Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants

Virus-induced gene silencing (VIGS) is an effective tool for studying the functions of plant genes, but only a few VIGS vectors available for woody plants were reported so far. Here we present an effective heterologous VIGS system in woody plants based on tobacco rattle virus (TRV) vectors. We first tested whether the TRV-vector can be directly applied to infect woody plant species, such as Vernicia fordii, Populus tomentosa Carr. and Camellia oleifera. The results revealed that TRV-mediated VIGS could be effectively elicited in V. fordii, weakly in P. tomentosa Carr., but not in C. oleifera. TRV-based VIGS vectors with heterologous phytoene desaturase (PDS) sequences from various woody plant species silenced successfully the endogenous PDS gene in Nicotina benthamiana and V. fordii. The photobleached leaf phenotype of silenced plants significantly correlated with the down-regulation of endogenous PDS as compared with controls. To further confirm the reliability of VIGS in V. fordii, we also isolated the cloroplastos alterados 1 gene from P. tomentosa Carr., and the silencing pheotypes of albino leaves were observed in V. fordii 2 weeks after inoculation using a heterologous TRV-based VIGS system. Taken together, we have successfully developed an Agrobacterium-mediated VIGS assay in V. fordii and demonstrated that V. fordii as a heterologous VIGS system provides a valuable tool for functional genomic analysis in woody plant species.     

Heterologous virus-induced gene silencing as a promising approach in plant functional genomics

VIGS (virus induced gene silencing) is considered as a powerful genomics tool for characterizing the function of genes in a few closely related plant species. The investigations have been carried out mainly in order to test if a pre-existing VIGS vector can serve as an efficient tool for gene silencing in a diverse array of plant species. Another route of investigation has been the constructing of new viral vectors to act in their hosts. Our approach was the creation of a heterologous system in which silencing of endogenous genes was achieved by sequences isolated from evolutionary remote species. In this study, we showed that a TRV-based vector cloned with sequences from a gymnosperm, Taxus baccata L. silenced the endogenous phytoene desaturase in an angiosperm, N. benthamiana. Our results showed that inserts of between 390 and 724 bp isolated from a conserved fragment of the Taxus PDS led to silencing of its homolog in tobacco. The real time analysis indicated that the expression of PDS was reduced 2.1- to 4.0-fold in pTRV-TbPDS infected plants compared with buffer treated plants. Once the best insert is identified and the conditions are optimized for heterologous silencing by pTRV-TbPDS in tobacco, then we can test if TRV can serve as an efficient silencing vector in Taxus. This strategy could also be used to silence a diverse array of genes from a wide range of species which have no VIGS protocol. The results also showed that plants silenced heterologously by the VIGS system a minimally affected with respect to plant growth which may be ideal for studying the genes that their complete loss of function may lead to decrease of plant growth or plant death.     

Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize

Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

A pseudorecombinant-chimeric Cucumber mosaic virus-based virus-induced gene silencing system rapidly and efficiently triggers persistent gene silencing in maize.     

Virus-Induced Gene Silencing as a Tool for Comparative Functional Studies in Thalictrum

Perennial woodland herbs in the genus Thalictrum exhibit high diversity of floral morphology, including four breeding and two pollination systems. Their phylogenetic position, in the early-diverging eudicots, makes them especially suitable for exploring the evolution of floral traits and the fate of gene paralogs that may have shaped the radiation of the eudicots. A current limitation in evolution of plant development studies is the lack of genetic tools for conducting functional assays in key taxa spanning the angiosperm phylogeny. We first show that virus-induced gene silencing (VIGS) of a PHYTOENE DESATURASE ortholog (TdPDS) can be achieved in Thalictrum dioicum with an efficiency of 42% and a survival rate of 97%, using tobacco rattle virus (TRV) vectors. The photobleached leaf phenotype of silenced plants significantly correlates with the down-regulation of endogenous TdPDS (P<0.05), as compared to controls. Floral silencing of PDS was achieved in the faster flowering spring ephemeral T. thalictroides. In its close relative, T. clavatum, silencing of the floral MADS box gene AGAMOUS (AG) resulted in strong homeotic conversions of floral organs. In conclusion, we set forth our optimized protocol for VIGS by vacuum-infiltration of Thalictrum seedlings or dormant tubers as a reference for the research community. The three species reported here span the range of floral morphologies and pollination syndromes present in Thalictrum. The evidence presented on floral silencing of orthologs of the marker gene PDS and the floral homeotic gene AG will enable a comparative approach to the study of the evolution of flower development in this group.     

Development of a virus-induced gene silencing (VIGS) system for <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L.

Virus-induced gene silencing (VIGS) is known as a rapid and efficient system for studying functions of interesting genes in plants. Tobacco rattle virus (TRV) is widely applied for the gene silencing of many plants. Although spinach is a TRV-susceptible plant, a TRV-based VIGS system has not yet been developed for spinach. In this study, we established a TRV-based VIGS system for spinach. To evaluate the functionality of the TRV-based VIGS system, the phytoene desaturase gene (SoPDS) was first isolated from spinach as a marker gene. Then, the VIGS vector pTRV2 was combined with the partial fragment of SoPDS gene in sense or antisense orientation. Using the Agrobacterium infiltration method, we introduced the pTRV2-SoPDS clone to silence the SoPDS gene in spinach. SoPDS was efficiently silenced, and consequently, greater than 90% of newly emerging leaves exhibited severe chlorosis symptoms in the treated plants. Levels of chlorosis symptoms were similar in both plants infected with pTRV2 vectors harboring sense (SoPDS_S) or antisense (SoPDS_A) gene fragments. Quantitative analysis of SoPDS gene expression by qRT-PCR revealed that gene expression was reduced by greater than 90% in both SoPDS_S and SoPDS_A VIGS plants. Chlorosis on leaves was prolonged up to 4~5 wk after Agrobacterium infiltration. The TRV-based VIGS system was effective in silencing the SoPDS gene in spinach, suggesting that it can be a useful reverse genetics tool for the functional study of spinach genes.     

An Optimized Protocol to Increase Virus-Induced Gene Silencing Efficiency and Minimize Viral Symptoms in Petunia

Virus-induced gene silencing (VIGS) is used to down-regulate endogenous plant genes. VIGS efficiency depends on viral proliferation and systemic movement throughout the plant. Although tobacco rattle virus (TRV)-based VIGS has been successfully used in petunia (Petunia?×?hybrida), the protocol has not been thoroughly optimized for efficient and uniform gene down-regulation in this species. Therefore, we evaluated six parameters that improved VIGS in petunia. Inoculation of mechanically wounded shoot apical meristems induced the most effective and consistent silencing compared to other methods of inoculation. From an evaluation of ten cultivars, a compact petunia variety, 'Picobella Blue', exhibited a 1.8-fold higher CHS silencing efficiency in corollas. We determined that 20 °C day/18 °C night temperatures induced stronger gene silencing than 23 °C/18 °C or 26 °C/18 °C. The development of silencing was more pronounced in plants that were inoculated at 3–4 versus 5 weeks after sowing. While petunias inoculated with pTRV2-NbPDS or pTRV2-PhCHS showed very minimal viral symptoms, plants inoculated with the pTRV2 empty vector (often used as a control) were stunted and developed severe necrosis, which often led to plant death. Viral symptoms were eliminated by developing a control construct containing a fragment of the green fluorescent protein (pTRV2-sGFP). These optimization steps increased the area of chalcone synthase (CHS) silencing by 69 % and phytoene desaturase (PDS) silencing by 28 %. This improved VIGS protocol, including the use of the pTRV2-sGFP control plants, provides stronger down-regulation for high-throughput analyses of gene function in petunia.     

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes

Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.

     

病毒诱导的基因沉默及其在植物基因功能研究中的应用

RNA介导的基因沉默是近年来在生物体中发现的一种基于核酸水平高度保守的特异性降解机制.病毒诱导的基因沉默（virus induced gene silencing, VIGS）是指携带植物功能基因cDNA的病毒在侵染植物体后,可诱导植物发生基因沉默而出现表型突变,进而可以研究该目的基因功能.至今,已经建立了以RNA病毒、DNA病毒、卫星病毒和DNA卫星分子为载体的VIGS体系,这些病毒载体能在多种寄主植物（包括拟南芥、番茄和大麦）上有效抑制功能基因的表达.VIGS已开始应用于N基因和Pto基因介导的抗性信号途径中关键基因的功能研究、抗病毒相关的寄主因子研究以及植物代谢和发育调控研究.在当前植物基因组或EST序列大量测定的情况下,VIGS为植物基因功能鉴定提供了有效的技术平台.     

Metabolomic profile of cacao cell suspensions growing in blue light/dark conditions with potential in food biotechnology

As one of the effective and powerful methods for gene function analysis in plants, transient overexpression and virus-induced gene silencing (VIGS) system were developed for oriental melon (Cucumis melo var. makuwa Makino). Here, the full-length of CmLOX10, the conserved domains of CmLOX10 and CmPDS were isolated from oriental melon. The vectors for overexpression of CmLOX10, and the VIGS vector of CmLOX10 and CmPDS were constructed and transformed into agrobacterium, respectively. Especially, the sprout absorption method was performed on oriental melon, and CmPDS was selected as a reporter gene in VIGS. The expression level of CmLOX10 significantly increased in the oriental melon with the CmLOX10 overexpression construct, and lipoxygenases (LOXs) contributed to the leaf cell death in the oriental melon. The expression levels of CmLOX10 and CmPDS were significantly decreased in plants with the VIGS construct, and there was albino phenotype in CmPDS silencing leaves. In this study, CmLOX10 gene was silenced using Tobacco rattle virus based VIGS system firstly. These results proved that the transient expression technology provides an excellent tool to explore gene function in oriental melon.

     

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.