<Emphasis Type="Italic">White clover mosaic virus</Emphasis>-induced gene silencing in pea

Double-stranded RNAs formed in secondary structures and replicative intermediates of viral genomes are thought to strongly elicit RNA silencing. This phenomenon is known as virus-induced gene silencing (VIGS). VIGS is a powerful tool for modifying gene expression in host plants. We constructed a virus vector based on White clover mosaic virus (WClMV) and demonstrated VIGS of phytoene desaturase (PDS) in pea. Photobleaching of tissues, caused by VIGS of PDS, was observed in restricted areas of upper leaves and stems. We confirmed that the PDS mRNA and subgenomic RNAs of WClMV were reduced in the photobleached tissues.     

刺萼龙葵PDS基因VIGS载体的构建

病毒诱导的基因沉默(VIGS)是通过插入目的基因片段的重组病毒来抑制植物内源基因表达的遗传技术,主要用于基因的功能分析。茄科植物刺萼龙葵(Solanum rostratum)是一种外来恶性杂草,研究证实,在农杆菌GV3101介导下,刺萼龙葵的八氢番茄红素脱氢酶(PDS)基因能被部分沉默,导致叶片和花朵出现白化表型。半定量RT-PCR检测显示,被侵染叶片和花朵的mRNA显著降解。VIGS沉默体系的建立可适用于研究刺萼龙葵的部分功能基因,有助于深入了解其生长发育调控的分子机制。     

Silencing of host basal defense response-related gene expression increases susceptibility of Nicotiana benthamiana to Clavibacter michiganensis subsp. michiganensis

Clavibacter michiganensis subsp. michiganensis is an actinomycete, causing bacterial wilt and canker disease of tomato (Solanum lycopersicum). We used virus-induced gene silencing (VIGS) to identify genes playing a role in host basal defense response to C. michiganensis subsp. michiganensis infection using Nicotiana benthamiana as a model plant. A preliminary VIGS screen comprising 160 genes from tomato known to be involved in defense-related signaling identified a set of 14 genes whose suppression led to altered host-pathogen interactions. Expression of each of these genes and three additional targets was then suppressed in larger-scale VIGS experiments and the effect of silencing on development of wilt disease symptoms and bacterial growth during an N. benthamiana-C. michiganensis subsp. michiganensis compatible interaction was determined. Disease susceptibility and in planta bacterial population size were enhanced by silencing genes encoding N. benthamiana homologs of ubiquitin activating enzyme, snakin-2, extensin-like protein, divinyl ether synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase 2, and Pto-like kinase. The identification of genes having a role in the host basal defense-response to C. michiganensis subsp. michiganensis advances our understanding of the plant responses activated by C. michiganensis subsp. michiganensis and raises possibilities for devising novel and effective molecular strategies to control bacterial canker and wilt in tomato.     

Construction of monopartite geminivirus-based virus-induced gene silencing (VIGS) vectors using a two-component strategy

Journal of General Plant Pathology - Virus-induced gene silencing (VIGS) is a rapid and efficient tool to elucidate plant gene functions by inserting a target gene fragment into a viral genome and...     

Virus-induced gene silencing in Solanum torvum

Journal of General Plant Pathology - Solanum torvum has broad resistance to wilt-causing bacteria and fungi and nematodes. Here, we applied virus-induced gene silencing (VIGS) in S. torvum and...     

番茄斑萎病毒N基因的VIGS载体构建

 番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)严重危害多种经济作物和园艺植物,其N基因与病毒的侵染力密切相关,将N基因直接构建到VIGS载体上研究其致病功能目前鲜见报道。本研究将TSWV N基因构建到pTRV-PTV00载体上,注射本氏烟,通过qRT-PCR定量分析发现先接种TSWV后注射沉默载体的植株抗TSWV效率达57.60%,先注射沉默载体后接种TSWV的植株抗TSWV效率达99.14%。结果表明先注射载体后接种TSWV的N基因沉默效率更高。TSWV N基因VIGS载体的构建可为研究N基因在病毒致病等方面的功能提供前期材料,并为TSWV抗病育种和田间绿色防控提供一定的理论依据。     

番茄斑萎病毒N基因的VIGS载体构建

 番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)严重危害多种经济作物和园艺植物,其N基因与病毒的侵染力密切相关,将N基因直接构建到VIGS载体上研究其致病功能目前鲜见报道。本研究将TSWV N基因构建到pTRV-PTV00载体上,注射本氏烟,通过qRT-PCR定量分析发现先接种TSWV后注射沉默载体的植株抗TSWV效率达57.60%,先注射沉默载体后接种TSWV的植株抗TSWV效率达99.14%。结果表明先注射载体后接种TSWV的N基因沉默效率更高。TSWV N基因VIGS载体的构建可为研究N基因在病毒致病等方面的功能提供前期材料,并为TSWV抗病育种和田间绿色防控提供一定的理论依据。     

香豆素合成途径关键酶基因Ghpal、Ghc4h和Gh4cl在棉花抗链格孢菌中的作用

链格孢菌Alternaria alternata引起的棉花轮纹斑病,是导致棉花早衰发生的主要成因之一,严重危害棉花生产。香豆素合成途径作为合成植保素的一条代谢支路,其产物能够抑制病原菌,增强植物的抗病性。为了揭示该途径在棉花抗链格孢菌中的作用,本文利用棉花皱缩病毒介导的基因沉默技术,通过对棉花香豆素合成途径中编码关键酶基因的沉默抑制,来解析其在棉花抗链格孢菌中的作用,所选择的基因有编码苯丙氨酸解氨酶(PAL)的基因Ghpal、肉桂-4-羟化酶(C4H)基因Ghc4h和4香豆酸-辅酶-A(4CL)的基因Gh4cl。结果显示,Ghpal、Ghc4h和Gh4cl基因受链格孢菌诱导转录,表明这些基因可能参与了棉花对链格孢菌的抗性反应。Ghpal、Ghc4h和Gh4cl基因分别被沉默后,结果显示各基因沉默植株对链格孢菌的抗性下降,表现为接种链格孢菌后,基因沉默植株的病情指数分别为43.36、38.27和44.78,显著高于野生型和VIGS空载体对照植株。这表明,Ghpal、Ghc4h和Gh4cl参与了棉花对链格孢菌的抗性反应。此外,离体条件下给Ghpal、Ghc4h和Gh4cl基因沉默植株叶片饲喂外源香豆素,结果发现基因沉默植株对链格孢菌的抗性得到恢复。这一结果进一步表明香豆素合成途径在棉花抗链格孢菌中起着重要作用。     

辣椒CaRKNIF2基因的诱导表达特征及RNAi效应分析

[目的]明确在多种植物病原物和植物激素处理下,辣椒CaRKNIF2基因的诱导表达特性,分析CaRKNIF2基因的抗根结线虫功能.[方法]以辣椒‘HDA149’为试材,分别接种南方根结线虫毒性群体、烟草花叶病毒(TMV)、辣椒青枯病菌、辣椒疫霉病菌以及信号分子水杨酸(SA)和茉莉酸甲酯(MeJA),实时荧光定量PCR分析了辣椒CaRKNIF2基因的诱导表达模式和特征;构建了含CaRKNIF2基因片段的RNAi载体,通过烟草脆裂病毒介导的基因沉默(VIGS)同源沉默辣椒CaRKNIF2基因,来评价CaRKNIF2基因沉默的辣椒植株对线虫侵染的应答效应.[结果]信号分子MeJA抑制CaRKNIF2的表达,但TMV、辣椒青枯病菌、辣椒疫霉病菌以及信号分子水杨酸(SA)能快速而显著地诱导CaRKNIF2的表达,而毒性南方根结线虫对CaRKNIF2的表达则基本无影响;CaRKNIF2基因沉默的辣椒植株接种非毒性南方根结线虫后根部卵块数明显增加.[结论]这些结果为进一步解析CaRKNIF2的调控功能提供了重要的证据.     

Transcriptome profile of acibenzolar-S-methyl-induced genes in tomato suggests a complex polygenic effect on resistance to Phytophthora infestans

Induced resistance by chemicals such as acibenzolar-S-methyl -ASM (commercialized as Actigard by Syngenta Inc) mimics the biological activation of systemic acquired resistance (SAR). ASM takes the place of salicylic acid (SA) in the SAR signal pathway inducing the same molecular markers and range of resistance. The goal of our work was to understand the downstream molecular events by which ASM confers resistance to Phytophthora infestans in tomatoes. To accomplish this goal we assayed gene expression in ASM-treated plants using a microarray with more than 12,000 tomato ESTs. As many as 300 genes were responsive to ASM. Of these, 117 were detected in most of the biological replications. Basal defense associated genes as well as SAR and disease resistance genes (R-like) involved in induced resistance and effector-triggered immunity were highly expressed. We attempted to determine the phenotype of 13 of these genes by virus induced gene silencing (VIGS). These 13 genes were selected on the basis of previous implication in plant defense response and by reliability of induction by ASM. VIGS was partially successful for three of the 13 genes, but this partial silencing did not lead to a significant reduction in the effect of ASM. The ethylene pathway was also activated in response to ASM, but a tomato mutant not responsive to ethylene remained responsive to ASM. It seems most likely that the ASM effect is complex and polygenic, depending on the effect of several genes.     

A new ZTL-type F-box functions as a positive regulator in disease resistance: VIGS analysis in barley against powdery mildew

In our previous studies, we found a new Zeitlupe (ZTL) type F-box protein which is expressed at a higher level upon avirulent pathogen infection (Bozkurt et al., 2007). F-box proteins mark the proteins to be degraded through 26S proteasome system by ubiquitination. Since the information on the role of ubiquitin mediated proteolysis in disease responses is advancing rapidly, we sought to understand the way which F-box functions in resistance response as part of ubiquitin–proteasome pathway. Interestingly, in response to silencing of this F-box gene via BSMV mediated virus induced gene silencing (VIGS) method, barley plants lost resistance towards avirulent pathogen race. The Pallas-01 line having Mla1 R-gene showed hyphae formations when inoculated with avirulent powdery mildew race, Bgh103, after 4-fold silencing. This observation suggests that F-box protein functions as a positive regulator in powdery mildew disease mechanism and broadens the function of ZTL-type F-box proteins, previously known to have roles only in circadian clocks, flowering time control, and phytochrome pathway.     

Insights into the role of ethylene perception in tomato resistance to vascular infection by Verticillium dahliae

A Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system was employed to investigate the role of the tomato ethylene receptor ETR4. By comparing wilting symptoms of verticillium wilt in wild-type, ethylene-insensitive Never ripe ( Nr ) mutant tomato plants and ETR4 -silenced plants, it was demonstrated that disease severity in the Nr and ETR4 -silenced plants was statistically reduced compared to wild-type plants. Disease incidence and severity were reduced by 11 and 20%, respectively, in the Nr plants compared to the wild-type plants, at 33 days post-inoculation (d.p.i.). In the ETR4 -silenced plants, disease incidence and severity were reduced by 14 and 15%, respectively, compared to the TRV -only-inoculated plants, at 37 d.p.i. Quantification of Verticillium dahliae by qPCR revealed that the reduction in symptom severity in the Nr plants was associated with significant reduction of growth of the pathogen in the vascular tissues of the Nr plants compared to that in the wild-type plants, suggesting that impaired perception of ethylene via the Never-ripe receptor results in increased disease resistance. Fungal reduction was evident at each sampling day in the Nr plants, ranging from 1·5 to 1·75 times less than that in the wild-type plants. Fungal quantification in the ETR4- silenced and TRV -only-inoculated plants showed similar levels of fungal biomass.     

番茄SlAGO1的表达分析及其抗番茄黄化曲叶病毒(TYLCV)功能研究

 本研究运用 MEGA 7.0 进行多序列比对并构建系统进化树,利用实时荧光定量 PCR(qRT-PCR)分析SlAGO1在番茄组织中的表达水平及接种TYLCV后的防御响应;通过沉默番茄八氢番茄红素脱氢酶基因SlPDS,建立‘矮番茄'沉默体系,采用 qRT-PCR 分析SlAGO1在沉默后对TYLCV表达的影响。系统进化树结果显示SlAGO1a/b(SlAGO1)氨基酸序列同源性极高,暗示其为同一个基因在染色体上的多个拷贝;qRT-PCR分析表明,SlAGO1在花、叶中显著表达,且受TYLCV的诱导表达;VIGS沉默SlAGO1后接种TYLCV侵染性克隆,植株表现出明显的病毒症状,qRT-PCR分析显示病毒含量显著高于对照,表明SlAGO1在番茄植株抗病毒中发挥重要作用。研究结果可为番茄抗病毒机制研究及番茄抗病毒育种奠定基础。     

南方根结线虫Col基因分子克隆及其VIGS效应分析

为了明确线虫角质层胶原蛋白基因(Col)与根结线虫病害的关系,利用从线虫基因组中预测的Col设计特异引物,克隆了南方根结线虫Meloidogyne incognitacol基因MiCol.该基因完整编码区长903bp,编码300个氨基酸.克隆的MiCol与预测的MiCol基因序列一致性高达99.67％,氨基酸序列一致性高达100％.利用病毒介导的基因沉默技术,将其导入番茄植株并接种南方根结线虫,60 d后,沉默载体pTV-MiColi处理番茄植株的根结数比空载体对照及清水对照分别减少49.4％和49.2％,表明MiCol基因沉默显著降低了南方根结线虫的侵染数量.     

GhMAPK参与调控棉花对链格孢的抗性

丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)级联途径,由三种级次磷酸化的MAPKKK (MAPK kinase kinase)、MAPKK (MAPK kinase)和MAPK蛋白激酶组成,广泛参与植物防御反应的调控。本文利用已发布的陆地棉Gossypium hirsutum L.全基因组数据,对其中的MAPK家族基因编码序列进行多序列比对和聚类分析。结果显示,53个GhMAPK大都含有植物MAPK保守的TXY基序,并且能够聚集到AtMAPK的A、B、C、D四个族中。进而利用病毒诱导的基因沉默(virus-induced gene silencing, VIGS)技术,研究GhMAPK是否参与调控棉花对链格孢Alternaria alternata抗性。链格孢是引起棉花轮纹斑病并导致棉花早衰的主要病原真菌。结果发现,沉默GhMAPK3、GhMAPK7、GhMAPK9及GhMAPK19的棉花植株,抗病性显著下降,表明这4种GhMAPK正调控棉花对链格孢的抗性。进一步利用靶向性沉默策略,验证这4种GhMAPK中各同源基因对棉花链格孢抗性的调控作用。结果显示,仅同源基因GhMAPK3b、GhMAPK7b、GhMAPK7c、GhMAPK9a、GhMAPK9b、GhMAPK19b和GhMAPK19c参与正调控棉花对链格孢的抗性,而同源基因GhMAPK3a、GhMAPK7a、GhMAPK7d和GhMAPK19a并不调控棉花对链格孢的抗性。     

SGT1 and HSP90 are essential for age-related non-host resistance of Nicotiana benthamiana against the oomycete pathogen Phytophthora infestans

The oomycete pathogen, Phytophthora infestans, is the causal agent of potato late blight, which is one of the most destructive and economically important plant diseases. We investigated the interaction between P. infestans and Solanaceous model plant Nicotiana benthamiana. Mature N. benthamiana plants were resistant to 8 isolates of P. infestans, whereas relatively young plants were susceptible to all isolates. Analysis with virus-induced gene silencing (VIGS) indicated that NbSGT1 and NbHSP90, genes essential for the function of R proteins, are required for the resistance of N. benthamiana to P. infestans. NbSGT1 was also required for the production of reactive oxygen species (ROS), hypersensitive cell death and expression of NbEAS, a gene for phytoalexin biosynthesis, induced by INF1, a secretory protein derived from P. infestans. These results suggested that N. benthamiana possibly possesses a broad-spectrum R protein against P. infestans, which requires an SGT1/HSP90-dependent mechanism, for the recognition of a conserved molecular pattern of P. infestans.     

Molecular cloning and virus-induced gene silencing of MiASB in the southern root-knot nematode, Meloidogyne incognita

Root-knot nematodes (Meloidogyne spp.), cause serious damage to agricultural production worldwide. In this study, we designed special primers based on the predicted Mitochondrial ATP synthase b subunit gene (ASB) sequence to clone the same gene in M. incognita (MiASB). The identity between the cloned MiASB and the predicted MiASB was as high as 100 %. Using the tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) system, we delivered MiASB RNAi triggers to the M. incognita feeding site on tomato seedlings, resulting in significantly fewer galls on the seedlings. Sixty days after inoculation with M. incognita, the number of root galls induced on the MiASB silence-treated seedlings was reduced by 64.3 % compared to that on the control seedlings, and reduced by 64.1 % compared to that on untreated control seedlings. This study revealed the MiASB silencing had a positive effect on the control of root-knot nematodes, and MiASB may be associated with the formation of galls caused by the nematode.     

本氏烟NbWRKY40亚家族转录因子抗病相关功能研究

 植物WRKY类转录因子的IIa亚类广泛参与调控植物的生物胁迫和非生物胁迫过程。本研究根据拟南芥和普通烟中具有抗病和抗胁迫功能的IIa亚类WRKY40转录因子的序列,利用基因同源克隆法,获得本氏烟中NbWRKY40基因。序列分析表明在本氏烟中有5个属于IIa亚类的基因,其序列之间具有高度相似性。qRT-PCR检测发现NbWRKY40基因是受寄生疫霉侵染诱导上调表达的基因,通过VIGS技术研究该类基因在本氏烟中的抗病相关功能,用半活体营养型寄生疫霉进行抗病性试验,结果表明NbWRKY40基因的沉默降低了本氏烟对寄生疫霉的抗性,且在侵染点的细胞坏死程度增强,过氧化氢积累和胼胝质沉积都明显减少。NbWRKY40基因沉默同样降低了本氏烟对死体营养型灰霉菌的抗性。检测NbWRKY40基因沉默后4个不同抗病信号通路下游基因的表达,发现基因沉默导致参与抗病和SA途径的PR1b、PR2b基因受疫霉侵染诱导表达的水平明显下降。综上,推测NbWRKY40基因可能依靠SA介导的信号通路参与调控本氏烟抗病性。