Inc (Coralville, IA, USA) Custom synthesized  
MES, Monohydrate VWR international (Radnor, PA, USA) CAS No. 145224-94-8  
Acetosyringone (Dimethoxy-4′-hydroxyacetophenone) Sigma Aldrich (St. Louis, MO, USA) D134406  
Vac-In-Stuff (Silwet L-77) Lehle Seeds (Round Rock, TX, USA) VIS-30  

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Senthil-Kumar, M., Lee, H., Mysore, K. S. VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance. J. Vis. Exp. (78), e51033, doi:10.3791/51033 (2013).

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