Functional Analysis of Gene ShARPC5 Involved in Tomato Resistance to Powdery Mildew
FENG ChanJing, SUN GuangZheng, WANG Yang,, MA Qing,College of Plant Protection, Northwest A&F University/State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, Shaanxi
Received:2019-07-10Accepted:2019-08-21Online:2020-01-01 作者简介 About authors 冯婵婧,E-mail:fcj413@163.com。
摘要 【目的】白粉病(powdery mildew)是番茄生产上的重要病害,严重影响番茄的产量。番茄基因组测序工作的完成为抗病基因挖掘提供了重要的信息资源。ARP2/3(actin-related protein 2 and 3)复合体是肌动蛋白微丝骨架动力学的主要调控因子,能够参与包括响应外界胁迫等多种细胞学过程。本研究通过对番茄ARPC5(actin-related protein C5)进行克隆和抗病功能验证,为番茄基因组信息完善、抗病机制解析和分子育种等方面打下基础。【方法】从番茄LA1777(Solanum habrochaites)cDNA中PCR扩增ShARPC5,使用DNAMAN 6.0进行多序列比对;MEGA 6.0构建系统发育树;应用在线工具ProtComp v. 9.0进行亚细胞定位预测。利用实时荧光定量PCR(qRT-PCR)比较接种白粉菌(Oidium neolycopersici,On-Lz)后高感品种Moneymaker(MM)和高抗品种LA1777中番茄ARPC5的表达特征,分析白粉菌侵染与ARPC5表达的相关性。应用病毒诱导的基因沉默(virus-induced gene silencing,VIGS)技术进一步验证该基因在番茄中的抗病功能,观察沉默株和野生型株系接种后表型变化,利用台盼蓝和DAB染色法检测植株产生过敏性坏死和H2O2的能力,并检测ShARPC5沉默后一些与植物抗病相关标记基因的表达变化。采用农杆菌浸花法遗传转化拟南芥过表达ShARPC5植株,观察转基因和野生型株系接种后表型变化,并统计单病斑分生孢子数。【结果】从番茄品种LA1777中克隆到ShARPC5,编码132个氨基酸残基,包含一个保守的P16-Arc结构域。与番茄MM-白粉菌亲和互作相比,非亲和互作的番茄品种LA1777在接种白粉菌后,ShARPC5显著上调表达,尤其在接种后18 h。在番茄上沉默ShARPC5能够增加植株对白粉菌On-Lz的敏感性,防卫反应基因PR1b1显著下调表达。组织学观察显示与对照植株相比,ShARPC5沉默植株接种后诱导产生过敏性坏死和活性氧减少。在烟草上瞬时过表达ShARPC5能够诱导产生坏死斑。相反,在拟南芥上过表达ShARPC5能够增加植物的抗病性。【结论】ShARPC5是番茄响应白粉菌侵染的重要基因,可减轻番茄白粉病的发病程度,在番茄抗白粉病机理研究方面具有较大的应用价值,可作为番茄抗白粉病分子育种的一个候选基因。 关键词:番茄白粉病;抗病性;肌动蛋白微丝骨架;ARP2/3复合体;ARPC5
Abstract 【Objective】Powdery mildew is an important disease in tomato production, and seriously affects the yield of tomato. Genome sequences of Solanum lycopersicum provide valuable information resources for disease-resistant gene searching. The actin- related protein 2 and 3 complex (ARP2/3 complex), a key regulator of actin cytoskeletal dynamics, has been linked to multiple cellular processes, including those associated with response to stress. In this study, tomato ARPC5, encoding a subunit protein of the ARP2/3 complex, was cloned and identified for disease resistance. The results will lay a foundation for upgrading tomato genomic information, further studying the resistance mechanism and molecular breeding.【Method】ShARPC5 was annotated from the genomic databases and cloned from tomato LA1777 (S. habrochaites). DNAMAN 6.0 was used for multiple sequence alignment and MEGA 6.0 was used for constructing phylogenetic tree. The prediction of protein subcellular localization was performed using ProtComp v. 9.0. The qRT-PCR was used to compare the expression of ARPC5 in the high-sensitive variety Moneymaker (MM) and high-resistant variety LA1777 induced by Oidium neolycopersici (On-Lz). The correlation between On-Lz infection and ARPC5 expression was analyzed. The function of ShARPC5 involving in tomato resistance to On-Lz was further verified by the technology of virus-induced gene silencing (VIGS). The phenotypic changes of ShARPC5-silenced and wild-type lines were observed. Hypersensitive response (HR) and H2O2 production were detected by trypan blue and DAB staining, respectively, and the expression of some marker genes related to plant disease resistance was assayed in ShARPC5-silenced plants. Additionally, genetic transformation of Arabidopsis thaliana was conducted by Agrobacterium-mediated floral-dip method. The phenotypic changes of transgenic and wild-type lines were observed, and the number of conidia per lesion was also counted.【Result】The S. habrochaites ARPC5 was identified and characterized. ShARPC5 encodes a 132-amino-acid protein possessing a conserved P16-Arc domain. Compared with the compatible interaction between tomato MM (Moneymaker) and On-Lz, the expression of ShARPC5 was significantly up-regulated in incompatible interaction between tomato LA1777 and On-Lz, especially at 18 hpi. Silencing of ShARPC5 in tomato could increase the susceptibility to the powdery mildew pathogen On-Lz. The expression of PR1b1, a maker gene related to signal regulation, was significantly down-regulated after silencing of ShARPC5. The histological observation showed that the induction of hypersensitive cell death and the generation of reactive oxygen were reduced in silenced-ShARPC5 tomato plants compared with wild types. Transient over-expression of ShARPC5 in tobacco could rapidly produce necrotic spots. Conversely, over-expression of ShARPC5 in A. thaliana, followed by inoculation with On-Lz, showed enhanced resistance.【Conclusion】ShARPC5 is an important gene which can reduce the incidence of tomato powdery mildew, and has a great application value in the mechanism study of tomato resistance to powdery mildew. At the same time, it can be used as a candidate gene for molecular breeding of tomato powdery mildew resistance. Keywords:tomato powdery mildew;resistance;actin cytoskeleton;ARP2/3 complex;ARPC5
PDF (1985KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 冯婵婧, 孙广正, 王阳, 马青. 番茄ShARPC5抗白粉病功能分析[J]. 中国农业科学, 2020, 53(1): 65-73 doi:10.3864/j.issn.0578-1752.2020.01.006 FENG ChanJing, SUN GuangZheng, WANG Yang, MA Qing. Functional Analysis of Gene ShARPC5 Involved in Tomato Resistance to Powdery Mildew[J]. Scientia Acricultura Sinica, 2020, 53(1): 65-73 doi:10.3864/j.issn.0578-1752.2020.01.006
0 引言
【研究意义】番茄白粉菌(Oidium neolycopersici)是一种活体营养寄生菌,能在番茄叶表面、背面、叶柄和花萼上产生白粉病斑,但不侵染果实。它通过影响植物的叶片使叶片失去生长活力,能导致产量(果实)损失高达50%[1]。目前,在温室和大田中对番茄白粉病的控制主要依赖于化学农药。然而,化学药剂不仅增加病原菌抗药性,也通过果实中农药的残留对环境和人体造成危害[2]。大多数栽培番茄易受白粉菌侵染,控制番茄白粉病的有效策略是培育抗性品种。【前人研究进展】目前,野生番茄品种中一些抗性基因已经被鉴定并转入栽培番茄,包括5个显性基因(Ol-1、Ol-3、Ol-4、Ol-5、Ol-6)、1个隐性基因(ol-2)和3个多基因抗性数量性状基因座(quantitative trait locus,QTLs)[3]。研究发现抗性基因介导的对番茄白粉病的抗病性与过敏性坏死(hypersensitive response,HR)有关[3]。当细胞产生HR时,未受感染的邻近细胞显示肌动蛋白微丝(actin filaments,AFs)聚集现象[4]。AFs是形成肌动蛋白细胞骨架的主要结构元件,既能将质膜和细胞壁上的物质通过囊泡的靶向输送转运到生长位点,也能特异性的转运防卫相关物质形成抵御病原菌侵入的屏障[5,6]。肌动蛋白组成的关键步骤是肌动蛋白成核,而肌动蛋白成核在很大程度上受ARP2/3(actin-related protein 2 and 3)复合体的活性调节,这能确保AFs正确形成[7]。ARP2/3复合体包含7个亚基,分别为ARP2、ARP3、ARPC1-ARPC5。该复合体是一种高度保守的肌动蛋白调节剂,能核化与质膜区域相关的分支状的肌动蛋白网络,也能促进特定细胞器和细胞骨架的耦合、在核内和细胞质间提供某种形式的物理连续性[8]。ARP2/3复合体不同亚基在发育过程中起着不同的作用。例如,ARP2对膜结合是必不可少的[9];ARP3在体内定位于肌动蛋白成核部位[10];ARPC1或ARPC2的缺失导致酿酒酵母的致死性和生存能力严重下降;拟南芥(Arabidopsis thaliana)ARP3或ARPC5的功能丧失导致根毛短小且弯曲[11,12];ARPC4是控制ARP2/3复合体组装和稳定性的最关键亚基[9];ARPC4和ARPC5在介导与ABA-和H2O2-诱导的保卫细胞肌动蛋白动力学中起关键作用[13]。【本研究切入点】ARP2/3复合体的亚基除了能够参与植物生长发育和结构组成,较少的研究证实其参与植物的抗病性[14]。西北农林科技大学植物保护学院植物病害综合治理团队发现白粉菌(On-Lz)与番茄Moneymaker(MM)(Solanum lycopersicum)形成亲和互作,而与番茄LA1777(S. habrochaites)非亲和互作,在非亲和互作体系中番茄ARPC5受白粉菌诱导显著上调表达,推测其可能参与抗白粉菌侵染的过程。【拟解决的关键问题】通过对ShARPC5进行克隆、生物信息学分析、差异性诱导表达、VIGS分析、烟草瞬时表达、拟南芥过量表达以及酵母突变体异源互补等方面的研究,为进一步揭示其抗病机制打下基础。
A:ShARPC5的多重氨基酸序列比对以及氨基酸序列的特征。氨基酸序列中黑色部分表示同源性达到100%,粉色表示同源性≥75%,青色表示同源性≥50% Multiple protein sequence alignment of ShARPC5 and characterized members of ARPC5 proteins. Black boxes indicate regions of 100% homology, pink boxes indicate ≥75% homology level, and cyan boxes highlight ≥50% homology level;B:ShARPC5系统进化分析,在不同的基因名称后显示各基因登录号 Evolutionary analysis of ShARPC5. Accession numbers are shown after the gene names Fig. 1Sequence analysis of tomato actin related protein 2 and 3 (ARP2/3) complex subunit 5 (ShARPC5)
A:不同处理(对照株、沉默ShPDS植株、沉默ShARPC5植株)接种白粉菌7 d后的表型观察Infection phenotypes of CK (TRV2), TRV2:ShPDS, TRV2:ShARPC5 tomato leaves at 7 dpi;B:对照株和沉默ShARPC5植株在接种白粉菌后7和14 d的病情指数Disease indexes of CK and TRV2:ShARPC5 plants at 7 and 14 dpi, respectively;C:对照株和沉默株接种白粉菌后24 h ShARPC5和PRs基因表达量变化Relative mRNA transcript levels of ShARPC5 and PRs at 24 hpi (determined by qRT-PCR) Fig. 3Effect of silencing of ShARPC5 in tomato LA1777 plants on the infection ability of On-Lz strain
A:对照植株和沉默植株接种白粉菌后24、48和72 h坏死产生率HR production rate of TRV2 (CK) or TRV2:ShARPC5 tomato leaves at 24, 48, and 72 hpi, respectively;B:对照植株和沉默植株接种白粉菌后24、48和72 h H2O2产生率H2O2 production rate of CK or TRV2:ShARPC5 tomato leaves at 24, 48, and 72 hpi, respectively Fig. 4Effect of silencing of ShARPC5 on tomato defense responses
A:农杆菌介导的瞬时表达在接种7 d后叶片的表型Leaf phenotypes at 7 dpi with Agrobacterium tumefaciens-expressing constructs;B:A中的烟草叶片用1﹕1的乙醇和冰醋酸脱色后的表型Leaf inoculation phenotypes after clearing in ethanol/acetic acid (1﹕1, v/v) for 2 days Fig. 5Hypersensitive necrosis induced by ShARPC5 in N. benthamiana
A:野生型Col-0转化pCAMBIA3301-ShARPC5植株上接种On-Lz 7 d后表型观察Infection phenotypes of WT Col-0 transgenic plants expressing pCAMBIA3301-ShARPC5. Images were taken at 7 dpi;B:野生型Col-0转化pCAMBIA3301-ShARPC5植株上接种On-Lz 7 d后植株上单病斑分生孢子数Quantitative assessment of conidiation on WT Col-0 plants expressing pCAMBIA3301-ShARPC5. Images were taken at 7 dpi Fig. 6Resistance of over-expression of ShARPC5 in A. thaliana against On-Lz
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