Virus-induced Gene Silencing Constructs Research Articles

An engineered citrus tristeza virus (T36CA)-based vector induces gene-specific RNA silencing and is graft transmissible to commercial citrus varieties.

A protein-expressing citrus tristeza virus (CTV)-based vector construct, pT36CA-V1.3, obtained from a California isolate of the T36 strain (T36CA), was retooled into a virus induced gene silencing (VIGS) system intended for use with studies of California citrus. VIGS constructs engineered with a truncated Citrus macrophylla (Cm) PHYTOENE DESATURASE (PDS) gene sequence in the sense or anti-sense orientation worked equally well to silence the endogenous CmPDS gene. In a parallel effort to optimize vector performance, two non-synonymous nucleotides in open reading frame 1a of pT36CA-V1.3 were replaced with those conserved in the reference sequences from the T36CA cDNA library. The resulting viruses, T36CA-V1.4 (with one amino acid modification: D760N) and T36CA-V1.5 (with two amino acid modifications: D760N and P1174L), along with T36CA-V1.3 were individually propagated in Nicotiana benthamiana and C. macrophylla plants. Enzyme-linked immunosorbent assay (ELISA) measurements of extracts of the newly emerged leaves suggested that all three viruses accumulated to similar levels in N. benthamiana plants at 5 week-post-inoculation. ELISA values of T36CA-V1.4- and -V1.5-infected C. macrophylla samples were significantly higher than that of T36CA-V1.3-infected samples within an 8 to 12 month-post-inoculation (mpi) window, suggesting a higher accumulation of T36CA-V1.4 and -V1.5 than T36CA-V1.3. However, at 36 mpi, the ELISA values suggested that all three viruses accumulated to similar levels. When C. macrophylla plants infected with each of the three viruses were grafted to commercial citrus varieties, a limited number of receptor plants became infected, demonstrating a weak but nonetheless (the first) successful delivery of T36CA to California-grown commercial citrus.

Virus induced gene silencing confirms oligogenic inheritance of brown stem rot resistance in soybean.

Brown Stem Rot (BSR), caused by the soil borne fungal pathogen Phialophora gregata, can reduce soybean yields by as much as 38%. Previous allelism studies identified three Resistant to brown stem Rot genes (Rbs1, Rbs2, and Rbs3), all mapping to large, overlapping regions on soybean chromosome 16. However, recent fine-mapping and genome wide association studies (GWAS) suggest Rbs1, Rbs2, and Rbs3 are alleles of a single Rbs locus. To address this conflict, we characterized the Rbs locus using the Williams82 reference genome (Wm82.a4.v1). We identified 120 Receptor-Like Proteins (RLPs), with hallmarks of disease resistance receptor-like proteins (RLPs), which formed five distinct clusters. We developed virus induced gene silencing (VIGS) constructs to target each of the clusters, hypothesizing that silencing the correct RLP cluster would result in a loss of resistance phenotype. The VIGS constructs were tested against P. gregata resistant genotypes L78-4094 (Rbs1), PI 437833 (Rbs2), or PI 437970 (Rbs3), infected with P. gregata or mock infected. No loss of resistance phenotype was observed. We then developed VIGS constructs targeting two RLP clusters with a single construct. Construct B1a/B2 silenced P. gregata resistance in L78-4094, confirming at least two genes confer Rbs1-mediated resistance to P. gregata. Failure of B1a/B2 to silence resistance in PI 437833 and PI 437970 suggests additional genes confer BSR resistance in these lines. To identify differentially expressed genes (DEGs) responding to silencing, we conducted RNA-seq of leaf, stem and root samples from B1a/B2 and empty vector control plants infected with P. gregata or mock infected. B1a/B2 silencing induced DEGs associated with cell wall biogenesis, lipid oxidation, the unfolded protein response and iron homeostasis and repressed numerous DEGs involved in defense and defense signaling. These findings will improve integration of Rbs resistance into elite germplasm and provide novel insights into fungal disease resistance.

Horizontally transferred genes as RNA interference targets for aphid and whitefly control.

RNA interference (RNAi)-based technologies are starting to be commercialized as a new approach for agricultural pest control. Horizontally transferred genes (HTGs), which have been transferred into insect genomes from viruses, bacteria, fungi or plants, are attractive targets for RNAi-mediated pest control. HTGs are often unique to a specific insect family or even genus, making it unlikely that RNAi constructs targeting such genes will have negative effects on ladybugs, lacewings and other beneficial predatory insect species. In this study, we sequenced the genome of a red, tobacco-adapted isolate of Myzus persicae (green peach aphid) and bioinformatically identified 30 HTGs. We then used plant-mediated virus-induced gene silencing (VIGS) to show that several HTGs of bacterial and plant origin are important for aphid growth and/or survival. Silencing the expression of fungal-origin HTGs did not affect aphid survivorship but decreased aphid reproduction. Importantly, although there was uptake of plant-expressed RNA by Coccinella septempunctata (seven-spotted ladybugs) via the aphids that they consumed, we did not observe negative effects on ladybugs from aphid-targeted VIGS constructs. To demonstrate that this approach is more broadly applicable, we also targeted five Bemisia tabaci (whitefly) HTGs using VIGS and demonstrated that knockdown of some of these genes affected whitefly survival. As functional HTGs have been identified in the genomes of numerous pest species, we propose that these HTGs should be explored further as efficient and safe targets for control of insect pests using plant-mediated RNA interference.

Imaging systemic calcium response and its molecular dissection using virus-induced gene silencing.

Many biotic and abiotic stimuli arrive locally on the plant. For example, attack by an insect or invasion by a fungal pathogen generally starts with a single leaf. However, the responses that are then elicited are often systemic, triggering effects throughout the entire plant body. One of the rapid signaling systems that helps coordinate these plant-wide response networks is changes in cytoplasmic Ca2+ that rapidly propagate throughout the plant. These Ca2+ signals are readily visualized using plants expressing green fluorescent protein-based Ca2+-sensitive bioreporters, such as those of the GCaMP and GECO families. Dissecting the underlying molecular machinery behind this systemic spread of information is often approached by imaging the Ca2+ response in mutants in candidate genes. Introducing the GFP sensor into the relevant genetic backgrounds and then selecting lines usable for imaging can be very time consuming. An alternative, more rapid approach to screening these candidates is through virus-induced gene silencing (VIGS), where target genes are suppressed in the wild-type bioreporter expressing plants. This chapter describes how to generate VIGS constructs targeted to candidate genes and then how to visualize wound-induced, systemic Ca2+ signaling in the VIGS suppressed plants.

Maize resistance to insect herbivory is enhanced by silencing expression of genes for jasmonate‐isoleucine degradation using sugarcane mosaic virus

AbstractPreviously, sugarcane mosaic virus (SCMV) was developed as a vector for transient expression of heterologous genes in Zea mays (maize). Here, we show that SCMV can also be applied for virus‐induced gene silencing (VIGS) of endogenous maize genes. Comparison of sense and antisense VIGS constructs targeting maize phytoene desaturase (PDS) showed that antisense constructs resulted in a greater reduction in gene expression. In a time course of gene expression after infection with VIGS constructs targeting PDS, lesion mimic 22 (Les22), and Iodent japonica 1 (Ij1), efficient expression silencing was observed 2, 3, and 4 weeks after infection with SCMV. However, at Week 5, expression of Les22 and Ij1 was no longer significantly reduced compared with control plants. The defense signaling molecule jasmonate‐isoleucine (JA‐Ile) can be inactivated by 12C‐hydroxylation and hydrolysis, and knockout of these genes leads to herbivore resistance. JA‐Ile hydroxylases and hydrolases have been investigated in Arabidopsis, rice, and Nicotiana attenuata. To determine whether the maize homologs of these genes function in plant defense, we silenced expression of ZmCYP94B1 (predicted JA‐Ile hydroxylase) and ZmJIH1 (predicted JA‐Ile hydrolase) by VIGS with SCMV, which resulted in elevated expression of two defense‐related genes, Maize Proteinase Inhibitor (MPI) and Ribosome Inactivating Protein 2 (RIP2). Although ZmCYP94B1 and ZmJIH1 gene expression silencing increased resistance to Spodoptera frugiperda (fall armyworm), Schistocerca americana (American birdwing grasshopper), and Rhopalosiphum maidis (corn leaf aphid), there was no additive effect from silencing the expression of both genes. Further work will be required to determine the more precise functions of these enzymes in regulating maize defenses.

Simultaneous gene expression and multi-gene silencing in Zea mays using maize dwarf mosaic virus

BackgroundMaize dwarf mosaic virus (MDMV), a member of the genus Potyvirus, infects maize and is non-persistently transmitted by aphids. Several plant viruses have been developed as tools for gene expression and gene silencing in plants. The capacity of MDMV for both gene expression and gene silencing were examined.ResultsInfectious clones of an Ohio isolate of MDMV, MDMV OH5, were obtained, and engineered for gene expression only, and for simultaneous marker gene expression and virus-induced gene silencing (VIGS) of three endogenous maize target genes. Single gene expression in single insertion constructs and simultaneous expression of green fluorescent protein (GFP) and silencing of three maize genes in a double insertion construct was demonstrated. Constructs with GFP inserted in the N-terminus of HCPro were more stable than those with insertion at the N-terminus of CP in our study. Unexpectedly, the construct with two insertion sites also retained insertions at a higher rate than single-insertion constructs. Engineered MDMV expression and VIGS constructs were transmissible by aphids (Rhopalosiphum padi).ConclusionsThese results demonstrate that MDMV-based vector can be used as a tool for simultaneous gene expression and multi-gene silencing in maize.

PssRNAit: A Web Server for Designing Effective and Specific Plant siRNAs with Genome-Wide Off-Target Assessment.

We report an advanced web server, the plant-specific small noncoding RNA interference tool pssRNAit, which can be used to design a pool of small interfering RNAs (siRNAs) for highly effective, specific, and nontoxic gene silencing in plants. In developing this tool, we integrated the transcript dataset of plants, several rules governing gene silencing, and a series of computational models of the biological mechanism of the RNA interference (RNAi) pathway. The designed pool of siRNAs can be used to construct a long double-strand RNA and expressed through virus-induced gene silencing (VIGS) or synthetic transacting siRNA vectors for gene silencing. We demonstrated the performance of pssRNAit by designing and expressing the VIGS constructs to silence Phytoene desaturase (PDS) or a ribosomal protein-encoding gene, RPL10 (QM), in Nicotiana benthamiana We analyzed the expression levels of predicted intended-target and off-target genes using reverse transcription quantitative PCR. We further conducted an RNA-sequencing-based transcriptome analysis to assess genome-wide off-target gene silencing triggered by the fragments that were designed by pssRNAit, targeting different homologous regions of the PDS gene. Our analyses confirmed the high accuracy of siRNA constructs designed using pssRNAit The pssRNAit server, freely available at https://plantgrn.noble.org/pssRNAit/, supports the design of highly effective and specific RNAi, VIGS, or synthetic transacting siRNA constructs for high-throughput functional genomics and trait improvement in >160 plant species.

Virus-Induced Gene Silencing and Transient Gene Expression in Soybean (Glycine max) Using Bean Pod Mottle Virus Infectious Clones.

Virus-induced gene silencing (VIGS) is a powerful and rapid approach for determining the functions of plant genes. The basis of VIGS is that a viral genome is engineered so that it can carry fragments of plant genes, typically in the 200 to 300 base pair size range. The recombinant viruses are used to infect experimental plants, and wherever the virus invades, the target gene or genes will be silenced. VIGS is thus transient, and in the span of a few weeks, it is possible to design VIGS constructs and then generate loss-of-function phenotypes through RNA silencing of the target genes. In soybean (Glycine max), the Bean pod mottle virus (BPMV) has been engineered to be valuable tool for silencing genes with diverse functions and also for over-expression of foreign genes. This protocol describes a method for designing BPMV constructs and using them to silence or transiently express genes in soybean. © 2016 by John Wiley & Sons, Inc.

Development of a new vector using Soybean yellow common mosaic virus for gene function study or heterologous protein expression in soybeans

A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5′ cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5′ terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3′ terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135–160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C) was constructed (pSYCMV-FMDV). Plants infiltrated with pSYCMV-FMDV were only detected via western blotting using the O1C antibody. Based on these results, we propose that the SYCMV-derived vector can be used for gene function study or expression of useful heterologous proteins in soybeans.

Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia.

BackgroundThe cereal rust fungi are destructive pathogens that affect grain production worldwide. Although the genomic and transcript sequences for three Puccinia species that attack wheat have been released, the functions of large repertories of genes from Puccinia still need to be addressed to understand the infection process of these obligate parasites. Host-induced gene silencing (HIGS) has emerged a useful tool to examine the importance of rust fungus genes while growing within host plants. In this study, HIGS was used to test genes from Puccinia with transcripts enriched in haustoria for their ability to interfere with full development of the rust fungi.ResultsApproximately 1200 haustoria enriched genes from Puccinia graminis f. sp. tritici (Pgt) were identified by comparative RNA sequencing. Virus-induced gene silencing (VIGS) constructs with fragments of 86 Puccinia genes, were tested for their ability to interfere with full development of these rust fungi. Most of the genes tested had no noticeable effects, but 10 reduced Pgt development after co-inoculation with the gene VIGS constructs and Pgt. These included a predicted glycolytic enzyme, two other proteins that are probably secreted and involved in carbohydrate or sugar metabolism, a protein involved in thiazol biosynthesis, a protein involved in auxin biosynthesis, an amino acid permease, two hypothetical proteins with no conserved domains, a predicted small secreted protein and another protein predicted to be secreted with similarity to bacterial proteins involved in membrane transport. Transient silencing of four of these genes reduced development of P. striiformis (Pst), and three of also caused reduction of P. triticina (Pt) development.ConclusionsPartial suppression of transcripts involved in a large variety of biological processes in haustoria cells of Puccinia rusts can disrupt their development. Silencing of three genes resulted in suppression of all three rust diseases indicating that it may be possible to engineer durable resistance to multiple rust pathogens with a single gene in transgenic wheat plants for sustainable control of cereal rusts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1791-y) contains supplementary material, which is available to authorized users.

A wheat ABC transporter contributes to both grain formation and mycotoxin tolerance.

The mycotoxin deoxynivalenol (DON) acts as a disease virulence factor for Fusarium fungi, and tolerance of DON enhances wheat resistance to Fusarium head blight (FHB) disease. Two variants of an ATP-binding cassette (ABC) family C transporter gene were cloned from DON-treated wheat mRNA, namely TaABCC3.1 and TaABCC3.2. These represent two of three putative genes identified on chromosomes 3A, 3B, and 3D of the wheat genome sequence. Variant TaABCC3.1 represents the DON-responsive transcript previously associated with DON resistance in wheat. PCR-based mapping and in silico sequence analyses located TaABCC3.1 to the short arm of wheat chromosome 3B (not within the FHB resistance quantitative trait locus Fhb1). In silico analyses of microarray data indicated that TaABCC3 genes are expressed in reproductive tissue and roots, and in response to the DON producer Fusarium graminearum. Gene expression studies showed that TaABCC3.1 is activated as part of the early host response to DON and in response to the FHB defence hormone jasmonic acid. Virus-induced gene silencing (VIGS) confirmed that TaABCC3 genes contributed to DON tolerance. VIGS was performed using two independent viral construct applications: one specifically targeted TaABCC3.1 for silencing, while the other targeted this gene and the chromosome 3A homeologue. In both instances, VIGS resulted in more toxin-induced discoloration of spikelets, compared with the DON effects in non-silenced spikelets at 14 d after toxin treatment (≥2.2-fold increase, P<0.05). Silencing by both VIGS constructs enhanced head ripening, and especially so in DON-treated heads. VIGS of TaABCC3 genes also reduced the grain number by more than 28% (P<0.05), both with and without DON treatment, and the effects were greater for the construct that targeted the two homeologues. Hence, DON-responsive TaABCC3 genes warrant further study to determine their potential as disease resistance breeding targets and their function in grain formation and ripening.

The Requirement of Multiple Defense Genes in SoybeanRsv1–Mediated Extreme Resistance toSoybean mosaic virus

Soybean mosaic virus (SMV) is a major viral pathogen of soybean. Among the three SMV resistance genes, Rsv1 mediates extreme resistance (ER) against most SMV strains, including the β-glucuronidase-tagged G2 isolate that was previously used in studies of Rsv1. Using virus-induced gene silencing (VIGS), we screened 82 VIGS constructs to identify genes that play a role in Rsv1-mediated ER to SMV infection. The target genes included putative Rsv1 candidate genes, soybean orthologs to known defense-signaling genes, and 62 WRKY transcription factors. We identified eight VIGS constructs that compromised Rsv1-mediated resistance when the target genes were silenced, including GmEDR1, GmEDS1, GmHSP90, GmJAR1, GmPAD4, and two WRKY transcription factors. Together, our results provide new insight into the soybean signaling network required for ER against SMV.

Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance

Although several wheat genes differentially expressed during the Russian wheat aphid resistance response have recently been identified, their requirement for and specific role in resistance remain unclear. Progress in wheat-aphid interaction research is hampered by inadequate collections of mutant germplasm and difficulty in transforming hexaploid wheat. Virus-induced gene silencing (VIGS) technology is emerging as a viable reverse genetics approach in cereal crops. However, the potential of VIGS for determining aphid defence gene function in wheat has not been evaluated. We report on the use of recombinant barley stripe mosaic virus (BSMV) to target and silence a WRKY53 transcription factor and an inducible phenylalanine ammonia-lyase (PAL) gene, both predicted to contribute to aphid defence in a genetically resistant wheat line. After inoculating resistant wheat with the VIGS constructs, transcript abundance was reduced to levels similar to that observed in susceptible wheat. Notably, the level of PAL expression was also suppressed by the WKRY53 construct, suggesting that these genes operate in the same defence response network. Both knockdowns exhibited a susceptible phenotype upon aphid infestation, and aphids feeding on silenced plants exhibited a significant increase in fitness compared to aphids feeding on control plants. Altered plant phenotype and changes in aphid behaviour after silencing imply that WKRY53 and PAL play key roles in generating a successful resistance response. This study is the first report on the successful use of VIGS to investigate genes involved in wheat-insect interactions.

Cell-free construction of disarmed Abutilon mosaic virus-based gene silencing vectors

The bipartite Abutilon mosaic virus (AbMV) was engineered as a versatile silencing vector in which the coat protein gene of DNA A was deleted and replaced by sequences of interest. Plants transgenic for the dimeric AbMV DNA B component were used as test hosts to minimize the risk of unintended release of the recombinant DNA. The vector construct was stable genetically upon systemic infection and, in common with the parental virus, the vector remained phloem-limited. For virus-induced gene silencing (VIGS), a phytoene desaturase gene fragment was isolated from Nicotiana benthamiana (NbPDS) and inserted into the vector. After agroinfection, phytoene desaturase silencing was triggered efficiently in all leaf tissues without interference by viral symptoms. In order to facilitate further the use of the system, a technique for cell-free construction of recombinants was established using rolling circle amplification and biolistic inoculation of DNA B-transgenic plants. This novel procedure provides a convenient and safe way for delivering VIGS constructs for functional genomics.

The Mi-1-Mediated Pest Resistance Requires Hsp90 and Sgt1

The tomato (Solanum lycopersicum) Mi-1 gene encodes a protein with putative coiled-coil nucleotide-binding site and leucine-rich repeat motifs. Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphids (Macrosiphum euphorbiae), and sweet potato whitefly (Bemisia tabaci). To identify genes required in the Mi-1-mediated resistance to nematodes and aphids, we used tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) to repress candidate genes and assay for nematode and aphid resistance. We targeted Sgt1 (suppressor of G-two allele of Skp1), Rar1 (required for Mla12 resistance), and Hsp90 (heat shock protein 90), which are known to participate early in resistance gene signaling pathways. Two Arabidopsis (Arabidopsis thaliana) Sgt1 genes exist and one has been implicated in disease resistance. Thus far the sequence of only one Sgt1 ortholog is known in tomato. To design gene-specific VIGS constructs, we cloned a second tomato Sgt1 gene, Sgt1-2. The gene-specific VIGS construct TRV-SlSgt1-1 resulted in lethality, while silencing Sgt1-2 using TRV-SlSgt1-2 did not result in lethal phenotype. Aphid and root-knot nematode assays of Sgt1-2-silenced plants indicated no role for Sgt1-2 in Mi-1-mediated resistance. A Nicotiana benthamiana Sgt1 VIGS construct silencing both Sgt1-1 and Sgt1-2 yielded live plants and identified a role for Sgt1 in Mi-1-mediated aphid resistance. Silencing of Rar1 did not affect Mi-1-mediated nematode and aphid resistance and demonstrated that Rar1 is not required for Mi-1 resistance. Silencing Hsp90-1 resulted in attenuation of Mi-1-mediated aphid and nematode resistance and indicated a role for Hsp90-1. The requirement for Sgt1 and Hsp90-1 in Mi-1-mediated resistance provides further evidence for common components in early resistance gene defense signaling against diverse pathogens and pests.