TRV-mediated Virus-induced Gene Silencing Research Articles

SlSERK3B Promotes Tomato Seedling Growth and Development by Regulating Photosynthetic Capacity.

Brassinosteroids (BRs) are a group of polyhydroxylated steroids for plant growth and development, regulating numerous physiological and biochemical processes and participating in multi-pathway signaling in plants. 24-Epibrassinolide (EBR) is the most commonly used BR for the investigation of the effects of exogenous steroidal phytohormones on plant physiology. Although SlSERK3B is considered a gene involved in the brassinosteroid (BR) signaling pathway, its specific role in plant growth and development has not been reported in detail. In this study, tomato (Solanum lycopersicum L.) seedlings treated with 0.05 μmol L-1 EBR showed a significant increase in plant height, stem diameter, and fresh weight, demonstrating that BR promotes the growth of tomato seedlings. EBR treatment increased the expression of the BR receptor gene SlBRI1, the co-receptor gene SlSERK3A and its homologs SlSERK3B, and SlBZR1. The SlSERK3B gene was silenced by TRV-mediated virus-induced gene silencing (VIGS) technology. The results showed that both brassinolide (BL) content and BR synthesis genes were significantly up-regulated in TRV-SlSERK3B-infected seedlings compared to the control seedlings. In contrast, plant height, stem diameter, fresh weight, leaf area and total root length were significantly reduced in silenced plants. These results suggest that silencing SlSERK3B may affect BR synthesis and signaling, thereby affecting the growth of tomato seedlings. Furthermore, the photosynthetic capacity of TRV-SlSERK3B-infected tomato seedlings was reduced, accompanied by decreased photosynthetic pigment content chlorophyll fluorescence, and photosynthesis parameters. The expression levels of chlorophyll-degrading genes were significantly up-regulated, and carotenoid-synthesising genes were significantly down-regulated in TRV-SlSERK3B-infected seedlings. In conclusion, silencing of SlSERK3B inhibited BR signaling and reduced photosynthesis in tomato seedlings, and this correlation suggests that SlSERK3B may be related to BR signaling and photosynthesis enhancement.

Development and application of virus-induced gene silencing (VIGS) for studying ApTT8 gene function in Agapanthus praecox ssp. orientalis

Agapanthus praecox ssp. orientalis is a famous summer-blooming perennial that produces hundreds of florets with blue or white tepal. In comparison, other colours are infrequent in the genus Agapanthus. Previous analysis revealed that delphinidin was the main but not the only anthocyanidin, petunidin and cyanidin also existed in the tepal. This indicated that the colouration and regulation mechanism in A. praecox was unique and worthy of study. However, the stable genetic transformation was low efficiency and time-consuming. A rapid and effective gene functional identification method was urgently needed. In this study, the first use of the VIGS system in A. praecox was described in detail. Tobacco rattle virus (TRV) caused systemic infection with mild symptoms in A. praecox, which was also confirmed by qRT-PCR. The reporter gene ApPDS (phytoene desaturase) was silenced using the injection and soaking method in basal plates of inflorescence, and the photo-bleaching symptoms were observed in tepal. The optimal Agrobacterium concentration was OD600 = 1.0. Then the VIGS system was used to silence a bHLH-like transcriptional factor, ApTT8, to identify its function in anthocyanin biosynthesis. The results showed that the total anthocyanin content was reduced to 12.21-fold compared with the empty control. Additionally, tepals were taken as recipients of VIGS using vacuum infiltration method. Total anthocyanin content was reduced to 3.44-fold in ApTT8-silenced tepal. The silencing efficiency of tepal was as high as 85.09 %. The structure genes, ApF3’H, ApF3’5’H, ApF3H, ApDFR, ApANS and ApUFGT were also significantly down-regulated. The successful silencing of the ApPDS and ApTT8 in this study indicated that the optimized TRV-mediated VIGS system could be used to target gene silencing in florets and tepal of A. praecox. This method will significantly shorten the time required for studying molecular mechanism of flower colouration and is of great significance for the research of A. praecox functional genomics.

An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits.

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia, tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS), suitable for the plant, was explored in this study. The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS (Forsythia phytoene desaturase) using stem infiltration and leaf infiltration methods. All the treated plants were pruned below the injection site after 7–15 d infection; the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level. Meanwhile, this system has been successfully tested and verified through virus detection and qRT-PCR analysis. After the optimization, Forsythia magnesium chelatase subunit H (FsChlH) was silenced successfully in Forsythia using this system, resulting in yellow leaves with decreased chlorophyll content. The system was stable, highly efficient and had greater rapidity and convenience, which made it suitable to study the function of genes related to physiological pathways such as growth and development, and metabolic regulation in Forsythia.

Use of TRV-mediated VIGS for functional genomics research in citrus

Virus induced gene silencing (VIGS) is a powerful tool for identifying gene functions in plants. Tobacco rattle virus (TRV)-mediated VIGS has been reported for silencing of endogenous genes in many plants. In citrus, analysis of gene function is mainly relied on the table genetic transformation, even though this technique is low-efficiency and time-consuming. In this study, a TRV-mediated VIGS was successfully applied for verification of gene function in citrus. A reporter gene, Magnesium chelatase subunit I (ChlI) from C. grandis was constructed into TRV2 vector to induce gene silencing with visible photobleaching symptom. TRV VIGS induced photobleaching symptom was observed on the leaves of Nicotiana benthamiana and Citrus after infiltration. The susceptibility to VIGS vector was found to be genotype dependent. Mexican lemon is the most susceptible one. The chlorosis area per leaf was 65.5% of the whole leaf area. GXSTY pummelo exhibited less susceptible to VIGS with only tiny patches of chlorosis appeared on the leaves of treated plants. The parameters were optimized to improve the efficiency. The tiny young leaves and half expanded leaves were more susceptible to VIGS infection. Young seedlings were highly susceptible to vacuum infiltration. The expression of fluorescence from GFP reporter gene was observed in the young seedlings of GXSTY after infiltration with TRV. Close to 100% of infiltration efficiency was achieved by vacuum infiltration. The results indicated that TRV mediated VIGS with vacuum infiltration could be an ideal tool for gene function studies in citrus. A TRV mediated VIGS has been optimized to use in the citrus for verification of gene function. The sensitivity to VIGS vector was genotype dependent. Young seedling and young leaf were the suitable materials for VIGS infection. Vacuum infiltration was a high efficiency infiltration method.

Vacuum and Co-cultivation Agroinfiltration of (Germinated) Seeds Results in Tobacco Rattle Virus (TRV) Mediated Whole-Plant Virus-Induced Gene Silencing (VIGS) in Wheat and Maize.

Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) has been frequently used in dicots. Here we show that it can also be used in monocots, by presenting a system involving use of a novel infiltration solution (containing acetosyringone, cysteine, and Tween 20) that enables whole-plant level VIGS of (germinated) seeds in wheat and maize. Using the established system, phytoene desaturase (PDS) genes were successfully silenced, resulting in typical photo-bleaching symptoms in the leaves of treated wheat and maize. In addition, three wheat homoeoalleles of MLO, a key gene repressing defense responses to powdery mildew in wheat, were simultaneously silenced in susceptible wheat with this system, resulting in it becoming resistant to powdery mildew. The system has the advantages generally associated with TRV-mediated VIGS systems (e.g., high-efficiency, mild virus infection symptoms, and effectiveness in different organs). However, it also has the following further advantages: (germinated) seed-stage agroinfiltration; greater rapidity and convenience; whole-plant level gene silencing; adequately stable transformation; and suitability for studying functions of genes involved in seed germination and early plant development stages.

Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus

Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus.

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.

Virus‐induced Gene Silencing in Eggplant (Solanum melongena)

Eggplant (Solanum melongena) is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing (VIGS) has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS (PHYTOENE DESATURASE) in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su (Sulfur), CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

Tobacco mosaic virus 126-kDa Protein Increases the Susceptibility of Nicotiana tabacum to Other Viruses and Its Dosage Affects Virus-Induced Gene Silencing

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein-green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.