Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a powerful tool for functional analysis of genes in plants. A wide-host-range VIGS vector, which was developed based on the Cucumber mosaic virus (CMV), was tested for its ability to silence endogenous genes involved in flavonoid biosynthesis in soybean. Symptomless infection was established using a pseudorecombinant virus, which enabled detection of specific changes in metabolite content by VIGS. It has been demonstrated that the yellow seed coat phenotype of various cultivated soybean lines that lack anthocyanin pigmentation is induced by natural degradation of chalcone synthase ( CHS ) mRNA. When soybean plants with brown seed coats were infected with a virus that contains the CHS gene sequence, the colour of the seed coats changed to yellow, which indicates that the naturally occurring RNA silencing is reproduced by VIGS. In addition, CHS VIGS consequently led to a decrease in isoflavone content in seeds. VIGS was also tested on the putative flavonoid 3'-hydroxylase ( F3'H ) gene in the pathway. This experiment resulted in a decrease in the content of quercetin relative to kaempferol in the upper leaves after viral infection, which suggests that the putative gene actually encodes the F3'H protein. In both experiments, a marked decrease in the target mRNA and accumulation of short interfering RNAs were detected, indicating that sequence-specific mRNA degradation was induced. The present report is a successful demonstration of the application of VIGS for genes involved in flavonoid biosynthesis in plants; the CMV-based VIGS system provides an efficient tool for functional analysis of soybean genes.     

Efficient virus-induced gene silencing in plants using a modified geminivirus DNA1 component

Virus‐induced gene silencing (VIGS) is currently recognized as a powerful reverse genetics tool for application in functional genomics. DNA1, a satellite‐like and single‐stranded DNA molecule associated with begomoviruses (Family Geminiviridae), has been shown to replicate autonomously but requires the helper virus for its dissemination. We developed a VIGS vector based on the DNA1 component of tobacco curly shoot virus (TbCSV), a monopartite begomovirus, by inserting a multiple cloning site between the replication‐associated protein open reading frame and the A‐rich region for subsequent insertion of DNA fragments of genes targeted for silencing. When a host gene (sulphur, Su) or transgene (green fluorescent protein, GFP) was inserted into the modified DNA1 vector and co‐agroinoculated with TbCSV, efficient silencing of the cognate gene was observed in Nicotiana benthamiana plants. More interestingly, we demonstrated that this modified DNA1 could effectively suppress GFP in transgenic N. benthamiana or endogenous Su in tobacco plants when co‐agroinoculated with tomato yellow leaf curl China virus (TYLCCNV), another monopartite begomovirus that does not induce any viral symptoms. A gene‐silencing system in Nicotiana spp., Solanum lycopersicum and Petunia hybrida plants was then established using TYLCCNV and the modified DNA1 vector. The system can be used to silence genes involved in meristem and flower development. The modified DNA1 vector was used to silence the AtTOM homologous genes (NbTOM1 and NbTOM3) in N. benthamiana. Silencing of NbTOM1 or NbTOM3 can reduce tobamovirus multiplication to a lower level, and silencing of both genes simultaneously can completely inhibit tobamovirus multiplication. Previous studies have reported that DNA1 is associated with both monopartite and bipartite begomoviruses, as well as curtoviruses. This vector system can therefore be applied for the study, analysis and discovery of gene function in a variety of important crop plants.     

Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencing

Jatropha curcas L. is a small, woody tree of the Euphorbiaceae family. This plant can grow on marginal land in the tropical and subtropical regions and produces seeds containing up to 30% oil. Several Asian countries have selected Jatropha for large scale planting as a biodiesel feedstock. Nevertheless, Jatropha also possesses several undesirable traits that may limit its wide adoption. An improved understanding of plant development and the regulation of fatty acid (FA) and triacylglyceride biosynthesis in Jatropha is particularly facilitative for the development of elite crops. Here, we show that a tobacco rattle virus (TRV) vector can trigger virus-induced gene silencing (VIGS) in Jatropha. Our optimized method produced robust and reliable gene silencing in plants agroinoculated with recombinant TRV harbouring Jatropha gene sequences. We used VIGS to investigate possible functions of 13 Jatropha genes of several functional categories, including FA biosynthesis, developmental regulation and toxin biosynthesis, etc. Based on the effects of VIGS on the FA composition of newly emerged leaves, we determined the function of several genes implicated in FA biosynthesis. Moreover, VIGS was able to discriminate independent functions of related gene family members. Our results show that VIGS can be used for high-throughput screening of Jatropha genes whose functions can be assayed in leaves.     

TECHNICAL ADVANCE: Transformation of the flax rust fungus,Melampsora lini: selection via silencing of an avirulence gene

Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium‐mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non‐transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.     

A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing

Virus-induced gene silencing (VIGS) is a rapid and robust method for determining and studying the function of plant genes or expressed sequence tags (ESTs). However, only a few plant species are amenable to VIGS. There is a need for a systematic study to identify VIGS-efficient plant species and to determine the extent of homology required between the heterologous genes and their endogenous orthologs for silencing. Two approaches were used. First, the extent of phytoene desaturase (PDS) gene silencing was studied in various Solanaceous plant species using Nicotiana benthamiana NbPDS sequences. In the second approach, PDS sequences from a wide range of plant species were used to silence the PDS gene in N. benthamiana. The results showed that tobacco rattle virus (TRV)-mediated VIGS can be performed in a wide range of Solanaceous plant species and that heterologous gene sequences from far-related plant species can be used to silence their respective orthologs in the VIGS-efficient plant N. benthamiana. A correlation was not always found between gene silencing efficiency and percentage homology of the heterologous gene sequence with the endogenous gene sequence. It was concluded that a 21-nucleotide stretch of 100% identity between the heterologous and endogenous gene sequences is not absolutely required for gene silencing.     

Bmi1 cooperates with Dnmt1-associated protein 1 in gene silencing

Polycomb group (PcG) proteins are involved in gene silencing through chromatin modifications. Among polycomb repressive complexes (PRCs), PRC1 exhibits H2A-K119 ubiquitin E3 ligase activity. However, the molecular mechanisms underlying PRC1-mediated gene silencing remain largely obscure. In this study, we found that Bmi1 directly interacts with Dnmt-associated protein 1 (Dmap1), which has been characterized to associate with the maintenance DNA methyltransferase, Dnmt1. Bmi1 was demonstrated to form a ternary complex with Dmap1 and Dnmt1 with Dmap1 in the central position. Chromatin immunoprecipitations confirmed the ternary complex formation within the context of the PRC1 at the Bmi1 target loci. Loss of Dmap1 binding to the Bmi1 target loci was tightly associated with derepressed gene expression in Bmi1-/- cells. Dmap1 knockdown exhibited the same impact as Bmi1 knockout did on the expression of Bmi1 targets, including Hox genes. Collectively, our findings suggest that Bmi1 incorporates Dmap1 in polycomb gene silencing.     

Host- and virus-induced gene silencing of HOG1-MAPK cascade genes in Rhizophagus irregularis inhibit arbuscule development and reduce resistance of plants to drought stress

Arbuscular mycorrhizal (AM) fungi can form beneficial associations with the most terrestrial vascular plant species. AM fungi not only facilitate plant nutrient acquisition but also enhance plant tolerance to various environmental stresses such as drought stress. However, the molecular mechanisms by which AM fungal mitogen-activated protein kinase (MAPK) cascades mediate the host adaptation to drought stimulus remains to be investigated. Recently, many studies have shown that virus-induced gene silencing (VIGS) and host-induced gene silencing (HIGS) strategies are used for functional studies of AM fungi. Here, we identify the three HOG1 (High Osmolarity Glycerol 1)-MAPK cascade genes RiSte11, RiPbs2 and RiHog1 from Rhizophagus irregularis. The expression levels of the three HOG1-MAPK genes are significantly increased in mycorrhizal roots of the plant Astragalus sinicus under severe drought stress. RiHog1 protein was predominantly localized in the nucleus of yeast in response to 1 M sorbitol treatment, and RiPbs2 interacts with RiSte11 or RiHog1 directly by pull-down assay. Importantly, VIGS or HIGS of RiSte11, RiPbs2 or RiHog1 hampers arbuscule development and decreases relative water content in plants during AM symbiosis. Moreover, silencing of HOG1-MAPK cascade genes led to the decreased expression of drought-resistant genes (RiAQPs, RiTPSs, RiNTH1 and Ri14-3-3) in the AM fungal symbiont in response to drought stress. Taken together, this study demonstrates that VIGS or HIGS of AM fungal HOG1-MAPK cascade inhibits arbuscule development and expression of AM fungal drought-resistant genes under drought stress.     

病毒诱导的基因沉默技术用于植物色素代谢机制的研究进展

色彩是评价园艺植物观赏性状的重要指标,而植物色素是影响植物色彩表型的关键因子。植物色素及其代谢产物在植物观赏器官颜色形成、植株生长发育调节及对逆境胁迫的响应等方面发挥着重要的作用,是植物研究领域长期关注的热点问题。病毒诱导基因沉默(virus-induced gene silencing,VIGS)是利用植物同源依赖性防御机制,特异性降低宿主内源性基因表达的一种重要基因组学工具,能够通过快速诱导植物基因沉默表型的产生,表征基因的功能,为缺乏遗传转化体系的植物的基因功能鉴定提供高效可行的替代方案。本文综述了VIGS技术在植物色素的生物合成、降解和调控机制上的应用现状,并探讨了VIGS技术在探究色素调控机制上的潜力和未来前景,以期进一步完善对不同植物色素的代谢过程和调控机制的理解,为改良植物色彩性状提供参考依据。     

Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats

Plant virus-based vectors carrying sequences homologous to endogenous genes trigger silencing through a homology-dependent RNA degradation mechanism. This phenomenon, called virus-induced gene silencing (VIGS), has potential as a powerful reverse-genetics tool in functional genomic programmes through transient, loss-of-function screens. Here, we describe a method to enhance the robustness of the VIGS phenotype by increasing the level of dsRNA molecule production, a critical step in the VIGS response. Incorporation of 40-60 base direct inverted-repeats into a plant viral vector generates RNA molecules that form dsRNA hairpins. A tobacco mosaic virus (TMV)-based vector carrying such inverted-repeats, homologous to a green fluorescent protein (gfp) transgene or an endogenous phytoene desaturase (pds) gene, generated a stronger and more pervasive VIGS phenotype than constructs carrying corresponding cDNA fragments in sense or antisense orientation. Real-time RT-PCR indicated that there was up to a threefold reduction in target mRNA accumulation in the tissues where VIGS was triggered by constructs carrying inverted-repeats compared to those where it was triggered by sense or antisense constructs. Moreover, an enhanced VIGS pds phenotype was observed using a different vector, based on barley stripe mosaic virus, in the monocotyledonous host barley. This demonstrates that VIGS can be significantly improved through the inclusion of small inverted-repeats in plant virus-based vectors, generating a more robust loss-of-function phenotype. This suggests that dsRNA formation can be a limiting factor in the VIGS phenomenon.     

Inactivation of a wheat protein kinase gene confers broad-spectrum resistance to rust fungi

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Reduction of Phakopsora pachyrhizi infection on soybean through host- and spray-induced gene silencing

Asian soybean rust (ASR), caused by the obligate fungal pathogen Phakopsora pachyrhizi, often leads to significant yield losses and can only be managed through fungicide applications currently. In the present study, eight urediniospore germination or appressorium formation induced P. pachyrhizi genes were investigated for their feasibility to suppress ASR through a bean pod mottle virus (BPMV)-based host-induced gene silencing (HIGS) strategy. Soybean plants expressing three of these modified BPMV vectors suppressed the expression of their corresponding target gene by 45%–80%, fungal biomass accumulation by 58%–80%, and significantly reduced ASR symptom development in soybean leaves after the plants were inoculated with P. pachyrhizi, demonstrating that HIGS can be used to manage ASR. In addition, when the in vitro synthesized double-stranded RNAs (dsRNAs) for three of the genes encoding an acetyl-CoA acyltransferase, a 40S ribosomal protein S16, and glycine cleavage system H protein were sprayed directly onto detached soybean leaves prior to P. pachyrhizi inoculation, they also resulted in an average of over 73% reduction of pustule numbers and 75% reduction in P. pachyrhizi biomass accumulation on the detached leaves compared to the controls. To the best of our knowledge, this is the first report of suppressing P. pachyrhizi infection in soybean through both HIGS and spray-induced gene silencing. It was demonstrated that either HIGS constructs targeting P. pachyrhizi genes or direct dsRNA spray application could be an effective strategy for reducing ASR development on soybean.     

Virus-induced gene silencing in Solanum species

Virus-induced gene silencing (VIGS) has been used routinely in Nicotiana benthamiana to assess functions of candidate genes and as a way to discover new genes required for diverse pathways, especially disease resistance signalling. VIGS has recently been shown to work in Arabidopsis thaliana and in tomato. Here, we report that VIGS using the tobacco rattle virus (TRV) viral vector can be used in several Solanum species, although the choice of vector and experimental conditions vary depending on the species under study. We have successfully silenced the phytoene desaturase (PDS) gene in the diploid wild species Solanum bulbocastanum and S. okadae, in the cultivated tetraploid S. tuberosum and in the distant hexaploid relative S. nigrum (commonly known as deadly nightshade). To test whether the system could be utilised as a rapid way to assess gene function of candidate resistance (R) genes in potato and its wild relatives, we silenced R1 and Rx in S. tuberosum and RB in S. bulbocastanum. Silencing of R1, Rx and RB successfully attenuated R-gene-mediated disease resistance and resulted in susceptible phenotypes in detached leaf assays. Thus, the VIGS system is an effective method of rapidly assessing gene function in potato.     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking CrylAb expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the crylAb gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of crylAb gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%-30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low con     

Dynamics of gene silencing by RNA interference

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.