Efficient Virus-induced Gene Silencing Research Articles

Integrating cotyledon-based virus-induced gene silencing with visual marker promises a rapid, highly effective validation of gene functions in Nepeta cataria.

Nepeta spp. generate volatile nepetalactone iridoids that have cat-attractant and insect-repellent activities. They differ from typical mint family (Lamiaceae) iridoids, which are non-volatile glucosides, and also vary from other species in the Nepetoideae sub-family, which do not generate iridoids. The chemistry and evolution of Nepeta make it suitable for further investigation. However, the lack of transgenic technology hampers the molecular and genetic investigations in Nepeta. Virus-induced gene silencing (VIGS) is a powerful tool to detect gene functions in vivo. Here, we constructed a modified VIGS method in Nepeta cataria, using cotyledon infiltration, with the gene silencing effect spreading to the first two pairs of true leaves. The VIGS efficiency reached as high as 84.4%, and the procedure takes only 3 weeks. We employed this method to validate the role of geraniol 8-hydroxylase in nepetalactone biosynthesis with ChlH as a visual marker in N. cataria. The method is also applicable to Nepeta mussinii. Thus, we developed an easy and effective VIGS approach, which will be advantageous for endogenous gene studies in two Nepeta species and holds the potential for application in other plants.

Efficient virus-induced gene silencing in Primulina using two virus vector systems

Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method and an effective technique for analyzing plant gene function. However, to date there have been no reports on the establishment of a VIGS system in Primulina, which includes over 200 species of plants, most of which have high ornamental value. In this study, we successfully established a VIGS system in P. 'Alfonso' and P. carinata using TRV vector system with phytoene desaturase (PDS) as marker gene. After being treated with pTRV2-PDS vector, both P. 'Alfonso' and P. carinata plants showed a pronounced photobleaching phenotype. The system was further optimized by exploring different infection conditions. The optimized TRV-based VIGS system was as follows: Agrobacterium OD600 = 0.5, leaf vacuum infiltration, and the silencing efficiency of the system can reach 75 %. Chlorata42 was used as another marker gene and further verified the establishment of TRV VIGS system in P. 'Alfonso'. In addition, the CaLCuV-based VIGS system was also established with PDS as marker gene in P. 'Alfonso'. The establishment of the TRV-based and CaLCuV-based VIGS systems provided a breakthrough in the validation methods for identifying gene function in Primulina genus.

Advancing virus-induced gene silencing in sunflower: key factors of VIGS spreading and a novel simple protocol

Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues.

Establishment and application of a root wounding-immersion method for efficient virus-induced gene silencing in plants.

In the post-genomic era, virus-induced gene silencing (VIGS) has played an important role in research on reverse genetics in plants. Commonly used Agrobacterium-mediated VIGS inoculation methods include stem scratching, leaf infiltration, use of agrodrench, and air-brush spraying. In this study, we developed a root wounding-immersion method in which 1/3 of the plant root (length) was cut and immersed in a tobacco rattle virus (TRV)1:TRV2 mixed solution for 30 min. We optimized the procedure in Nicotiana benthamiana and successfully silenced N. benthamiana, tomato (Solanum lycopersicum), pepper (Capsicum annuum L.), eggplant (Solanum melongena), and Arabidopsis thaliana phytoene desaturase (PDS), and we observed the movement of green fluorescent protein (GFP) from the roots to the stem and leaves. The silencing rate of PDS in N. benthamiana and tomato was 95-100%. In addition, we successfully silenced two disease-resistance genes, SITL5 and SITL6, to decrease disease resistance in tomatoes (CLN2037E). The root wounding-immersion method can be used to inoculate large batches of plants in a short time and with high efficiency, and fresh bacterial infusions can be reused several times. The most important aspect of the root wounding-immersion method is its application to plant species susceptible to root inoculation, as well as its ability to inoculate seedlings from early growth stages. This method offers a means to conduct large-scale functional genome screening in plants.

An efficient virus-induced gene silencing of PDS gene in Solanum lycopersicum (cv. Rio Grande) and its functional analysis

An efficient virus-induced gene silencing of PDS gene in Solanum lycopersicum (cv. Rio Grande) and its functional analysis

Visible gland constantly traces virus-induced gene silencing in cotton.

A virus-induced gene silencing (VIGS) system was established to induce endogenous target gene silencing by post-transcriptional gene silencing (PTGS), which is a powerful tool for gene function analysis in plants. Compared with stable transgenic plant via Agrobacterium-mediated gene transformation, phenotypes after gene knockdown can be obtained rapidly, effectively, and high-throughput through VIGS system. This approach has been successfully applied to explore unknown gene functions involved in plant growth and development, physiological metabolism, and biotic and abiotic stresses in various plants. In this system, GhCLA1 was used as a general control, however, silencing of this gene leads to leaf albino, wilting, and plant death ultimately. As such, it cannot indicate the efficiency of target gene silencing throughout the whole plant growth period. To address this question, in this study, we developed a novel marker gene, Gossypium PIGMENT GLAND FORMATION GENE (GoPGF), as the control to trace the efficiency of gene silencing in the infected tissues. GoPGF has been proved a key gene in gland forming. Suppression of GoPGF does not affect the normal growth and development of cotton. The number of gland altered related to the expression level of GoPGF gene. So it is a good marker that be used to trace the whole growth stages of plant. Moreover, we further developed a method of friction inoculation to enhance and extend the efficiency of VIGS, which facilitates the analysis of gene function in both the vegetative stage and reproductive stage. This improved VIGS technology will be a powerful tool for the rapid functional identification of unknown genes in genomes.

Efficient virus-induced gene silencing system in pumpkin (Cucurbita maxima) using apple latent spherical virus vector

Crude-sap of apple latent spherical virus (ALSV)-infected Chenopodium quinoa leaves was rub-inoculated on the expanded cotyledons of various Cucurbitaceae plants. Most of the species were systemically infected with the virus without obvious symptoms, except pumpkin (Cucurbita maxima). In pumpkin, the ALSV infection was restricted to inoculated cotyledons; it did not spread to the upper true leaves. In situ hybridization showed that the ALSV was confined to part of the cotyledon tissues and it did not invade the phloem tissue, when inoculated at the expanded cotyledon stage. However, when total RNAs from ALSV-infected C. quinoa leaves were inoculated into the cotyledons immediately after germination (folded cotyledon stage) using particle bombardment, ALSV efficiently caused systemic infection. Systemic infection of pumpkin seedlings occurred only when the cotyledons were inoculated within a few days after germination. No systemic infection was observed in the seedlings 4 days after germination. In the grafting test, ALSV was not transmitted from the infected rootstocks to the healthy scions of pumpkins. An efficient virus-induced gene silencing system for pumpkins was established, in which infection with ALSV vectors harboring the phytoene desaturase or sulfur gene fragments resulted in a uniform phenotype in the true leaves of pumpkin seedlings.

Virus-Induced Gene Silencing as a Tool to Study Regulation of Alkaloid Biosynthesis in Medicinal Plants.

Advancements in genomics and transcriptomics have generated invaluable resources for the discovery of novel genes related to complex specialized metabolic pathways in plants. Virus-induced gene silencing (VIGS) has emerged as a powerful tool that is widely used for rapid functional characterization of genes in planta. VIGS has advantages over other reverse genetic approaches, such as RNAi-mediated suppression or T-DNA knockout, because it does not require the development of stable transgenic lines which is technically challenging and time consuming. Catharanthus roseus is an important medicinal plant that produces more than a hundred monoterpenoid indole alkaloids (MIAs), including the antineoplastic drugs vincristine and vinblastine. Biosynthesis of these alkaloids is strikingly complex, resulting in MIA accumulation in low quantities. Jasmonic acid (JA) is an elicitor of the MIA biosynthesis. Exogenous application of JA in C. roseus induces MIA pathway gene expression and increases MIA accumulation. The core JA signaling module comprises multiple components including the JA coreceptor Coronatine-Insensitive 1(COI1). COI1 plays a key role in JA-responsive gene expression in plants. Because generation of stable transgenic C. roseus plants is challenging, VIGS is being used for functional characterization of genes in the MIA pathway. Here we describe a detailed method for the VIGS-mediated suppression of C. roseus COI1(CrCOI1) expression to decipher the regulatory mechanism of JA-induced elicitation of MIA biosynthesis. When performing VIGS, gene silencing efficiency and the viral spread are monitored by the development of visible phenotype in the control plants. We use the C. roseus phytoene desaturase (CrPDS) and Protoporphyrin IX Mg-chelatase subunit H (CrChlH) as visual markers to access VIGS efficiency and viral spread. The protocol described here could be used for the functional characterization of genes involved in other metabolic pathways and in other medicinal plants.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin

BackgroundLupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV).ResultsWe identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)—a gene likely to be involved in toxic alkaloid biosynthesis—we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a ‘PDS co-silencing’ approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL.ConclusionsWe provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

Promoting virus-induced gene silencing of pepper genes by a heterologous viral silencing suppressor.

Pepper is an important vegetable cultivated worldwide. Pepper fruits accumulate unique metabolites, capsanthin and capsaicin, which are important raw materials for natural pigment and medicine. Pepper plants are rich in genetic diversity and are attractive subjects for fruit developmental studies. The release of pepper reference genomes provided key genetic information for dissecting function of pepper genes underlying various interesting phenotypes (Kim et al., 2014; Qin et al., 2014). However, due to the difficulty in transformation, dissecting genetic mechanisms in pepper remained technically challenging.

Temperature modulates virus-induced transcriptional gene silencing via secondary small RNAs.

Virus-induced gene silencing (VIGS) can be harnessed to sequence-specifically degrade host transcripts and induce heritable epigenetic modifications referred to as virus-induced post-transcriptional gene silencing (ViPTGS) and virus-induced transcriptional gene silencing (ViTGS), respectively. Both ViPTGS and ViTGS enable manipulation of endogenous gene expression without the need for transgenesis. Although VIGS has been widely used in many plant species, it is not always uniform or highly efficient. The efficiency of VIGS is affected by developmental, physiological and environmental factors. Here, we use recombinant Tobacco rattle viruses (TRV) to study the effect of temperature on ViPTGS and ViTGS using GFP as a reporter gene of silencing in N.benthamiana 16c plants. We found that unlike ViPTGS, ViTGS was impaired at high temperature. Using a novel mismatch-small interfering RNA (siRNA) tool, which precisely distinguishes virus-derived (primary) from target-generated (secondary) siRNAs, we demonstrated that the lack of secondary siRNA production/amplification was responsible for inefficient ViTGS at 29°C. Moreover, inefficient ViTGS at 29°C inhibited the transmission of epigenetic gene silencing to the subsequent generations. Our finding contributes to understanding the impact of environmental conditions on primary and secondary siRNA production and may pave the way to design/optimize ViTGS for transgene-free crop improvement.

An efficient papaya leaf distortion mosaic potyvirus vector for virus-induced gene silencing in papaya

Papaya (Carica papaya L.) is regarded as an excellent model for genomic studies of tropical trees because of its short generation time and its small genome that has been sequenced. However, functional genomic studies in papaya depend on laborious genetic transformations because no rapid tools exist for this species. Here, we developed a highly efficient virus-induced gene silencing (VIGS) vector for use in papaya by modifying an artificially attenuated infectious clone of papaya leaf distortion mosaic virus (PLDMV; genus: Potyvirus), PLDMV-E, into a stable Nimble Cloning (NC)-based PLDMV vector, pPLDMV-NC, in Escherichia coli. The target fragments for gene silencing can easily be cloned into pPLDMV-NC without multiple digestion and ligation steps. Using this PLDMV VIGS system, we silenced and characterized five endogenous genes in papaya, including two common VIGS marker genes, namely, phytoene desaturase, Mg-chelatase H subunit, putative GIBBERELLIN (GA)-INSENSITIVE DWARF1A and 1B encoding GA receptors; and the cytochrome P450 gene CYP83B1, which encodes a key enzyme involved in benzylglucosinolate biosynthesis. The results demonstrate that our newly developed PLDMV VIGS vector is a rapid and convenient tool for functional genomic studies in papaya.

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.

Virus-induced gene silencing as a tool for functional studies in Cleome violacea.

PremiseCleomaceae is emerging as a promising family to investigate a wide range of phenomena, such as C4 photosynthesis and floral diversity. However, functional techniques are lacking for elucidating this diversity. Herein, we establish virus‐induced gene silencing (VIGS) as a method of generating functional data for Cleome violacea, bolstering Cleomaceae as a model system.MethodsWe leveraged the sister relationship of Cleomaceae and Brassicaceae by using constructs readily available for Arabidopsis thaliana to provide initial information about the feasibility of VIGS in C. violacea. We then developed endogenous constructs to optimize VIGS efficiency and viability for fruit development.Results PHYTOENE DESATURASE was successfully downregulated in C. violacea using both heterologous and endogenous constructs. The endogenous construct had the highest degree of downregulation, with many plants displaying strong photobleaching. FRUITFULL‐treated plants were also successfully downregulated, with a high rate of survival but less effective silencing; only a small percentage of survivors showed a strong phenotype.DiscussionOur optimized VIGS protocol in C. violacea enables functional gene analyses at different developmental stages. Additionally, C. violacea is amenable to heterologous knockdown, which suggests that a first pass using non‐endogenous constructs is a possible route to test additional species of Cleomaceae.

Identification of citrus immune regulators involved in defence against Huanglongbing using a new functional screening system.

SummaryHuanglongbing (HLB) is the most devastating citrus disease in the world. Almost all commercial citrus varieties are susceptible to the causal bacterium, Candidatus Liberibacter asiaticus (CLas), which is transmitted by the Asian citrus psyllid (ACP). Currently, there are no effective management strategies to control HLB. HLB‐tolerant traits have been reported in some citrus relatives and citrus hybrids, which offer a direct pathway for discovering natural defence regulators to combat HLB. Through comparative analysis of small RNA profiles and target gene expression between an HLB‐tolerant citrus hybrid (Poncirus trifoliata × Citrus reticulata) and a susceptible citrus variety, we identified a panel of candidate defence regulators for HLB‐tolerance. These regulators display similar expression patterns in another HLB‐tolerant citrus relative, with a distinct genetic and geographic background, the Sydney hybrid (Microcitrus virgata). Because the functional validation of candidate regulators in tree crops is always challenging, we developed a novel rapid functional screening method, using a C. Liberibacter solanacearum (CLso)/potato psyllid/Nicotiana benthamiana interaction system to mimic the natural transmission and infection circuit of the HLB complex. When combined with efficient virus‐induced gene silencing in N. benthamiana, this innovative and cost‐effective screening method allows for rapid identification and functional characterization of regulators involved in plant immune responses against HLB, such as the positive regulator BRCA1‐Associated Protein, and the negative regulator Vascular Associated Death Protein.

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.

An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)-based vector.

CaLCuV-based VIGS effectively works in cabbage and contributes to efficient functional genomics research in Brassica crop species. Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, is an effective technique for analysing the functions of genes in plants. However, no VIGS vectors have been available for Brassica oleracea until now. Here, tobacco rattle virus (TRV), pTYs and cabbage leaf curl virus (CaLCuV) gene-silencing vectors (PCVA/PCVB) were chosen to improve the VIGS system in cabbage using the phytoene desaturase (PDS) gene as an efficient visual indicator of VIGS. We successfully silenced the expression of PDS and observed photobleaching phenomena in cabbage in response to pTYs and CaLCuV, with the latter being more easy to operate and less expensive. The parameters potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including the targeting fragment strategy, inoculation method and incubation temperature, were then compared. The optimized CaLCuV-based VIGS system involves the following: an approximately 500bp insert sequence, an Agrobacterium OD600 of 1.0, use of the vacuum osmosis method applied at the bud stage, and an incubation temperature of 22°C. Using these parameters, we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected the Mg-chelatase H subunit (ChlH) gene in cabbage and knocked down its expression, and we observed yellow leaves, as expected. We successfully applied the CaLCuV-based VIGS system to two other representative Brassica crop species, B. rapa and B. nigra, and thus expanded the application scope of this system. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crop species.

Development of an efficient virus-induced gene silencing method in petunia using the pepper phytoene desaturase (PDS) gene

Virus-induced gene silencing (VIGS) is widely used for functional analysis of genes in plants. Because of the variation in its effectiveness among plant species, the VIGS system is normally optimized using the phytoene desaturase (PDS) gene as a visible indicator. In this study, we optimized an efficient VIGS system for petunias using the PDS gene isolated from pepper (Capsicum annuum) plants as a visible indicator. Application of the CaPDS gene effectively induced gene silencing in the petunia cultivars Mirage Rose, Mirage Pink, and Picobella Blue. However, silencing effectiveness was observed to be limited by genotypes and inoculation methods; apical meristem application was the appropriate method, whereas the highest silencing effectiveness among the cultivars was found in Picobella Blue. In addition, it was found that higher silencing effectiveness was associated with higher degradation of the endogenous PhPDS mRNA. Moreover, in terms of plant age and temperatures, 3-week-old plants grown at 20 °C day/18 °C night showed high silencing effectiveness for all cultivars. Taken together, infection of apical meristem of 3-week-old plants grown at 20 °C day/18 °C night with pTRV2-CaPDS exhibited the highest silencing effectiveness of all cultivars. Therefore, this study indicated the effectiveness of the CaPDS in the VIGS experiment with petunias, as well as the involvement of different factors in the mechanism influencing silencing effectiveness in petunias. These results will enable high-throughput functional analysis of genes associated with commercially important traits in petunias. CaPDS gene effectively induced gene silencing in the petunia cultivars, however, higher silencing effectiveness was observed to be limited by genotypes, inoculation methods, plant age, inoculation temperature, and presence of endogenous PhPDS mRNA.

Chinese Wheat Mosaic Virus-Induced Gene Silencing in Monocots and Dicots at Low Temperature.

Virus-induced gene silencing (VIGS) is an important tool for functional genomics studies in plants. With this method, it is possible to target most endogenous genes and downregulate the messenger RNA (mRNA) in a sequence-specific manner. Chinese wheat mosaic virus (CWMV) has a bipartite, single-strand positive RNA genome, and can infect both wheat and Nicotiana benthamiana, and the optimal temperature for systemic infection in plants is 17°C. To assess the potential of the virus as a vector for gene silencing at low temperature, a fragment of the N. benthamiana or wheat phytoene desaturase (PDS) gene was expressed from a modified CWMV RNA2 clone and the resulting photo bleaching in infected plants was used as a reporter for silencing. Downregulation of PDS mRNA was also measured by quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR). In experiments using fragments of PDS ranging from 500 to 1500 nucleotides, insert length influenced the stability and the efficiency of VIGS. The CWMV induced silencing system was also used to suppress miR165/166 and miR3134a through expression of miRNA target mimics. The relative expression levels of mature miR165/166 and miR3134a decreased whereas the transcript levels of their target genes increased. Interestingly, we also found the CWMV-induced silencing system was more efficient compare with the vector based on Barley stripe mosaic virus (BSMV) or Foxtail mosaic virus (FoMV) in wheat or the vector based on TRV in N. benthamiana at 17°C. In summary, the CWMV vector is effective in silencing endogenous genes and miRNAs at 17°C, thereby providing a powerful tool for gene function analysis in both N. benthamiana and wheat at low temperature.

An efficient and improved method for virus-induced gene silencing in sorghum

BackgroundAlthough the draft genome of sorghum is available, the understanding of gene function is limited due to the lack of extensive mutant resources. Virus-induced gene silencing (VIGS) is an alternative to mutant resources to study gene function. This study reports an improved and efficient method for Brome mosaic virus (BMV)-based VIGS in sorghum.MethodsSorghum plants were rub-inoculated with sap prepared by grinding 2 g of infected Nicotiana benthamiana leaf in 1 ml 10 mM potassium phosphate buffer (pH 6.8) and 100 mg of carborundum abrasive. The sap was rubbed on two to three top leaves of sorghum. Inoculated plants were covered with a dome to maintain high humidity and kept in the dark for two days at 18 °C. Inoculated plants were then transferred to 18 °C growth chamber with 12 h/12 h light/dark cycle.ResultsThis study shows that BMV infection rate can be significantly increased in sorghum by incubating plants at 18 °C. A substantial variation in BMV infection rate in sorghum genotypes/varieties was observed and BTx623 was the most susceptible. Ubiquitin (Ubiq) silencing is a better visual marker for VIGS in sorghum compared to other markers such as Magnesium Chelatase subunit H (ChlH) and Phytoene desaturase (PDS). The use of antisense strand of a gene in BMV was found to significantly increase the efficiency and extent of VIGS in sorghum. In situ hybridization experiments showed that the non-uniform silencing in sorghum is due to the uneven spread of the virus. This study further demonstrates that genes could also be silenced in the inflorescence of sorghum.ConclusionIn general, sorghum plants are difficult to infect with BMV and therefore recalcitrant to VIGS studies. However, by using BMV as a vector, a BMV susceptible sorghum variety, 18 °C for incubating plants, and antisense strand of the target gene fragment, efficient VIGS can still be achieved in sorghum.