Virus-induced Gene Silencing Vector Research Articles

Construction of an Infectious Poa semilatent virus cDNA Clone and Comparisons of Hordeivirus Cytopathology and Pathogenicity.

Poa semilatent virus (PSLV), Lychnis ringspot virus (LRSV), and Barley stripe mosaic virus (BSMV) are members of the genus Hordeivirus in the family Virgaviridae. However, the biological properties and molecular genetics of PSLV have not been compared with other hordeiviruses. Here, we have constructed an infectious cDNA clone of the PSLV Canadian strain and provided evidence that PSLV differs from BSMV and LRSV. First, unlike the other two hordeiviruses that replicate in chloroplasts, PSLV induces dramatic structural changes in peroxisome during its infection in barley. The αa replication protein also localizes to peroxisomes, suggesting that PSLV replication occurs in peroxisomes. Second, PSLV encodes a γb protein that shares 19 to 23% identity with those of other hordeiviruses, and its activity as a viral suppressor of RNA (VSR) silencing is distinct from those of BSMV and LRSV. Substitution of the BSMV γb protein with that of PSLV or LRSV revealed a negative correlation between VSR activity and symptom severity of the recombinant BSMV derivatives. Intriguingly, the Ser-Lys-Leu (SKL) peroxisome-targeting signals differ among γb proteins of various hordeiviruses, including some BSMV strains. The presence of the C-terminal SKL motif in the γb protein impairs its silencing suppressor activity and influences symptoms. Finally, we developed a PSLV-based virus-induced gene silencing vector that induced strong and effective silencing phenotypes of endogenous genes in barley, wheat, and millet. Our results shed new light on hordeivirus pathogenesis and evolution, and provide an alternative tool for genomics studies of model hosts and economically important monocots.

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.

A Plant Virus-Based Vector System for Gene Function Studies in Pepper.

While pepper (Capsicum annuum) is a highly recalcitrant species for genetic transformation studies, plant virus-based vectors can provide alternative and powerful tools for transient regulation and functional analysis of genes of interest in pepper. In this study, we established an effective virus-based vector system applicable for transient gain- and loss-of-function studies in pepper using Broad bean wilt virus2 (BBWV2). We engineered BBWV2 as a dual gene expression vector for simultaneous expression of two recombinant proteins in pepper cells. In addition, we established enhanced and stable expression of recombinant proteins from the BBWV2-based dual vector via coexpression of a heterologous viral suppressor of RNA silencing. We also developed a BBWV2-based virus-induced gene silencing (VIGS) vector, and we successfully silenced the phytoene desaturase gene (PDS) using the BBWV2-based VIGS vector in various pepper cultivars. Additionally, we optimized the BBWV2-based VIGS system in pepper by testing the efficiency of PDS gene silencing under different conditions. This BBWV2-based vector system represents a convenient approach for rapid and simple analysis of gene functions in pepper.

Cucumber mosaic virus-induced gene silencing in banana

Banana (Musa spp.) is one of the world’s most important staple and cash crops. Despite accumulating genetic and transcriptomic data, low transformation efficiency in agronomically important Musa spp. render translational researches in banana difficult by using conventional knockout approaches. To develop tools for translational research in bananas, we developed a virus induced-gene silencing (VIGS) system based on a banana-infecting cucumber mosaic virus (CMV) isolate, CMV 20. CMV 20 genomic RNA 1, 2, and 3, were separately cloned in Agrobacterium pJL89 binary vectors, and a cloning site was introduced on RNA 2 immediately after the 2a open reading frame to insert the gene targeted for silencing. An efficient Agrobacterium inoculation method was developed for banana, which enabled the CMV 20 VIGS vector infection rate to reach 95% in our experiments. CMV 20-based silencing of Musa acuminata cv. Cavendish (AAA group) glutamate 1-semialdehyde aminotransferase (MaGSA) produced a typical chlorotic phenotype and silencing of M. acuminata phytoene desaturase (MaPDS) produced a photobleachnig phenotype. We show this approach efficiently reduced GSA and PDS transcripts to 10% and 18% of the control, respectively. The high infection rate and extended silencing of this VIGS system will provide an invaluable tool to accelerate functional genomic studies in banana.

Phosphatase GhDsPTP3a interacts with annexin protein GhANN8b to reversely regulate salt tolerance in cotton (Gossypium spp.).

Salinity is among the major factors limiting crop production worldwide. Despite having moderate salt-tolerance, cotton (Gossypium spp.) suffers severe yield losses to salinity stresses, largely due to being grown on saline-alkali and dry lands. To identify genetic determinants conferring salinity tolerance in cotton, we deployed a functional genomic screen using a cotton cDNA library in a virus-induced gene silencing (VIGS) vector. We have revealed that silencing of GhDsPTP3a, which encodes a protein phosphatase, increases cotton tolerance to salt stress. Yeast two-hybrid screens indicated that GhDsPTP3a interacts with GhANN8b, an annexin protein, which plays a positive role in regulating cotton response to salinity stress. Salt stress induces GhANN8b phosphorylation, which is subsequently dephosphorylated by GhDsPTP3a. Ectopic expression of GhDsPTP3a and GhANN8b oppositely regulates plant salt tolerance and calcium influx. In addition, we have revealed that silencing of GhDsPTP3a or GhANN8b exerts opposing roles in regulating GhSOS1 transcript levels, and ectopic expression of GhANN8b elevates Na+ efflux in Arabidopsis under salinity stress. Our study demonstrates that a cotton phosphatase GhDsPTP3a and an annexin protein GhANN8b interact and reversely modulate Ca2+ and Na+ fluxes in cotton salinity responses.

Plant Virus Vectors 3.0: Transitioning into Synthetic Genomics.

Plant viruses were first implemented as heterologous gene expression vectors more than three decades ago. Since then, the methodology for their use has varied, but we propose it was the merging of technologies with virology tools, which occurred in three defined steps discussed here, that has driven viral vector applications to date. The first was the advent of molecular biology and reverse genetics, which enabled the cloning and manipulation of viral genomes to express genes of interest (vectors 1.0). The second stems from the discovery of RNA silencing and the development of high-throughput sequencing technologies that allowed the convenient and widespread use of virus-induced gene silencing (vectors 2.0). Here, we briefly review the events that led to these applications, but this treatise mainly concentrates on the emerging versatility of gene-editing tools, which has enabled the emergence of virus-delivered genetic queries for functional genomics and virology (vectors 3.0).

Gene silencing approaches through virus-based vectors: speeding up functional genomics in monocots.

The design and use of existing VIGS vectors for revealing monocot gene functions are described and potential new vectors are discussed, which may expand their repertoire. Virus induced gene silencing (VIGS) is a method of transient gene silencing in plants, triggered by the use of modified viral vectors. VIGS has found widespread use in deciphering the functions of plant genes, mainly for dicots. In the last decade, however, its use in monocots has increased noticeably, involving not only previously described viruses for monocots, but also those described for dicots. Additional viruses have been modified for VIGS to bring a larger collection of monocots under the ambit of this method. For monocots, new methods of inoculation have been tried to obtain increased silencing efficiency. The issue of insert stability and duration of silencing have also been addressed by various research groups. VIGS has been used to unravel the functions of a fairly large collection of monocot genes. This review summarizes the above developments, bringing out some of the gaps in our understanding and identifies directions to develop this technology further in the coming years.

Overexpression and VIGS system for functional gene validation in oriental melon (Cucumis melo var. makuwa Makino)

As one of the effective and powerful methods for gene function analysis in plants, transient overexpression and virus-induced gene silencing (VIGS) system were developed for oriental melon (Cucumis melo var. makuwa Makino). Here, the full-length of CmLOX10, the conserved domains of CmLOX10 and CmPDS were isolated from oriental melon. The vectors for overexpression of CmLOX10, and the VIGS vector of CmLOX10 and CmPDS were constructed and transformed into agrobacterium, respectively. Especially, the sprout absorption method was performed on oriental melon, and CmPDS was selected as a reporter gene in VIGS. The expression level of CmLOX10 significantly increased in the oriental melon with the CmLOX10 overexpression construct, and lipoxygenases (LOXs) contributed to the leaf cell death in the oriental melon. The expression levels of CmLOX10 and CmPDS were significantly decreased in plants with the VIGS construct, and there was albino phenotype in CmPDS silencing leaves. In this study, CmLOX10 gene was silenced using Tobacco rattle virus based VIGS system firstly. These results proved that the transient expression technology provides an excellent tool to explore gene function in oriental melon. Transient over-expression and virus-induced gene silencing (VIGS) system could be performed successfully in oriental melon by leaf agro-infiltration and sprout absorption methods, respectively.

Applications of Virus Induced Gene Silencing (VIGS) in Plant Functional Genomics Studies

Virus induced gene silencing (VIGS) is an effective technology that exploits an antiviral defense mechanism in plants. It is a recently developed gene transcript suppression technique for characterizing the function of plant genes. VIGS is rapid, efficient and specific system for transient gene silencing. The major steps in VIGS includes; engineering viral genomes to the appropriate viral vector to incorporate fragments of host genes that are targeted to be silenced, infecting the appropriate plant hosts and silencing the target genes as part of the defense mechanism of the plant against virus infection. The VIGS vector is a recombinant virus engineered to be able to carry a piece an endogenous gene from the host. During infection with the modified vector, the host’s defense reaction will be induced against the cloned host gene; a loss of function phenotype makes it possible to identify the function of the gene. The recombinant virus is introduced into plant cells through Agrobacterium tumefaciens mediated transient expression or in vitro transcribed RNA inoculation or direct DNA inoculation. The Trans gene is amplified along with the viral RNA by RNA dependent RNA polymerase generating dsRNA molecules. dsRNA is the triggering molecule of Post transcriptional gene silencing. VIGS as a reverse genetics tool for functional genomics studies presenting several advantages. Despite its great potential, many limitations remain to be overcome. In this review, the molecular mechanism in VIGS technology, its advanced application in plant functional genomics studies and the major limitation and potential future prospects were briefly discussed.

RNA Silencing-Mediated Apple Latent Spherical Virus Vaccine in Plants.

The apple latent spherical virus (ALSV), originally isolated from an apple tree in Japan, is a small spherical virus with a diameter of 25 nm and comprises a bisegmented, single-stranded RNA genome (RNA1 and RNA2) and three different capsid proteins (Vp25, Vp20, and Vp24). The virus can experimentally infect a broad range of plants including, not only model plants (Arabidopsis thaliana and Nicotiana species) but also economically important crops such as cucumber, soybean, tomato, fruit trees, and flowers. ALSV has been used as an effective plant virus vector for virus-induced gene silencing (VIGS) to assess gene functions because the virus infects most of the host plants without showing any symptoms and induces a uniform knockout phenotype in infected plants. Moreover, the VIGS persists throughout plant growth in infected plants. Here, we show that genetically engineered ALSV vectors (ALSV vaccines) containing a partial genome sequence of pathogenic viruses display a high degree of cross-protection against the challenge inoculation of the corresponding pathogenic viruses. Treatment effects can also be expected in virus-infected plants by subsequent inoculation with ALSV vaccine.

Analysis of Antiviral Resistance Signaling Pathways by Virus-Induced Gene Silencing in Nicotiana benthamiana.

Owing to the absence of antiviral chemicals, strategies to deploy antiviral resistance in plants are desirable. Deciphering antiviral resistance mechanisms has been extensively promoted by virus-induced gene silencing (VIGS) technique, which decreases the transcriptional level of the gene of interest via RNA silencing machinery triggered by the partial gene fragment inserted into a viral vector. This technique has contributed to addressing the function of a number of host genes, which are involved in signaling pathways leading to Resistance (R) protein-mediated resistance and the viral disease symptoms such as systemic necrosis. Here we describe the general VIGS protocol and its tips to analyze antiviral resistance mechanism using Tobacco rattle virus (TRV)-based VIGS vector in tobacco plant Nicotiana benthamiana. In most cases, the knockdown of host genes by TRV vector is highly efficient and uniform at the whole plant level but is not associated with any severe symptoms. Using this method together with inoculation of a challenge virus and transient overexpression of a viral elicitor or another host signaling component by agroinfiltration, as well as measurement of hallmarks of antiviral responses, we can address the role of the host factors and the epistatic relationship between several host factors in the resistance signaling pathway against plant virus infection. The protocol described here using the highly susceptible host N. benthamiana to a variety of plant viruses provides the opportunity to study resistance mechanisms underlying many host-virus interactions.

Virus-induced gene silencing: empowering genetics in non-model organisms.

Virus-induced gene silencing (VIGS) is an RNA interference-based technology used to transiently knock down target gene expression by utilizing modified plant viral genomes. VIGS can be adapted to many angiosperm species that cover large phylogenetic distances, allowing the analysis of gene functions in species that are not amenable to stable genetic transformation. With a vast amount of sequence information already available and even more likely to become available in the future, VIGS provides a means to analyze the functions of candidate genes identified in large genomic or transcriptomic screens. Here, we provide a comprehensive overview of target species and VIGS vector systems, assess recent key publications in the field, and explain how plant viruses are modified to serve as VIGS vectors. As many reports on the VIGS technique are being published, we also propose minimal reporting guidelines for carrying out these experiments, with the aim of increasing comparability between experiments. Finally, we propose methods for the statistical evaluation of phenotypic results obtained with VIGS-treated plants, as analysis is challenging due to the predominantly transient nature of the silencing effect.

Phloem-Triggered Virus-Induced Gene Silencing Using a Recombinant Polerovirus.

The phloem-limited poleroviruses infect Arabidopsis thaliana without causing noticeable disease symptoms. In order to facilitate visual infection identification, we developed virus-induced gene silencing (VIGS) vectors derived from Turnip yellows virus (TuYV). Short sequences from the host gene AtCHLI1 required for chlorophyll biosynthesis [42 nucleotides in sense or antisense orientation or as an inverted-repeat (IR), or an 81 nucleotide sense fragment] were inserted into the 3′ non-coding region of the TuYV genome to screen for the most efficient and robust silencing vector. All recombinant viruses produced a clear vein chlorosis phenotype on infected Arabidopsis plants due to the expression inhibition of the AtCHLI1 gene. The introduction of a sense-oriented sequence into TuYV genome resulted in a virus exhibiting a more sustainable chlorosis than the virus containing an IR of the same length. This observation was correlated with a higher stability of the sense sequence insertion in the viral genome. In order to evaluate the impact of the TuYV silencing suppressor P0 in the VIGS mechanism a P0 knock-out mutation was introduced into the recombinant TuYV viruses. They induced a similar but milder vein clearing phenotype due to lower viral accumulation. This indicates that P0 does not hinder the performances of the TuYV silencing effect and confirms that in the viral infection context, P0 has no major impact on the production, propagation and action of the short distance silencing signal in phloem cells. Finally, we showed that TuYV can be used to strongly silence the phloem specific AtRTM1 gene. The TuYV-derived VIGS vectors therefore represent powerful tools to easily detect and monitor TuYV in infected plants and conduct functional analysis of phloem-restricted genes. Moreover this example indicates the potential of poleroviruses for use in functional genomic studies of agronomic plants.

Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots

AimWe report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots.BackgroundVIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function.ResultsWe developed an African cassava mosaic virus—based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots.ConclusionsThe African cassava mosaic virus—based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.

A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response.

A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress. Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching phenotype in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12-14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

Barley stripe mosaic virus (BSMV) as a virus-induced gene silencing vector in maize seedlings.

Barley stripe mosaic virus (BSMV) was the first reported and still widely used virus-induced gene silencing (VIGS) vector for monocotyledons including wheat and barley. Despite BSMV's reported infectivity on maize (Zea mays), the use of the virus as a vector in maize has not been optimized. Here, we assayed infectivity of BSMV in different maize cultivars by vascular puncture inoculation. Through knockdown of the endogenous host phytoene desaturase gene, we demonstrate for the first time that BSMV can be used as a VIGS vector in maize. This adds BSMV to the repertoire of tools available for functional studies in maize.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors.

Use of a Virus Gene Silencing Vector for Maize Functional Genomics Research.

Virus-induced gene silencing (VIGS) is a genetic technology that exploits the RNA-mediated defense against virus. The method has great potential for plant reverse genetics as it could knock down gene expression in a rapid way, which is triggered by a replicating viral genome engineered to carry a fragment of host gene to be silenced. A number of efficient VIGS vectors are available for dicots, such as for model plant Nicotiana benthamiana; however, only a few of VIGS vectors for monocotyledonous cereal crops. Here, we describe the method for the use of a newly developed VIGS vector based on a maize-infecting Cucumber mosaic virus (CMV) strain ZMBJ-CMV for maize. The RNA2 of ZMBJ-CMV was modified as a vector pCMV201-2bN81 having multiple cloning sites for the insert of 100-300bp fragment of target gene. Using a method of vascular puncture inoculation of maize seeds with crude sap prepared from Agrobacterium-infiltrated N. benthamiana leaves, silencing of target genes could be obtained in 4weeks.

Virus-Induced Gene Silencing in Maize with a Foxtail mosaic virus Vector.

Virus-induced gene silencing (VIGS) is a powerful technology for rapidly and transiently knocking down the expression of plant genes to study their functions. A VIGS vector for maize derived from Foxtail mosaic virus (FoMV), a positive-sense single-stranded RNA virus, was recently developed. A cloning site created near the 3' end of the FoMV genome enables insertion of 200-400 nucleotide fragments of maize genes targeted for silencing. The recombinant FoMV clones are inoculated into leaves of maize seedlings by biolistic particle delivery, and silencing is typically observed within 2 weeks after inoculation. This chapter provides a protocol for constructing FoMV VIGS clones and inoculating them into maize seedlings.

Enhanced Host-Parasite Resistance Based on Down-Regulation of Phelipanche aegyptiaca Target Genes Is Likely by Mobile Small RNA.

RNA silencing refers to diverse mechanisms that control gene expression at transcriptional and post-transcriptional levels which can also be used in parasitic pathogens of plants that Broomrapes (Orobanche/Phelipanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops and cause severe negative effects on the yield and yield quality of those crops. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we suggest an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipanche aegyptiaca genes PaACS, PaM6PR, and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing vector and stable expression in transgenic tomato Solanum lycopersicum (Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACS and PaM6PR transcripts from P. aegyptiaca tubercles grown on transgenic tomato or on TRV-infected Nicotiana benthamiana plants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamiana plants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes.