In plants, RNA silencing plays a key role in antiviral defense. To counteract host defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that interfere with the cellular silencing machinery through various mechanisms not always well understood1.Mungbean yellow mosaic virus (MYMV) is a plant bipartite geminivirus responsible for a devastating plant disease in some areas of tropics and sub-tropics where its natural host, Vigna mungo, is one of the major sources of food. In some bipartite Begomoviruses both AC2 and AC4 viral proteins act as VSRs, albeit with different efficiency in the same viral species2. Previous studies demonstrated that the MYMV-encoded AC2, which transactivates transcription of late viral genes, functions as a strong VSR3. We examined the role of MYMV AC4 and showed by quantitative real-time PCR that it is essential for infectivity but not for virus replication. MYMV AC4 acts as a determinant of pathogenicity and when expressed from the heterologous Potato virus X (PVX) increases the severity of viral-induced symptom in Nicotiana benthamiana. Importantly, we showed that AC4 counteracts virus induced gene silencing (VIGS) in PVX-GFP-infected N. benthamiana 16c lines, and strongly suppresses the systemic phase of silencing but is an inefficient suppressor of local gene silencing in Agrobacterium co-infiltration experiments. These results demonstrate that MYMV hosts two silencing suppressors, AC2 and AC4, that, probably target distinct steps of the silencing machinery. Interestingly, while most of the known VSRs have cytoplasmic localization we observed that MYMV AC4 specifically accumulates to the plasma membrane (PM) and secondarily to the nucleus. Despite its hydrophilic nature, MYMV AC4 is not predicted to be a globular protein and, probably because of its short length (only 100 aminoacids), it is very poorly structured. AC4 has no trans-membrane domain but hosts a typical nuclear localization signal consensus sequence (KRR) and a cysteine (C11), flanked by two nonpolar phenylalanines, that is predicted to be palmitoylated. Palmitoylation is a post-translational modification that regulates membrane-protein interactions and serves to increase relative membrane affinity. It consists of the reversible linkage of palmitate to proteins at cysteine residues belonging to a variety of sequence motifs. Site-specific mutagenesis experiment revealed that these two domains are functional and responsible for the subcellular localizations of AC4 to the PM (C11) and for nuclear targeting (KRR). Furthermore, the evidence that the N-terminal 12 aminoacids of AC4 expressed in fusion with GFP specifically redirect the cytosolic protein to the PM, supports the indication that C11 mediates protein binding to PM via a process of S-palmitoylation previously unreported for any plant virus protein. We obtained biochemical confirmation of AC4 palmitoylation by means of biotin switch assay4.Remarkably, replacement of C11 with

Virus-induced gene silencing (VIGS) is a well-established reverse genetics technology for assessment of gene functions in plants. VIGS is a transient loss-of-function assay that involves three steps: engineering viral genomes to include fragments of host genes that are targeted to be silenced, infecting the plant hosts and suppressing the target genes expression by post-transcriptionalgene silencing (PTGS).Suppression of specific mRNA accumulation allows correlation between gene silencing and the deriving phenotype, providing clues on gene functions.However, the efficiency of this technology may be scarce. In these cases, weak and/or non-homogeneous distribution of VIGS through the plant may generate results not fully coherent,. This often limits the extensive application of the technique to more permissive plant species such as Nicotiana benthamiana.Aiming at increasing VIGS efficiency in functional studies,particularly in key crop species, we produced and tested new constructs using a Tobacco rattle virus (TRV)-based vector in tomato (Solanum lycopersicum) and other solanaceous crops. This innovative approach consisted in cloning into the TRV vector a short fragment of a host gene containing at its termini mutations designed forthe expression of small interfering RNAs (siRNAs) that mimicked a microRNA(miRNA) structure.The recently developed artificial microRNAs (amiRNAs) technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites.We designed a vector where amiRNA-like small RNAs are generated when viral intermediate dsRNA forms are targeted by the host PTGS machinery in the cytoplasm.In the viral vector, we inserted mutant sequences designed to contain at both their 5' and 3' termini one or two mismatches at selected positions. Mismatched sequences were computed by the WMD3 web tool (wmd3.weigelworld.org), an algorithm that generates all possible amiRNAs using full-length target gene sequences as input.Short (110nt) amiRNA-like containing sequences were compared for their VIGS efficiency with wild-type sequences, shorter- or longer-sized inserts and inverted-repeat constructs. Upon inoculation of our constructs , VIGS established earlier and more extensively than its wild-type counterpart in tomato, N. benthamiana and N. tabacum. For instance, suppression of the tomato reporter gene magnesium chelatase (ChlI or SU) with the VIGS-amiRNA-like construct produced the typical yellow phenotype earlier and more extensively than the standard TRV-PDS (phytoene desaturase) VIGS vector. Quantitative RT-PCR confirmed the efficiency of our VIGSamiRNA-like constructs in terms of post-transcriptional suppression of host target mRNAs. Our results are discussed in the light of their beneficial contribution t

Plant viruses move through plasmodesmata (PD) either as nucleoprotein complexes (NPCs) or as tubuleguidedencapsidated particles with the help of movement proteins (MPs). To explore how and why MPsspecialize in one mechanism or the other, we tested the exchangeability of MPs encoded by DNA and RNA virusgenomes by means of an engineered alfalfa mosaic virus (AMV) system. We show that Caulimoviridae (DNAgenome virus) MPs are competent for RNA virus particle transport but are unable to mediate NPC movement,and we discuss this restriction in terms of the evolution of DNA virus MPs as a means of mediating DNA viralgenome entry into the RNA-trafficking PD pathway.

Virus-induced gene silencing (VIGS) is a well-established reverse genetics technology for assessment of gene functions in plants. VIGS is a transient loss-of-function assay that involves three steps: engineering viral genomes to include fragments of host genes that are targeted to be silenced, infecting the plant hosts and suppressing the target genes expression by post-transcriptional gene silencing (PTGS), the defense mechanism deployed by plants against virus infections. Suppression of specific mRNA accumulation allows correlation between gene silencing and the deriving phenotype, providing clues on gene functions. However, the efficiency of this technology may be affected by various factors, including virus vector properties and susceptibility of plant host species. In several cases, weak and/or non-homogeneous distribution in the plant (or in the single leaf) of VIGS may generate results not fully coherent, particularly in terms of correlation between phenotype and accumulation levels of the specifically suppressed RNA. This often limits the extensive application of the technique to more permissive plant species such as Nicotiana benthamiana. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop species, we produced and tested new constructs using a Tobacco rattle virus (TRV)-based vector in tomato (Solanum lycopersicum) and other solanaceous crops. This innovative approach consisted in cloning into the TRV vector a short fragment of a host gene containing at its termini mutations designed for the expression of small interfering RNAs (siRNAs) that mimicked a microRNA (miRNA) structure. The recently developed artificial microRNAs (amiRNAs) technology modifies an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs targeting transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed to contain mismatches at specific nucleotides with respect to their target sites. Unlike the conventional amiRNA strategy, where target-specific 21nt small silencing RNAs derive from longer double-stranded RNA (dsRNA) precursors that are processed in the nucleus by DCL1, we designed a vector where amiRNA-like small RNAs are generated when viral intermediate dsRNA forms are targeted by the host PTGS machinery in the cytoplasm. In the viral vector, we inserted mutant sequences designed to contain at both their 5' and 3' termini one or two mismatches at selected positions. Mismatched sequences were computed by the WMD3 web tool (wmd3.weigelworld.org), an algorithm that generates all possible amiRNAs using full-length target gene sequences as input. Short (110nt) amiRNA-like containing sequences were compared for their VIGS efficiency with wild-type sequences, shorter- or longer-sized inserts and inverted-repeat constructs. Upon inoculation of our constructs , VIGS established earlier and more extensively than its wild-type counterpart in tomato, N.

Cytosine methylation is a stable and heritable modification of the DNA that imparts epigenetic control throughout the genome, including regulation of coding and noncoding elements. In plants, previous studies have contributed to shape the epigenetic landscape in developmental processes, whereas the potential for these pathways to be dynamically regulated during non-developmental processes, such as stress responses, has not been investigated thoroughly at date. Recently, it has been demonstrated that DNA methylation is involved in controlling the Arabidopsis thaliana defence response against bacterial pathogens, and several lines of evidence suggest that plant immune response to viral infections imply an involvement of DNA methylation. It has been shown that plant viruses have the capability to modify the methylation profile of the host genome, although with scarce loci-specific methylation. However a high-resolution quantitative analysis of DNA methylation changes, a technology now available for genome-wide investigations (also known as BS-Seq) is still missing in the case of plant-virus interactions. With the aim of investigating how epigenetic mechanisms contribute to the onset and progression of diseases, we used BS-Seq to analyse methylation processes on the host genome induced by a RNA virus (Cucumber mosaic virus, CMV) and a DNA virus (Cauliflower mosaic virus, CaMV). First analysis of the BS-seq data demonstrates that infection of each virus modifies the methylation landscape of all 5 chromosomes. Our data strongly indicate that the mechanism induced by RNA- and DNA-virus infection must be different. Interestingly, the RNA-virus infection mainly induces de-methylation whereas CaMV infection triggers a general hyper-methylation. Investigations on the genomic localization of these modifications result in a similar list of regions affected by the different viruses. On the other side, analysis of the differentially methylated sites according to annotated genes reveals significantly more differentially methylated genes in CMV-than in CaMV-infected plants. Furthermore, gene feature analysis shows prevalent promoter regions de-methylation upon infection of both viruses. However, we find many hypermethylated coding regions in CMV infected plants versus hardly differentially methylated coding regions in DNA-virus infected Arabidopsis.Finally, perhaps the most intriguing concluding remark of this preliminary analysis is that despite we find differentially methylated regions within gene features, such as transcripts, transposon and promoter, the large majority of methylation modifications are located in intergenic (not yet annotated) regions and might cover and regulate still unknown non-coding RNA products.

The establishment and maintenance of DNA methylation are relatively well understood whereas little is known about their dynamics and biological relevance in innate immunity [1-2]. In plants, modulation of DNA methylation might be an effective mechanism to regulate gene expression in response to abiotic and biotic stresses. Recent evidences from large-scale epigenomic approaches indicate that dynamic DNA methylation changes are not limited to gene imprinting but can regulate the plant's immune system in response to pathogens.In plants, virus infections trigger the expression of non-coding small RNAs (smRNAs) by also influencing the epigenetic status of the host genome; however, the involvement of DNA methylation in regulation of plant immune system in response to virus infection has not been so far investigated. In this context, we are carrying out a study aiming to elucidate the impact of DNA and RNA virus infections on genomic DNA methylation in plants, and their correlation with also the expression of smallRNA, by integrating the analysis of multiple "omics" datasets obtained by using next-generation sequencing technologies.In this paper we present the results of the analysis on the methylation modifications induced by the viruses infection on the whole genome and on coding and non-coding gene regions.

The transport of a viral genome from cell to cell is enabled by movement proteins (MPs) targeting the cell periphery to mediate the gating of plasmodesmata. Given their essential role in the development of viral infection, understanding the regulation of MPs is of great importance. Here, we show that cauliflower mosaic virus (CaMV) MP contains three tyrosine-based sorting signals that interact with an Arabidopsis (Arabidopsis thaliana) mA-adaptin subunit. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide. The presence of at least one of the three endocytosis motifs is essential for internalization of the protein from the plasma membrane to early endosomes, for tubule formation, and for CaMV infection. In addition, we show that MP colocalizes in vesicles with the Rab GTPase AtRAB-F2b, which is resident in prevacuolar late endosomal compartments that deliver proteins to the vacuole for degradation. Altogether, these results demonstrate that CaMV MP traffics in the endocytic pathway and that virus viability depends on functional host endomembranes. © 2014 American Society of Plant Biologists. All rights reserved.

Virus-induced gene silencing (VIGS) is a transient loss-of-function assay that involves threesteps: engineering the genome of a viral vector to include a fragment of host gene that is targeted tobe silenced, infecting the plant hosts and suppressing the target gene expression by posttranscriptionalgene silencing (PTGS). VIGS is a well-established reverse genetics technology forassessment of gene functions in plants. However, the efficiency of this technology may be low insome plant species, and this often limits the application of the technique to more permissive modelhosts. Aiming at increasing VIGS efficiency in functional studies, particularly in key crop speciessuch as tomato (Solanum lycopersicum), we tested an innovative approach that consisted in: a)enhancement of the target gene cleavage efficiency by exploiting the artificial microRNA(amiRNA) technology; and b) validation of a bioinformatic method for selecting the most suitablegene fragments for induction of gene silencing.The recently developed amiRNA technology modifies an endogenous gene silencingmechanism that processes natural miRNA precursors to small silencing RNAs targeting specifictranscripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designedto contain mismatches at specific nucleotides with respect to their target sites, thus increasingeffectiveness of target gene cleavage as compared to RNA silencing processes guided by otherperfectly matching small RNAs.The WMD3 software (wmd3.weigelworld.org) was used for both identification of putativeamiRNA sequences and selection of suitable gene regions. In WMD3, an algorithm generates insilico all possible amiRNAs putatively able to anneal to full-length target mRNA. We selected andcompared cDNA fragments (110-120 nt) from gene regions with either high or low content ofputative amiRNAs, inserted point mutations to express amiRNA-like small RNAs from the viralvectors, and cloned these cDNAs into tobacco rattle virus (TRV)-based VIGS vectors. The variableVIGS effects of such vectors were analyzed on two tomato reporter genes, phytoene desaturase(PDS) and magnesium chelatase (ChlI or SU), whose VIGS phenotypes consist in leaf bleachingand yellowing, respectively, and therefore could be visually assessed. VIGS efficiency by severaldifferent inserts was compared by evaluating intensity of VIGS phenotype, target mRNA levels andaccumulation of VIGS-target specific small interfering RNAs. Overall, our results clearly indicatedthat: i) VIGS efficiency increased when gene sequences inserted in TRV vectors included amiRNAlikepoint mutations; ii) VIGS efficiency was significantly reduced when cDNA fragments fromgene regions with low amiRNA content were expressed in TRV vectors; and iii) WMD3 wasproved an effective bioinformatic tool to select proper target gene sequences in VIGS experiment.Our results are discussed in the light of their beneficial contri

Plant virus genomes cross the barrier of the host cell wall and move to neighboring cells either in the form of nucleoprotein complex or encapsidated into virions. Virus transport is facilitated by virus-encoded movement proteins (MP), which are different from one another in number, size, sequence, and in the strategy used to overcome the size exclusion limit (SEL) of plasmodesmata (PD). 1 A group of them forms tubules inside the lumen of highly modified PDs upon removal of the desmotubule. To date the molecular mechanism(s) and the host factors involved in the assembly of MP tubules as well as the mechanistic aspects of virus particle transport throughout them remain substantially unknown. In a recent study, we showed that Cauliflower mosaic virus (CaMV) MP traffics in the endocytic pathway with the help of three tyrosine-sorting signals, which are not required to target MP to the plasma membrane but are essential for tubule formation. 2 This evidence unravels a previously unknown connection between the plant endosomal system and tubule-mediated virus movement that is here supported by demonstration of hindrance of tubule assembly upon Brefeldin A (BFA) treatment. We discuss the implications of our data on the mechanisms of viral transport through tubules and draw parallels with plant mechanisms of polarized growth.

The establishment and maintenance of DNA methylation are relatively well understood whereas little is known about their dynamics and biological relevance in innate immunity. In plants, modulation of DNA methylation might be an effective mechanism to regulate gene expression in response to abiotic and biotic stresses. Recent evidence through large-scale epigenomicapproaches indicate that dynamic DNA methylation changes are not limited to gene imprinting but can regulate the plant's immune system in response to pathogens. In plants, virus infections trigger expression and regulation of non-coding smallRNAs, and genomic regions are epigenetically modified through the action of the same molecules; however, the involvement of DNA methylation in regulation of plant immune system in response to virus infection was not investigated before. We have examined for the first time the impact of virus infections on genomic DNA methylation and the correlation with smallRNA regulation and gene expression by integrating together analysis of multiple "omics" datasets based on next-generation sequencing platforms. To investigate the possibility that DNA methylation dynamically responds to virus infection, we performed whole-genome bisulfite sequencing on Arabidopsis leaves systemically infected with either the DNA genome virus Cauliflower mosaic virus (CaMV-Arabidopsis) or the RNA virus Cucumber mosaic virus (CMV-Arabidopsis). Single-base resolution methylome analysis revealed more than 3.7million methyl-cytosines (mCs) for the control plant. Interestingly in CMV Arabidopsis we found 300.000 more mCs (hypermethylated) and in CaMV-Arabidopsis 700.000 mCs less (hypomethylated). Focusing on differentially methylated regions (DMR, 250nt in length) we observed a balanced distribution of hyper- and hypomethylation in CG and CHH context in CMV-Arabidopsis (total DMRs 2700) but in CaMV-Arabidopsis we have predominantly hypomethylated DMRs in CHH context (total DMRs 5600). Gene features including coding, non-coding and promoter sequences were assigned to unique gene identifiers according to the TAIR nomenclature. Among differentially methylated gene features, promoter regions were the vast majority, accounting, in specific mCs contexts, for up to 80% of the total. The whole gene ID dataset was subjected to gene functional enrichment analysis by using the DAVID package tool. Interestingly, definite functional categories such as "plant defense" and "auxin signalling pathway" resulted significantly enriched. The correlation between the DNA methylation status and the transcriptional modulation of those genes is under investigation. A comparison between methylation profiles induced by either CaMV or CMV infections revealed conspicuous qualitative and quantitative differences. Taken together our results indicate that RNA- and DNA-genome virus infection induce different regulation of DNA methylation and, at least in part, different immune response in Arabidopsis.

Mulberry is a deciduous tree belonging to the Moraceae family. Although its economical importance and spreading all over the world, due mainly to the domesticated silkworm (Bombyx mori L.) breeding, to date, only few information are available about viral and virus-like diseases affecting this plant. In 2012 a small fragment of a Badnavirus DNA genome was sequenced after a DOP-PCR assay conducted on a mulberry plant, originated from Lebanon, showing symptoms of leaf mottling and vein yellowing. The virus, whose particles were observed with electron microscopy from a partially purified preparation and in tissue thin sections, was provisionally named Mulberry badnavirus-1 (MBV-1). Since different attempts of completing the full-length genome sequence through a conventional approach failed, a small RNA library was constructed for deep sequencing and run according to Illumina protocol ssRNAs analysis allowed the design of a set of primers used in PCR for the achievement of the full length sequence. The complete genome contains all the sequence features and the characteristic functional domains of the genus Badnavirus (highest nucleotide similarity shared with FBV-1 at 54%). By contrast to the badnaviruses, with genomes encoding for 3-4 ORFs, MBV-1 resembles genome organization of Petunia vein clearing virus (PVCV), which bears a single ORF. The study of the distribution of sRNA on the MBV-1 complete genome showed a tidy prevalence of 21- and 22-nt reads, differently from other viruses in the Caulimoviridae family, featuring a nuclear replication and typically supporting an accumulation of the 24-nt sRNAs involved in methylation processes.

Some plant viruses, with RNA and DNA genomes, move from cell to cell in a tubule-guided fashion. The movement protein (MP) is the essential structural component of these tubules crossing highly modified plasmodesmata (PD) upon removal of the desmotubule. Entire virus particles move throughout tubules to adjacent cells. While the ultrastructural properties of MP tubules have been largely studied, the molecular mechanism of assembly and the mechanistic aspects of virus particle transport through them remain completely unknown.The MP of Cauliflower mosaic virus (CaMV) forms tubules that guide the movement of encapsidated virus particles via coiled-coil interaction with the virion-associated protein (VAP)1. We used scanning deletion mutagenesis starting from the MP C-terminus to determine the shortest sequence required for tubule formation. Expression of the deletion mutants in fusion with the green fluorescent protein (GFP) in Nicotiana benthamiana protoplasts revealed that the C-terminal coiled-coil domain (32-terminal amino acids), mediating interaction with VAP, is involved neither in tubule assembly nor in their stabilization. All mutants longer than 280 aminoacids (full length MP, 327 amino acids) formed protruding tubular structures whereas deletion or mutation of the two leucines occupying the positions 279 (L279) and 280 (L280) abolished tubule formation but did not affect protein targeting to plasma membrane (PM). Noticeably, dileucine motifs are cargo selection and sorting signals for recruitment to clathrin-coated vesicles2. Consistently with this observation, we also demonstrated that tubule assembly of MP is hindered upon treatment of N. benthamiana protoplasts with Brefeldin A, which inhibits anterograde vesicle transport including recycling from endosomes back to PM. These observations support the hypothesis that CaMV MP may use vesicular trafficking not only to be correctly supplied and targeted to the PM3 but also specifically for tubule assembly. To gain more insight into the arrangement of MP molecules within the tubule structure, we examined a possibility of MP self-interaction other than via the coiled-coil domain1. To this aim, the N-terminal (MPa), central (MPb), and C-terminal (MPc) fragments of the MP sequence were expressed in fusion to GST and used in GST-pull down experiments to test their interaction with a full-length CaMV MP tagged with the Hemagglutinin epitope (MP-HA). This experiment revealed the presence of binding regions in the MPa and MPc fragments. Analysis of another mutant of the GST-C-terminal fragment (MPc?cc) confirmed that the domain of self-interaction in the MPc fragment was distinct from the coiled-coil domain. Further investigations contributed to demonstrate the homo-interaction character of the fragments and allowed to form a hypothesis on how CaMV MP monomers oligomerize in the tubule structure. Finally, we demonstrated that self-interaction of the MPc fragment is not hindered upon deletion of

Membrane trafficking is essential in eukaryotic cells as a delivery system for newly synthesized proteins from the endoplasmic reticulum (ER) to reach the plasma membrane (PM) or the tonoplast via intermediate endomembrane compartments. The selective transport of macromolecules between different compartments of the endomembrane system is mediated by small vesicles via adaptor complexes (AP-1-4). The ? subunit of AP complexes is devoted to cargo protein selection via a specific and well characterized interaction with a tyrosin-sorting signal (YXX?, where ? is a bulky hydrophobic residue and X is any amino acid). In plant systems, ER and PM provide membrane continuity between cells through the connections made by plasmodesmata (PD). Virus movement, which requires passage of macromolecules through PD connections, is mediated by one or more virus-encoded MPs with the help of the host cytoskeleton and/or endomembranes. The MP encoded by Cauliflower mosaic virus (CaMV) forms tubules guiding encapsidated virus particle cell-cell transport via an indirect MP-virion interaction. CaMV MP does not require an intact cytoskeleton for both PM-targeting and tubule formation. However, how this and the other tubule-forming MPs targets the PM and form tubules remains to be elucidated. In this study, we examined the three tyrosine-sorting motifs in CaMV MP and showed that each of them interacts directly with subunit ? of an Arabidopsis AP-complex. Fluorophore-tagged MP is incorporated into vesicles labeled with the endocytic tracer FM4-64 and the pharmacological interference of tyrphostin A23, an inhibitor of endocytosis, confirmed that MP traffics in the endocytic pathway. Mutations in the three endocytosis domains revert in the viral context suggesting that vesicle carrier activity is essential for CaMV viability. In our system, mutation of the three tyr-signals blocks internalization of the protein from the plasma membrane to early endosomes, PD localization and tubule formation, but does not prevent targeting of newly synthesized MP to the plasma membrane, at least in the early stages of infection. The evidence we provide that upon mutation of all three YXX?-signals MP can efficiently interact with PDLP1, a PD protein involved in assembly of CaMV MP into tubules, indicates that this MP mutant is competent to form tubules and its failure to accumulate in PD and to form tubules more probably depends on the inability to target PDs. This suggests that after targeting the plasma membrane (via as yet unknown strategy), MP might use a recycling pathway for specific targeting of PD via constitutive cycling between EE and PD. As constitutive cycling of plasma membrane proteins is blocked by BFA, recycling of MP is supported here by the demonstration that formation of foci (and tubules) is inhibited upon treatment of protoplasts with BFA. Tyrosine-signals can interact with several ?-adaptins and help the same protein to traffic in different compartments of the end

Plant viruses are obligate parasites that exploit host components for replication and spread inside the host. Transport of the viral genome is enabled by movement proteins (MPs) targeting the cell periphery to mediate passage throughout plasmodesmata (PD). Pectin methylesterase (PME) is one of the critical host factors facilitating MPs in PD gating, and a direct interaction of PME with Tobacco mosaic virus (TMV) MP is required for viral movement and in turn for virus viability. PME is a critical enzyme for host development and defence, acting via complex mechanisms involving multigenic and tissue specific isoforms and endogenous inhibitors. This composite activity of PME suggests that level and timing of protein accumulation, with respect to virus inoculation and MP expression, can be critical for the functional outcome of the PME-MP interaction and in turn for the success of a viral infection. Based on this notion, we tested different experimental conditions to evaluate the beneficial effect of the downregulation of PME gene expression on the development of TMV-induced disease and on plant protection. We used virus induced gene silencing technology (VIGS) to downregulate PME gene expression, which resulted in a 30-45 % reduction of TMV symptom severity and, correspondingly, to a 60 % reduction of TMV RNA accumulation in systemic leaves. VIGS proved to be a rapid and effective technology for PME gene silencing in functional assays and for plant defence from viral infection. Our findings indicate that N. benthamiana plants with hindered expression of PME survive a TMV infection, which kills non-silenced plants within a week.