Virus-induced Gene Silencing Vector Research Articles

Comprehensive analysis of pepper (Capsicum annuum) RAV genes family and functional identification of CaRAV1 under chilling stress

BackgroundDespite its known significance in plant abiotic stress responses, the role of the RAV gene family in the response of Capsicum annuum to chilling stress remains largely unexplored.ResultsIn this study, we identified and characterized six members of the CaRAV gene subfamily in pepper plants through genome-wide analysis. Subsequently, the CaRAV subfamily was classified into four branches based on homology with Arabidopsis thaliana, each exhibiting relatively conserved domains within the branch. We discovered that light response elements accounted for the majority of CaRAVs, whereas low-temperature response elements were specific to the NGA gene subfamily. After pepper plants were subjected to chilling stress, qRT‒PCR analysis revealed that CaRAV1, CaRAV2 and CaNGA1 were significantly induced in response to chilling stress, indicating that CaRAVs play a role in the response to chilling stress. Using virus-induced gene silencing (VIGS) vectors, we targeted key members of the CaRAV gene family. Under normal growth conditions, the MDA content and SOD enzyme activity of the silenced plants were slightly greater than those of the control plants, and the REC activity was significantly greater than that of the control plants. The levels of MDA and electrolyte leakage were greater in the silenced plants after they were exposed to chilling stress, and the POD and CAT enzyme activities were significantly lower than those in the control, which was particularly evident under repeated chilling stress. In addition, the relative expression of CaPOD and CaCAT was greater in V2 plants upon repeated chilling stress, especially CaCAT was significantly greater in V2 plants than in the other two silenced plants, with 3.29 and 1.10 increases within 12 and 24 h. These findings suggest that CaRAV1 and CaNGA1 positively regulate the response to chilling stress.ConclusionsSilencing of key members of the CaRAV gene family results in increased susceptibility to chilling damage and reduced antioxidant enzyme activity in plants, particularly under repeated chilling stress. This study provides valuable information for understanding the classification and putative functions of RAV transcription factors in pepper plants.

Umbravirus-like RNA viruses are capable of independent systemic plant infection in the absence of encoded movement proteins.

The signature feature of all plant viruses is the encoding of movement proteins (MPs) that supports the movement of the viral genome into adjacent cells and through the vascular system. The recent discovery of umbravirus-like viruses (ULVs), some of which only encode replication-associated proteins, suggested that they, as with umbraviruses that lack encoded capsid proteins (CPs) and silencing suppressors, would require association with a helper virus to complete an infection cycle. We examined the infection properties of 2 ULVs: citrus yellow vein associated virus 1 (CY1), which only encodes replication proteins, and closely related CY2 from hemp, which encodes an additional protein (ORF5CY2) that was assumed to be an MP. We report that both CY1 and CY2 can independently infect the model plant Nicotiana benthamiana in a phloem-limited fashion when delivered by agroinfiltration. Unlike encoded MPs, ORF5CY2 was dispensable for infection of CY2, but was associated with faster symptom development. Examination of ORF5CY2 revealed features more similar to luteoviruses/poleroviruses/sobemovirus CPs than to 30K class MPs, which all share a similar single jelly-roll domain. In addition, only CY2-infected plants contained virus-like particles (VLPs) associated with CY2 RNA and ORF5CY2. CY1 RNA and a defective (D)-RNA that arises during infection interacted with host protein phloem protein 2 (PP2) in vitro and in vivo, and formed a high molecular weight complex with sap proteins in vitro that was partially resistant to RNase treatment. When CY1 was used as a virus-induced gene silencing (VIGS) vector to target PP2 transcripts, CY1 accumulation was reduced in systemic leaves, supporting the usage of PP2 for systemic movement. ULVs are therefore the first plant viruses encoding replication and CPs but no MPs, and whose systemic movement relies on a host MP. This explains the lack of discernable helper viruses in many ULV-infected plants and evokes comparisons with the initial viruses transferred into plants that must have similarly required host proteins for movement.

Identification and Functional Analysis of 1-Deoxy-D-xylulose-5-phosphate Synthase Gene in Tomatoes (Solanum lycopersicum)

1-deoxy-D-xylulose-5-phosphate synthase (DXS) is a rate-limiting enzyme in terpene synthesis that can affect the accumulation of secondary metabolites in plants. In this study, three DXS gene family members were identified in the tomato genome-wide database. Using bioinformatics methods, we analyzed the gene structure, evolutionary affinities, and cis-acting elements of the SlDXS gene family members. Promoters of SlDXS genes contain plant hormone-responsive elements such as the CGTCA-motif, TGACG-motif, ABRE, TCA-element, TGA-element, ERE, CAT-box, and AACA-motif, which suggested that the SlDXS gene family may play an important role in hormone response. The RT-qPCR analysis showed that the tomato DXS2 gene was able to respond upon exposure to methyl jasmonate (MeJA). The construction of a virus-induced gene silencing (VIGS) vector for the SlDXS gene showed that the SlDXS2 gene was also able to respond to MeJA in silenced plants, but the induction level was lower relative to that of wild-type plants. The SlDXS1 gene is associated with the synthesis of photosynthetic pigments. This study provides a reference for the further elucidation of the DXS gene’s biological function in the terpenoid synthesis pathway in tomatoes.

The Resistance of Soybean Variety Heinong 84 to Apple Latent Spherical Virus Is Controlled by Two Genetic Loci.

Apple latent spherical virus (ALSV) is widely used as a virus-induced gene silencing (VIGS) vector for function genome study. However, the application of ALSV to soybeans is limited by the resistance of many varieties. In this study, the genetic locus linked to the resistance of a resistant soybean variety Heinong 84 was mapped by high-throughput sequencing-based bulk segregation analysis (HTS-BSA) using a hybrid population crossed from Heinong 84 and a susceptible variety, Zhonghuang 13. The results showed that the resistance of Heinong 84 to ALSV is controlled by two genetic loci located on chromosomes 2 and 11, respectively. Cleaved amplified polymorphic sequence (CAPS) markers were developed for identification and genotyping. Inheritance and biochemical analyses suggest that the resistance locus on chromosome 2 plays a dominant dose-dependent role, while the other locus contributes a secondary role in resisting ALSV. The resistance locus on chromosome 2 might encode a protein that can directly inhibit viral proliferation, while the secondary resistance locus on chromosome 11 may encode a host factor required for viral proliferation. Together, these data reveal novel insights on the resistance mechanism of Heinong 84 to ALSV, which will benefit the application of ALSV as a VIGS vector.

Rapid Construction and Application of a Vector for Tobacco Ringspot Virus-Induced McPDS Silencing in Bitter Gourd

The aim of this study is to facilitate the construction of virus-induced gene silencing vectors and to provide a reference or positive control for gene silencing in bitter gourd. A recombinant TRSV (tobacco ringspot virus) containing two components, pTRSV1 and pTRSV2, was used in this study. The fragment of the McPDS target was cloned into pTRSV2 via combined enzymic ligation during digestion. The TRSV components were agro-infiltrated into tobacco leaves to grow virus particles, which were then extracted and mechanically inoculated into the bitter gourd plants. The effect of TRSV-McPDS-mediated McPDS gene silencing was evaluated by observing the photo-bleaching phenotype, detecting the TRSV virus, and quantifying the downregulation of MCPDS gene expression and chlorophyll contents. The results showed that all bitter gourd plants infected with the empty TRSV or TRSV-McPDS virus grew and developed normally, with no visible signs of viral disease. However, after seven days of inoculation, only the bitter gourd plants that were inoculated with TRSV-McPDS showed obvious photobleaching in the leaves, stems, and buds. The TRSV-specific fragments were tested out in the systemically infected leaves of bitter gourd. The transcription level of the McPDS gene in the leaves dropped by 84.7%. The chlorophyll content also dropped significantly. These data suggest that the rapidly constructed VIGS vector TRSV-McPDS successfully induced McPDS silencing in bitter gourd. Taken together, the results of this study provide a practical method for vector construction in various VIGS applications, as well as a reference and a positive control for TRSV-induced gene silencing in bitter gourd.

Identification of WRKY gene family members in amaranth based on a transcriptome database and functional analysis of AtrWRKY42-2 in betalain metabolism.

WRKY TFs (WRKY transcription factors) contribute to the synthesis of secondary metabolites in plants. Betalains are natural pigments that do not coexist with anthocyanins within the same plant. Amaranthus tricolor ('Suxian No.1') is an important leaf vegetable rich in betalains. However, the WRKY family members in amaranth and their roles in betalain synthesis and metabolism are still unclear. To elucidate the molecular characteristics of the amaranth WRKY gene family and its role in betalain synthesis, WRKY gene family members were screened and identified using amaranth transcriptome data, and their physicochemical properties, conserved domains, phylogenetic relationships, and conserved motifs were analyzed using bioinformatics methods. In total, 72 WRKY family members were identified from the amaranth transcriptome. Three WRKY genes involved in betalain synthesis were screened in the phylogenetic analysis of WRKY TFs. RT-qPCR showed that the expression levels of these three genes in red amaranth 'Suxian No.1' were higher than those in green amaranth 'Suxian No.2' and also showed that the expression level of AtrWRKY42 gene short-spliced transcript AtrWRKY42-2 in Amaranth 'Suxian No.1' was higher than that of the complete sequence AtrWRKY42-1, so the short-spliced transcript AtrWRKY42-2 was mainly expressed in 'Suxian No.2' amaranth. Moreover, the total expression levels of AtrWRKY42-1 and AtrWRKY42-2 were down-regulated after GA3 treatment, so AtrWRKY42-2 was identified as a candidate gene. Therefore, the short splice variant AtrWRKY42-2 cDNA sequence, gDNA sequence, and promoter sequence of AtrWRKY42 were cloned, and the PRI 101-AN-AtrWRKY42-2-EGFP vector was constructed to evaluate subcellular localization, revealing that AtrWRKY42-2 is located in the nucleus. The overexpression vector pRI 101-AN-AtrWRKY42-2-EGFP and VIGS (virus-induced gene silencing) vector pTRV2-AtrWRKY42-2 were transferred into leaves of 'Suxian No.1' by an Agrobacterium-mediated method. The results showed that AtrWRKY42-2 overexpression could promote the expression of AtrCYP76AD1 and increase betalain synthesis. A yeast one-hybrid assay demonstrated that AtrWRKY42-2 could bind to the AtrCYP76AD1 promoter to regulate betalain synthesis. This study lays a foundation for further exploring the function of AtrWRKY42-2 in betalain metabolism.

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.

Establishment of virus-induced gene silencing (VIGS) system in Luffa acutangula using Phytoene desaturase (PDS) and tendril synthesis related gene (TEN)

BackgroundVirus-induced gene silencing (VIGS) is a reverse genetics technology that can efficiently and rapidly identify plant gene functions. Although a variety of VIGS vectors have been successfully used in plants, only a few reports on VIGS technology in Luffa exist.ResultsIn the present study, a new cucumber green mottle mosaic virus (CGMMV)-based VIGS vector, pV190, was applied to establish the CGMMV-VIGS to investigate the feasibility of the silencing system for Luffa. Phytoene desaturase (PDS) gene was initially selected as a VIGS marker gene to construct a recombinant vector. Plants infected with Agrobacterium harboring pV190-PDS successfully induced effective silencing in Luffa, and an effective gene silencing phenotype with obvious photobleaching was observed. To further validate the efficiency, we selected TEN for gene-silencing, which encodes a CYC/TB1-like transcription factor and is involved in tendril development. Luffa plants inoculated with the pV190-TEN exhibited shorter tendril length and nodal positions where tendrils appear are higher compared to those of non-inoculated plants. RT-qPCR showed that the expression levels of PDS and TEN were significantly reduced in the CGMMV-VIGS plants. Moreover, we evaluated the CGMMV-VIGS efficiency in three cucurbits, including cucumber, ridge gourd, and bottle gourd.ConclusionWe successfully established a CGMMV-based VIGS system on ridge gourd and used marker genes to identify the feasibility of the silencing system in Luffa leaves and stems.

A Mycovirus VIGS Vector Confers Hypovirulence to a Plant Pathogenic Fungus to Control Wheat FHB.

Mycovirus-mediated hypovirulence has the potential to control fungal diseases. However, the availability of hypovirulence-conferring mycoviruses for plant fungal disease control is limited as most fungal viruses are asymptomatic. In this study, the virus-induced gene silencing (VIGS) vector p26-D4 of Fusarium graminearum gemytripvirus 1 (FgGMTV1), a tripartite circular single-stranded DNA mycovirus, is successfully constructed to convert the causal fungus of cereal Fusarium head blight (FHB) into a hypovirulent strain. p26-D4, with an insert of a 75-150bp fragment of the target reporter transgene transcript in both sense and antisense orientations, efficiently triggered gene silencing in Fusarium graminearum. Notably, the two hypovirulent strains, p26-D4-Tri101, and p26-D4-FgPP1, obtained by silencing the virulence-related genes Tri101 and FgPP1 with p26-D4, can be used as biocontrol agents to protect wheat from a fungal disease FHB and mycotoxin contamination at the field level. This study not only describes the first mycovirus-derived VIGS system but also proves that the VIGS vector can be used to establish multiple hypovirulent strains to control pathogenic fungi.

Development of cassava common mosaic virus-based vector for protein expression and gene editing in cassava

BackgroundPlant virus vectors designed for virus-mediated protein overexpression (VOX), virus-induced gene silencing (VIGS), and genome editing (VIGE) provide rapid and cost-effective tools for functional genomics studies, biotechnology applications and genome modification in plants. We previously reported that a cassava common mosaic virus (CsCMV, genus Potexvirus)-based VIGS vector was used for rapid gene function analysis in cassava. However, there are no VOX and VIGE vectors available in cassava.ResultsIn this study, we developed an efficient VOX vector (CsCMV2-NC) for cassava by modifying the CsCMV-based VIGS vector. Specifically, the length of the duplicated putative subgenomic promoter (SGP1) of the CsCMV CP gene was increased to improve heterologous protein expression in cassava plants. The modified CsCMV2-NC-based VOX vector was engineered to express genes encoding green fluorescent protein (GFP), bacterial phytoene synthase (crtB), and Xanthomonas axonopodis pv. manihotis (Xam) type III effector XopAO1 for viral infection tracking, carotenoid biofortification and Xam virulence effector identification in cassava. In addition, we used CsCMV2-NC to deliver single guide RNAs (gMePDS1/2) targeting two loci of the cassava phytoene desaturase gene (MePDS) in Cas9-overexpressing transgenic cassava lines. The CsCMV-gMePDS1/2 efficiently induced deletion mutations of the targeted MePDS with the albino phenotypes in systemically infected cassava leaves.ConclusionsOur results provide a useful tool for rapid and efficient heterologous protein expression and guide RNA delivery in cassava. This expands the potential applications of CsCMV-based vector in gene function studies, biotechnology research, and precision breeding for cassava.

Transcriptome Analysis and VIGS Identification of Key Genes Regulating Citric Acid Metabolism in Citrus.

Citrus (Citrus reticulata) is one of the world's most widely planted and highest-yielding fruit trees. Citrus fruits are rich in a variety of nutrients. The content of citric acid plays a decisive role in the flavor quality of the fruit. There is a high organic acid content in early-maturing and extra-precocious citrus varieties. Reducing the amount of organic acid after fruit ripening is significant to the citrus industry. In this study, we selected a low-acid variety, "DF4", and a high-acid variety, "WZ", as research materials. Through WGCNA analysis, two differentially expressed genes, citrate synthase (CS) and ATP citrate-pro-S-lyase (ACL), were screened out, which related to the changing citric acid. The two differentially expressed genes were preliminarily verified by constructing a virus-induced gene-silencing (VIGS) vector. The VIGS results showed that the citric acid content was negatively correlated with CS expression and positively correlated with ACL expression, while CS and ACL oppositely control citric acid and inversely regulate each other. These results provide a theoretical basis for promoting the breeding of early-maturing and low-acid citrus varieties.

A cowpea severe mosaic virus-based vector simplifies virus-induced gene silencing and foreign protein expression in soybean

BackgroundSoybean gene functions cannot be easily interrogated through transgenic disruption (knock-out) of genes-of-interest, or transgenic overexpression of proteins-of-interest, because soybean transformation is time-consuming and technically challenging. An attractive alternative is to administer transient gene silencing or overexpression with a plant virus-based vector. However, existing virus-induced gene silencing (VIGS) and/or overexpression vectors suitable for soybean have various drawbacks that hinder their widespread adoption.ResultsWe describe the development of a new vector based on cowpea severe mosaic virus (CPSMV), a plus-strand RNA virus with its genome divided into two RNA segments, RNA1 and RNA2. This vector, designated FZ, incorporates a cloning site in the RNA2 cDNA, permitting insertion of nonviral sequences. When paired with an optimized RNA1 construct, FZ readily infects both Nicotiana benthamiana and soybean. As a result, FZ constructs destined for soybean can be first delivered to N. benthamiana in order to propagate the modified viruses to high titers. FZ-based silencing constructs induced robust silencing of phytoene desaturase genes in N. benthamiana, multiple soybean accessions, and cowpea. Meanwhile, FZ supported systemic expression of fluorescent proteins mNeonGreen and mCherry in N. benthamiana and soybean. Finally, FZ-mediated expression of the Arabidopsis transcription factor MYB75 caused N. benthamiana to bear brown leaves and purple, twisted flowers, indicating that MYB75 retained the function of activating anthocyanin synthesis pathways in a different plant.ConclusionsThe new CPSMV-derived FZ vector provides a convenient and versatile soybean functional genomics tool that is expected to accelerate the characterization of soybean genes controlling crucial productivity traits.

Watermelon goes viral: introducing a vector for virus-induced gene silencing in cucurbits.

Watermelon goes viral: introducing a vector for virus-induced gene silencing in cucurbits.

Characterization of a foxtail mosaic virus vector for gene silencing and analysis of innate immune responses in Sorghum bicolor.

Sorghum is vulnerable to many biotic and abiotic stresses, which cause considerable yield losses globally. Efforts to genetically characterize beneficial sorghum traits, including disease resistance, plant architecture, and tolerance to abiotic stresses, are ongoing. One challenge faced by sorghum researchers is its recalcitrance to transformation, which has slowed gene validation efforts and utilization for cultivar development. Here, we characterize the use of a foxtail mosaic virus (FoMV) vector for virus-induced gene silencing (VIGS) by targeting two previously tested marker genes: phytoene desaturase (PDS) and ubiquitin (Ub). We additionally demonstrate VIGS of a subgroup of receptor-like cytoplasmic kinases (RLCKs) and report the role of these genes as positive regulators of early defence signalling. Silencing of subgroup 8 RLCKs also resulted in higher susceptibility to the bacterial pathogens Pseudomonas syringae pv. syringae (B728a) and Xanthomonas vasicola pv. holcicola, demonstrating the role of these genes in host defence against bacterial pathogens. Together, this work highlights the utility of FoMV-induced gene silencing in the characterization of genes mediating defence responses in sorghum. Moreover, FoMV was able to systemically infect six diverse sorghum genotypes with high efficiency at optimal temperatures for sorghum growth and therefore could be extrapolated to study additional traits of economic importance.

Virus-induced gene silencing for in planta validation of gene function in cucurbits.

Virus-induced gene silencing (VIGS) is a powerful tool for high-throughput analysis of gene function. Here, we developed the VIGS vector pCF93, from which expression of the cucumber fruit mottle mosaic virus genome is driven by the cauliflower mosaic virus 35S promoter to produce viral transcripts in inoculated plants. To test the utility of the pCF93 vector, we identified candidate genes related to male sterility (MS) in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. Specifically, we exploited previously reported reference-based and de novo transcriptome data to define 38 differentially expressed genes between a male-sterile line and its fertile near-isogenic line in the watermelon cultivar DAH. We amplified 200- to 300-bp fragments of these genes, cloned them into pCF93, and inoculated DAH with the resulting VIGS clones. The small watermelon cultivar DAH enabled high-throughput screening using a small cultivation area. We simultaneously characterized the phenotypes associated with each of the 38 candidate genes in plants grown in a greenhouse. Silencing of 8 of the 38 candidate genes produced male-sterile flowers with abnormal stamens and no pollen. We confirmed the extent of gene silencing in inoculated flowers using reverse transcription-qPCR. Histological analysis of stamens from male-fertile and male-sterile floral buds and mature flowers revealed developmental defects and shrunken pollen sacs. Based on these findings, we propose that the pCF93 vector and our VIGS system will facilitate high-throughput analysis for the study of gene function in watermelons.

A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity

Plants have evolved defense mechanisms to suppress viral transcription and replication by transcriptional and post-transcriptional gene silencing mediated by virus-derived small interfering RNAs (vsiRNAs). Based on this response, virus-induced gene silencing (VIGS)-based technology has been developed to silence target genes on either host plants or insect pests. This mechanism could also be used for the silencing of genes of interest in the medical field. We used the VIGS vector pEuMV-YP:Krt18, which was obtained by inserting the Mus musculus (M. musculus) Krt18 sequence into pEuMV-YP:ΔAV1. The objective was to evaluate the capacity of pEuMV-YP:Krt18 to induce Nicotiana benthamiana (N. benthamiana) production of vsiRNAs of a specific sequence that belongs to neither the plant genome nor the wild virus genome, which were used to induce cross-kingdom gene silencing between plants and mammals. The percentage of vsiRNA for each viral gene was calculated from an sRNA library of N. benthamiana plants infected by pEuMV-YP: Krt18. When the vsiRNAs were characterized, it was found that they corresponded to all the genes of the pEuMV-YP:Krt18 vector. These vsiRNAs induced the silencing of the Krt18 gene in M. musculus macrophages, supporting the ability to use VIGS vectors in plants as biofactories for the production of sRNAs that induce gene silencing in mammals.

Construction and Characterization of a Botrytis Virus F Infectious Clone.

Botrytis virus F (BVF) is a positive-sense, single-stranded RNA (+ssRNA) virus within the Gammaflexiviridae family of the plant-pathogenic fungus Botrytis cinerea. In this study, the complete sequence of a BVF strain isolated from B. cinerea collected from grapevine fields in Spain was analyzed. This virus, in this work BVF-V448, has a genome of 6827 nt in length, excluding the poly(A) tail, with two open reading frames encoding an RNA dependent RNA polymerase (RdRP) and a coat protein (CP). The 5′- and 3′-terminal regions of the genome were determined by rapid amplification of cDNA ends (RACE). Furthermore, a yet undetected subgenomic RNA species in BVF-V448 was identified, indicating that the CP is expressed via 3′ coterminal subgenomic RNAs (sgRNAs). We also report the successful construction of the first BVF full-length cDNA clone and synthesized in vitro RNA transcripts using the T7 polymerase, which could efficiently transfect two different strains of B. cinerea, B05.10 and Pi258.9. The levels of growth in culture and virulence on plants of BVF-V448 transfected strains were comparable to BVF-free strains. The infectious clones generated in this work provide a useful tool for the future development of an efficient BVF foreign gene expression vector and a virus-induced gene silencing (VIGS) vector as a biological agent for the control of B. cinerea.

Virus-Induced Gene Silencing in Chrysanthemum seticuspe Using the Tomato Aspermy Virus Vector.

Chrysanthemum is one of the most economically important flowers globally due to its high ornamental value. In recent years, a large percentage of the Chrysanthemum seticuspe genome has been determined, making this species useful as a model chrysanthemum plant. To fully utilize the genome’s information, efficient and rapid gene functional analysis methods are needed. In this study, we optimized the tomato aspermy virus (TAV) vector for virus-induced gene silencing (VIGS) in C. seticuspe. Conventional plant virus inoculation methods, such as the mechanical inoculation of viral RNA transcripts and agroinoculation into leaves, did not achieve successful TAV infections in C. seticuspe, but vacuum infiltration into sprouts was successful without symptoms. The TAV vector harboring 100 nucleotides of the phytoene desaturase (PDS) gene caused photobleaching phenotypes and a reduction in CsPDS expression in C. seticuspe. To our knowledge, this is the first report of VIGS in chrysanthemums.

RNAi Expression in Cotton for Control of Herbivorous Insects.

Cultivated cotton (Gossypium hirsutum) is heavily attacked by various species of insects worldwide and breeding of new varieties resistant to pests is still a hard battle to win. RNAi technology is an important reverse genetics tool to induce gene silencing in eukaryotic organisms and produce phenotypic modifications. In cotton, RNAi was applied to investigate gene function and enhance resistance to insects and pathogens. Different methods and techniques can be used to synthetize double stranded RNA (dsRNA) into plant cells. The Agrobacterium-mediated transformation is a common method to introduce RNAi binary plasmids into cotton genome and obtain stable transgenics plants. This methodology includes the coculture of cotton tissues with Agrobacterium cultures, selection of transgenic cells and induction of somatic embryogenesis to finally obtain transgenic plants after a relatively long period of time. The transient synthesis of dsRNA mediated by virus-induced gene silencing (VIGS) in cotton is an alternative to anticipate the silencing effect of a specific RNA sequence, prior to the development of a stable transgenic plant. VIGS vectors are incorporated into the plant by agroinfiltration technique. During VIGS replication inside plant cells, synthetized dsRNA allows the study on specific heterologous gene expression including the phenotypic effect on herbivorous target pests, thus facilitating a rapid evaluation of dsRNA expressed in cotton plants against individual insect target genes. Here we describe the complementation of these two techniques to evaluate RNAi-based cotton plant protection against insect pests.

RTBV-Based VIGS Vector for Functional Genomics in Rice: Methodology, Advances, Challenges, and Future Implications.

The availability of protocols for virus-induced gene silencing (VIGS) in rice has opened up an important channel for the elucidation of gene functions in this important crop plant. Here, we present an updated protocol of a VIGS system based on Rice tungro bacilliform virus (RTBV) for gene silencing in rice. We present complete updated protocols for VIGS in rice, compare the system with other existing ones for monocots, identify some of the challenges faced by this system and discuss ways in which the vector could be improved for better silencing efficiency.