Article

Infection of Tobacco orArabidopsisPlants by CMV Counteracts Systemic Post-transcriptional Silencing of Nonviral (Trans)Genes

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Abstract

Cucumber mosaic cucumovirus (CMV) infection but not tomato black ring nepovirus infection counteracted post-transcriptional gene silencing (PTGS) of nitrate reductase (Nia) or beta-glucuronidase (uidA) transgenes in newly developing leaves of tobacco and Arabidopsis plants. PTGS did not affect meristems of noninfected silenced plants, indicating that the interfering effect of CMV is not likely to occur in the meristem. Models are proposed to explain how CMV (which has no sequence similarity to the Nia or uidA transgenes) can inhibit cellular factors involved in the RNA degradation step of PTGS and/or inhibit the systemic spread of the silencing signal to tissues emerging from the meristem.

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... To explain this synergistic interaction, Pruss et al. suggested that one of the viruses suppresses a host defense mechanism that normally limits the accumulation of the second virus. PTGS has been implicated in this antiviral defense by the finding that the virus-encoded proteins, which are responsible for synergism, also have the ability to suppress PTGS (Anandalakshmi et al., 1998;Beclin et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998). Moreover, because suppressors of gene silencing enhance the accumulation of diverse virus types, these findings provide indirect evidence that the PTGS-like resistance is a generalized defense mechanism in plants. ...
... A straightforward counterdefense strategy, used by potyviruses (Kasschau et al., 1997;Anandalakshmi et al., 1998;Brigneti et al., 1998) and cucumber mosaic virus (Beclin et al., 1998;Brigneti et al., 1998), employs virus-encoded suppressors of PTGS that also suppress the RNA-mediated defense response. In a second type of counterdefense strategy, the virus may evade the RNA-mediated mechanism by replicating rapidly. ...
Article
Previously, it was shown that the upper leaves of plants infected with nepoviruses and caulimoviruses are symptom free and contain reduced levels of virus. These leaves are said to be recovered. Recovery is associated with RNA-mediated cross-protection against secondary virus infection. Here, by analyzing plants infected with viruses that are quite distinct from the nepovirus or caulimovirus groups, we demonstrate that this RNA-mediated defense is a general response to virus infection. Upon infection with a tobravirus, plants exhibited RNA-mediated cross-protection and recovery, as occurs in nepovirus-infected plants. However, upon infection with a potexvirus, plants exhibited RNA-mediated cross-protection without recovery. In both instances, a transient gene expression assay showed that RNA-mediated cross-protection was functionally equivalent to post-transcriptional gene silencing. Combined, these data provide direct evidence that post-transcriptional gene silencing of nuclear genes is a manifestation of a natural defense mechanism that is induced by a wide range of viruses.
... In Arabidopsis thaliana, FT is involved in the control of long-days-induced flowering [19, 27±29]. FT also regulates stomatal opening [30]. ...
... Of note, a faint, but visible, GFP expression level was observed in the apical meristems and, on occasions, in the tip of some rootlets of the GFFG/GFP or GFFG+GFP lines ( Figure 1C and data not shown). Thus, as previously observed in other plant species, IR-constructs trigger in Vitis a potent RNA silencing reaction in somatic tissues but is, somehow, less efficient in actively dividing cells [29][30][31] . ...
Article
In grapevine, gibberellins activate latent bud and stimulate the formation of tendrils but in contrast to Arabidopsis, they appear to inhibit the formation of inflorescences. Moreover, as the flowering of the grapevine is not sensitive ta photoperiod, one might wonder whether the ortholog of the FTgene (VvFT) still has an integrative raie in leaves and it activates the expression of the ortholog ofLFY gene (VFL). ln the first part of my thesis, we conducted a molecular analysis with original material: a rootstock 41 B transformed with a construct containing the VvFT gene under the control of the 35S promoter and a derivative of the plant Pinot Meunier, carrying a mutation in the GA-INSENSITIVE gene (GAl). Our study shows that gibberellins and 1 or the gene VvFT activate genes in flowering as a VFL, but with very different responses between the tendril, latent buds and inflorescences.ln the second part of my thesis, we are interested in silencing. We produced transgenic plants of the PN40024 which line containing either the gene encoding GFP, a stem-loop structure GF-FG, orbath. The embryogenic callus transgenic GFP and GFP + GF-FG fluoresce. We observed acomplete disappearance of fluorescence in PN40024 GFP + GF-FG, from the first leaves appear and in the whole plant Molecular analysis revealed small RNAs of 21 nt and 24nt produced from the stem-loop structure GF-FG. Small secondary 21 nt RNAs produced from the sequence of the GFP were also detected.
... In our research, we found that the accumulation of ORMV and TuMV increased accompanied by the up-regulated expression of BnSGS3, suggesting that these two viruses can successfully inhibit RNA silencing in plants [28]. So far, many viruses have been reported to have this function [29][30][31][32][33], which may be relevant to the virus suppressors of RNA silencing (VSRs). Among these VSRs, HC-Pro protein encoded by potyviruses (TuMV also encodes this HC-Pro) is a highly effective VSR that not only inhibits silencing but can also reverse an already established RNA silencing [34,35]. ...
... Correlation analysis revealed a negative linear correlation between the two factors ( Figure 6G-I). Previous studies also showed 5-fold over accumulation of CMV RNA in sgs3 mutant [4] and CMV 2b deficient mutant failed to infect wild-type Arabidopsis but was highly virulent in sgs3 mutant [40], which suggested that the CMV 2b protein did not target the silencing mechanism in the same way with HC-Pro [30,31]. During infection, CMV 2b directly interacted with AGO1 and specifically inhibited its slicing activity [41][42][43][44]. ...
Article
Full-text available
Virus diseases greatly affect oilseed rape (Brassica napus) production. Investigating antiviral genes may lead to the development of disease-resistant varieties of oilseed rape. In this study, we examined the effects of the suppressor of gene silencing 3 in Brassica napus (BnSGS3, a putative antiviral gene) with different genus viruses by constructing BnSGS3-overexpressing (BnSGS3-Ov) and BnSGS3-silenced (BnSGS3-Si) oilseed rape (cv. Zhongshuang No. 6) plants. These three viruses are Oilseed rape mosaic virus (ORMV), Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV). The native BnSGS3 expressed in all examined tissues with the highest expression in siliques. All three viruses induced BnSGS3 expression, but ORMV induced a dramatic increase in the BnSGS3-Ov plants, followed by TuMV and CMV. Upon inoculation with three different viruses, transcript abundance of BnSGS3 gene follows: BnSGS3-Ov > non-transgenic plants > BnSGS3-Si. The accumulation quantities of ORMV and TuMV exhibited a similar trend. However, CMV accumulation showed an opposite trend where virus accumulations were negatively correlated with BnSGS3 expression. The results suggest that BnSGS3 selectively inhibits CMV accumulation but promotes ORMV and TuMV accumulation. BnSGS3 should be used in different ways (up- and down-regulation) for breeding virus-resistant oilseed rape varieties.
... RNA silencing operates at several levels including transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). The former involves methylation of DNA cytosine bases by RNA-directed DNA methylation (RdDM) (Jones et al., 1998;Mette et al., 2000;Wassenegger et al., 1994), while the latter occurs either by cleavage (Baumberger and Baulcombe, 2005) or translational inhibition (Brodersen et al., 2008) of homologous RNA transcripts. Broadly, sRNAs can be divided into microRNAs (miRNAs) and short-interfering RNAs (siRNAs), of which there are several types (Table 1). ...
... The spread of silencing into upper leaves closely resembles the venous spread of phloem-specific dyes (Roberts et al., 1997). The use of nontoxic concentrations of cadmium to block phloem transport has repeatedly been shown to inhibit systemic silencing spread (B eclin et al., 1998;Ghoshroy et al., 1998;Ueki and Citovsky, 2001). Finally, a more recent study analysing xylem and phloem exudate in Brassica napus revealed the presence of both long RNAs and sRNAs in phloem sap, while RNAs of any size could not be detected in the xylem (Buhtz et al., 2008). ...
Article
RNA silencing is a form of genetic regulation, which is conserved across eukaryotes and has wide ranging biological functions. Recently, there has been a growing appreciation for the importance of mobility in RNA silencing pathways, particularly in plants. Moreover, in addition to the importance for mobile RNA silencing in an evolutionary context, the potential for utilizing mobile short silencing RNAs in biotechnological applications is becoming apparent. This review aims to set current knowledge of this topic in a historical context and provides examples to illustrate the importance of mobile RNA silencing in both natural and artificially engineered systems in plants. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
... RNA silencing is known to be strongly suppressed in apical tissue and newly emerged leaves after CMV infection (Béclin et al. 1998;Brigneti et al. 1998). In the present study, we observed that suppression of RNA silencing was induced mostly in the new developing leaves. ...
... Breakdown of TuMV resistance by CMV-KT must have occurred in the new, developing leaves because CMV-KT cannot suppress the RNA silencing machinery in such developing tissues before viral invasion. The presence of CMV was essential for the suppression of silenced sense transgenes in new leaves (Béclin et al. 1998). These results suggest that the breakdown of TuMV resistance by CMV-KT may be due to the viral movement and distribution after infection. ...
Article
Full-text available
The ability of Cucumber mosaic virus (CMV) subgroup II strain CMV-KT to suppress RNA silencing via posttranscriptional gene silencing (PTGS) was determined using Turnip mosaic virus (TuMV)-resistant transgenic Arabidopsis thaliana (TuR-At) carrying TuMV coat protein (CP) transgenes. CMV-KT induced only slight growth retardation in TuR-At plants, but no dwarf or mosaic symptoms, while subgroup I strain Y (CMV-Y) caused severe symptoms. Expression of the RNA-silenced TuMV-CP transgene resumed and produced TuMV-CP in either CMV-KT- or CMV-Y-infected TuR-At plants, indicating that the RNA silencing of the TuMV-CP gene was suppressed. Challenge inoculation experiments revealed that RNA silencing of the TuMV-CP transgene by CMV-KT infection is suppressed mostly in young developing leaves, but not in mature leaves, of TuR-At plants. These results demonstrated that transgene-derived virus resistance in transgenic Arabidopsis plants can be compromised by even a mild strain of CMV infection.
... In 1998, four groups of researchers demonstrated, virtually at the same time, that PTGS is indeed a plant defense mechanism by showing that some viruses have a counterdefensive strategy involving the suppression of gene silencing (Anandalakshmi et al., 1998;Beclin et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998). The discovery that gene silencing is involved in defense against viruses provided some explanation to the phenomenon that had been heavily associated with transgenic plants. ...
... Certain plant viruses encode proteins that can suppress PTGS (Anandalakshmi et al., 1998;Beclin et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998;Voinnet et al., 1999Voinnet et al., , 2000. The identification of such proteins provides a novel approach to understanding silencing in plants. ...
Article
Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component–proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs.
... Certain viruses encode proteins that act as viral suppressors of gene silencing (Anandalakshmi et al., 1998;Béclin et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998). For example, the HC-Pro protein of potyviruses can reverse silencing in tissues in which silencing already has been set, and the 2b protein of cucumber mosaic virus (CMV) prevents initiation of gene silencing at the growing points of the plant. ...
Article
Post-transcriptional gene silencing (PTGS) is a homology-dependent process that reduces cytoplasmic RNA levels. In several experimental systems, there is also an association of PTGS with methylation of DNA. To investigate this association, we used plants carrying a transgene encoding the green fluorescent protein (GFP). Gene silencing was induced using potato virus X RNA vectors carrying parts of the coding sequence or the promoter of the GFP transgene. In each instance, homology-based, RNA-directed methylation was associated with silencing. When the GFP-transcribed region was targeted, PTGS affected both transgene and viral RNA levels. When methylation was targeted to a promoter region, transgene RNA levels were reduced; however, viral RNA levels were unaffected. For comparison, we induced PTGS of the gene encoding the endogenous ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) small subunit (rbcS) by inoculation with potato virus X–rbcS. In this example, no methylation of the rbcS DNA was associated with the reduction in rbcS transcript levels, and viral RNA levels were unaffected. Finally, we investigated DNA methylation by using GFP-transformed plants in which PTGS was induced by localized introduction of a T-DNA carrying GFP sequences. In these plants, there was methylation of a GFP transgene associated with systemic spread of a gene-silencing signal from the infiltrated part of the plant. This transgene methylation was not affected when systemic PTGS was blocked by suppressors of silencing encoded by potato virus Y and cucumber mosaic virus. Combined, these data support an epigenetic model of PTGS in which transgene methylation is associated with an RNA–DNA interaction that ensures that PTGS is maintained.
... These findings have been extended to an unrelated virus, cucumber mosaic virus. Infection of tobacco or Arabidopsis plants with this virus can relieve post-transcriptional silencing in newly developing leaves [16], and Brigneti at al. [15] have now shown that such suppression depends on the 2b protein of the virus. Significantly, this protein was already known to be a suppressor of the host plant's defense mechanisms against viruses in general. ...
Article
Plants can become 'immune' to attck by virsus by degrading specific viral RNA, but some plant virsues have evolved the general capacity to suppress this resistance mechanism.
... As a counterdefense, certain plant viruses encode proteins that can suppress the RNA silencing in order to overcome the defense mechanism. Cucumber mosaic virus (CMV)-contains the 2b protein [47], which inhibits post-transcriptional gene silencing (PTGS). The 2b protein is a protein that contains a nuclear localization signal required for long distance viral movement in some hosts and it suppresses silencing only in newly emerged tissues that develop after infection [48]. ...
Article
Full-text available
Cucumber mosaic virus is one of the most constraints to the production of tomato and other vegetable crops worldwide. Here, we generated an RNAi construct containing inverted repeat of 1138 bp fragment of a partial replicase gene of CMV-O and used it to produce transgenic tomato plants expressing CMV-specific dsRNA of the replicase gene. Inoculation of transgenic plants with CMV strain O discriminated three categories of plants: plants that showed complete resistance, which were free of symptoms; highly resistant plants, which had mild symptoms , but later recovered because new leaves that emerged were free of symptoms; and susceptible plants, which showed severe symptoms similar to wild-type plants. The completely resistant lines were selected and challenged with a closely related strain, CMV-Y. Interestingly, the transgenic plant lines either remained immune or showed high levels of resistance to the strain. No virus could be detected in uninoculated new leaves of the resistant lines after RT-PCR and Dot immunobinding assay (DIBA) analyses. We could show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgenic specific siRNA.
... To counteract RNAi, many plant viruses encode some specific proteins called suppressors, which are produced in response to natural protection of plants against viruses. The discovery that plant viruses encode suppressors of gene silencing (Anandalakshmi et al. 1998; Beclin et al. 1998; Brigneti et al. 1998; Kasschau and Carrington, 1998; Vaucheret et al. 1998) provided a strong support that RNAi functions as a natural defense mechanism against viruses (Lindbo et al. 1993; Ratcliff et al. 1999). These virus-encoded suppressors act at various components of the RNAi pathway (specifically at DICER, Ago and RISC), allowing the virus to replicate and suppressing natural defense of plants (Li and Ding, 2006). ...
... N-terminal region of 1a protein contains putative methyltransferase domain [39] and C-terminal region shows sequence similarity to viral helicases [16]. RNA 2 encodes for another protein 2b, which is expressed from the subgenomic RNA 4A and acts as suppressor of gene silencing [4,5,27]; plays a role in long distance movement of the virus [9,23,43,54] and also behave as pathogenicity determinant [10,41,43]. A recent finding suggested that this suppressor protein is indirectly involved in aphid transmission [56]. ...
Article
Cucumber mosaic virus (CMV) has a wide host range causing severe damage in many important agricultural and ornamental crops. Earlier reports showed the prevalence of CMV subgroup I isolates in India. However, some recent reports point towards increasing incidence of subgroup II isolates in the country. The complete genome of a CMV isolate causing severe mosaic in cucumber was characterized and its phylogenetic analysis with other 21 CMV isolates reported worldwide clustered it with subgroup II strains. The genome comprised of RNA 1 (3,379 nucleotides), RNA 2 (3,038 nucleotides) and RNA 3 (2,206 nucleotides). The isolate showed highest homology with subgroup II isolates: 95.1–98.7, 87.7–98.0, and 85.4–97.1 % within RNA1, RNA2, and RNA3, respectively. RNA1 and RNA2 were closely related to the Japanese isolate while RNA3 clustered with an American isolate. Host range studies revealed that isolate showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The isolate induced leaf deformation and mild filiform type symptoms in tomato. To best of our knowledge this is the first report of complete genome of CMV subgroup II isolate from India.
... Apart from this, CMV-2b protein was found to manipulate viral cell to cell movement in plants (Soards et al. 2002). First report of functional characterization of CMV-2b as a RSS CMV 2b was shown to avert the initiation of RNA silencing in newly emerging tissue but it cannot reverse established RNA silencing (Beclin et al. 1998;Brigneti et al. 1998). This result advocated that 2b might be potentially required for preventing the cell to cell spread of the silencing signal, from the locally-infected parts to the rest of the plant to promote further virus spread (Goto et al. 2007). ...
Chapter
Plant small RNAs, namely si- and miRNAs, control a gamut of biological functions by regulating gene expressions. One of the major functions is to protect the host genome from molecular parasites, including the viruses. The virus-infected plants allow generating the siRNAs from all over the viral genomes that, in turn, control viral gene expressions post-transcriptionally leading to inhibition of viral growth and spread. In the case of DNA viruses, the siRNAs also exert transcriptional control of viral gene expression in an epigenetic manner by promoting methylation of the promoter of viral genes. Further, transcriptional gene silencing (TGS) mechanism has also been shown to be involved in symptom remission. DNA viruses also interfere with the methyl cycle to prevent the availability of methyl donor (S-adenosyl methionine) for methylating viral DNAs. However, in the battles between the host and viruses, the viruses have also evolved to encode few proteins from their genomes that counteract the RNAi-mediated host defense reactions. Such group of proteins is collectively known as RNAi suppressors which also participate in viral life cycle in manifold ways besides thwarting the host RNAi activities towards the viruses. In addition, these virus-encoded proteins also manipulate the components of TGS machinery such as histone and/or DNA methyl transferases, to combat the antiviral silencing mechanism. These are also called the pathogenicity factors as they principally govern the disease symptoms in the host. The mechanistic action of a few of the viral-encoded suppressors has been dealt in some detail within the text. These proteins deregulate the host miRNAs during the expression of disease. Several studies have now shown that transgenic expression of viral suppressors can alter the accumulation and/or functioning of miRNAs leading to developmental abnormalities. Molecules like HC-Pro, P19, etc. were shown to affect the processing and activity of miRNAs. Hence the antiviral strategies could be developed by silencing these viral suppressors. Our laboratories have developed tomato transgenics expressing miRNAs and tasiRNAs which can efficiently silence the RNAi suppressors of tomato leaf curl viruses and offer a high degree of tolerance towards the viruses. The future direction of research including the biotechnological usages of the viral suppressors has been discussed.
... These silencing suppressor proteins may act at different steps in the PTGS pathway. Thus, (i) the potyvirus helper component-proteinase (HCPro) interferes with initiation and maintenance of silencing at a step coincident with or upstream of siRNA production (Anandalakshmi et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998;Llave et al., 2000;Mallory et al., 2001), (ii) the 2b protein of cucumber mosaic virus (CMV) prevents initiation of PTGS in new growth by inhibiting the long-range PTGS signaling activity (Béclin et al., 1998;Brigneti et al., 1998;Guo and Ding, 2002), and (iii) p25 from potato virus X suppresses production or action of the mobile silencing signal (Voinnet et al., 2000). It has been shown that the viral suppressors identified to date target distinct stages of PTGS. ...
Thesis
Full-text available
The sequence of the coat protein gene (CP) of potato virus X (PVX) was determined from cDNA clones. The CP gene was cloned into the pBAD-TOPO expression vector upon induction; the viral protein was expressed as 6XHis-tagged PVX fusion protein in Escherichia coli (E. coli) BL21 cells. The fusion protein was confirmed by western blot analysis using antibodies specific for the PVX. The predicted length of the coat protein gene was 714 nucleotides, corresponding to a 238 amino acid coat protein of Mr 25 KD. The nucleotide sequence of the coat protein gene of PVX (PVX-CP) was compared to the sequences of the coat protein genes of other potexviruses. The PVX-CP gene of the Egyptian isolate was found to be 80 to 96 % homologous to those of other potexviruses at the nucleotide level. Antiserum obtained from rabbit after injection with 6XHis-tagged PVX fusion protein was immuno-reactive towards the PVX in indirect ELISA, immunocapture RT-PCR (IC-RT-PCR) and dot blot immunobinding assay (DBIA). The recent development of gene transfer approaches in plants has revealed that transgene can undergo silencing after integration in the genome. More investigations have demonstrated that double-stranded RNA (dsRNA) can silence genes by triggering degradation of homologous RNA in the cytoplasm and by directing methylation of homologous nuclear DNA sequences. In this study, the efficiency of the dsRNA for the ability to trigger resistance against the Egyptian isolate of PVX (PVX-Eg2) has been investigated. A new, fast and easy method for gene delivery was proposed to transform potato and Nicotiana benthamiana plants with different constructs. PCR was carried out to detect the viral genome in challenged plants; infiltrated plants with the pFGC5491 vector without any insert, sense construct, and antisense construct; indicating the presence of viral genome in challenged plants. Infiltrated tobacco and potato plants with sense/antisense construct gave negative amplification indicating the absence of the viral genome. However, 3/10 tobacco plants infiltrated with sense/antisense construct, were able to amplify the expected fragments. While 1/10 plants gave positive PCR product in case of potato plants infiltrated with sense/antisense construct.
... There is the additional concern of PTGS/RNAi suppressor proteins that have been found in several plant and animal viruses. Viruses encoding proteins that are suppressors of RNA silencing was first reported from plant viruses in 1998 [8,14,21]. A number of plant [146,169,193] and insect [103] viruses have since been reported to express different RNAi suppressor proteins. ...
Article
RNA interference has evolved from being a nuisance biological phenomenon to a valuable research tool to determine gene function and as a therapeutic agent. Since pioneering observations regarding RNA interference were first reported in the 1990s from the nematode worm, plants and Drosophila, the RNAi phenomenon has since been reported in all eukaryotic organisms investigated from protozoans, plants, arthropods, fish and mammals. The design of RNAi therapeutics has progressed rapidly to designing dsRNA that can specifically and effectively silence disease related genes. Such technology has demonstrated the effective use of short interfering as therapeutics. In the absence of a B cell lineage in arthropods, and hence no long term vaccination strategy being available, the introduction of using RNA interference in crustacea may serve as an effective control and preventative measure for viral diseases for application in aquaculture.
... N-terminal region of 1a protein has putative methyltransferase domain [20] and C-terminal is homologous to viral helicases [11]. The 2b gene expressed from subgenomic RNA4A acts as suppressor of post transcriptional gene silencing [2,6,16] besides its role in long distance movement [9,14,31,35] and aphid transmission [36]. The 3a gene encodes the movement protein (MP) and 3b, the coat protein (CP) expressed through subgenomic RNA (RNA4). ...
Article
The complete genome of cucumber mosaic virus (CMV) from black pepper was sequenced and compared with CMV isolates from subgroups I and II reported worldwide. Percent identity and phylogenetic analyses clearly indicated that the CMV isolate from black pepper (BP) belongs to subgroup IB. Sequence analyses also showed the presence of a rare deletion of nine nucleotides in the putative methyltransferase domain of 1a gene which was observed only in two more isolates of CMV among one hundred 1a gene sequences of CMV for which sequence information is available in the database. Interestingly this deletion is not present in the black pepper isolate of CMV from China (WN1) and from Indian long pepper that is closely related to black pepper. Percent identity analyses showed that the 3′untranslated region (UTR) of the three RNAs of the BP isolate were conserved with 91% identity whereas the 5′UTR of three RNAs showed 52–80% identity. The level of gene conservation among the subgroups was highest in coat protein and lowest in 2b. The values of nucleotide diversity studies were further consistent with the above data. The ratio of non-synonymous to the synonymous substitution of the five genes of three RNAs was in the order 1a > 2a > 2b > 3a > 3b and less than one for all the genes, indicating purifying selection. These clearly reflect that the protein encoded by RNA1 is highly tolerant to amino acid changes followed by that of RNA2 and, RNA3 is the least tolerant correlating to its functional importance.
... PVX-GF, a virus that does not enter the meristem (Schwach et al., 2005), induced ubiquitous virus-induced gene silencing (VIGS) of an ectopically expressed GFP in N. benthamiana plants, except in the apical growth point (Ruiz et al., 1998). RNAi-based silencing of transgenes was also shown to occur throughout the plant but not in meristems in other cases (Béclin et al., 1998;Voinnet et al., 1998), suggesting that small RNAs are unable to enter and/or trigger silencing in this tissue. However, in two other studies, meristem-excluded viruses were able to trigger VIGS within meristems (Jones et al., 1998;Peele et al., 2001). ...
Article
Full-text available
In the arms race between plants and viruses, two frontiers have been utilized for decades to combat viral infections in agriculture. First, many pathogenic viruses are excluded from plant meristems, which allows the regeneration of virus-free plant material by tissue culture. Second, vertical transmission of viruses to the host progeny is often inefficient, thereby reducing the danger of viral transmission through seeds. Numerous reports point to the existence of tightly linked meristematic and transgenerational antiviral barriers that remain poorly understood. In this review, we summarize the current understanding of the molecular mechanisms that exclude viruses from plant stem cells and progeny. We also discuss the evidence connecting viral invasion of meristematic cells and the ability of plants to recover from acute infections. Research spanning decades and performed on a variety of virus/host combinations has made clear that, beside morphological barriers, RNA interference (RNAi) plays a crucial role in preventing - or allowing - meristem invasion and vertical transmission. How a virus interacts with plant RNAi pathways in the meristem has profound effects on its symptomatology, persistence, replication rates, and, ultimately, entry into the host progeny.
... The PTGS suppressor activity of potyviruses and Cucumber mosaic virus (CMV) has been extensively studied (Anandalakshimi et al., 1998;Béclin et al., 1998;Brigneti et al., 1998;Lucy et al., 2000). Indeed, it is known that the HC-Pro and the 2b proteins encoded by Potato virus Y (PVY) and CMV, respectively, break PTGS by interfering with different steps of the specific RNA degradation pathway. ...
Article
Full-text available
SUMMARY Plants expressing viral sequences may show homolo- gy-dependent virus resistance (HDR), which is charac- terized by specific degradation of transgenic RNA and viral RNAs homologous to the transgene. This resis- tance mechanism is a form of post-transcriptional gene silencing (PTGS), which can be suppressed by virus-en- coded proteins. We analysed the effect of Potato virus Y, Cucumber mosaic virus and Potato virus X infections on HDR to Cymbidium ringspot virus (CymRSV) shown by Nicotiana benthamiana transgenic line 92KA1 ex- pressing the full-length replicase gene of CymRSV. We observed breaking of resistance in transgenic plants doubly infected with PTGS suppressor viruses and CymRSV. The need of considering the effect of PTGS suppressor viruses on resistance or PTGS-dependent phenotype in transgenic crops is discussed.
... Perhaps the most significant recent discovery in potyvirus research is the demonstration that HC-Pro can act as a negative regulator of a plant defense mechanism based upon PTGS. Since orthologs of HC-Pro are being discovered in other viruses (e.g., CMV; Béclin et al. 1998;Brigneti et al. 1998), it may be that viruses generally need to counteract PTGS to develop systemic infections. It will be intriguing to see whether PTGS and its regulation by viral proteins could be connected with latency in plants and animals, or even to the changes in host gene expression associated with virus replication. ...
Article
Full-text available
In recent years, researchers have adopted many new technologies to help understand potyvirus pathogenesis. Their findings have illuminated key aspects of the interactions between the host and the virus, and between the virus and its aphid vector. This review focuses on advances in our understanding of the molecular determinants of systemic infection, symptom expression, aphid and seed transmission, and natural and engineered resistance to potyviruses. Very recent developments in the area of post-transcriptional gene silencing indicate not only that the process is fundamental to engineered resistance, but may also underlie many aspects of the biology of plant viruses.
... RNA1 encodes protein 1a, which is necessary for viral replication and contains helicase and methyltransferase motifs (Kadaré and Haenni 1997). RNA2 encodes protein 2a, the viral polymerase (O'Reilly and Kao 1998), as well as protein 2b, which is involved in the long-distance movement of CMV in the plant (Soards et al. 2002) and the suppression of gene silencing (Béclin et al. 1998). RNA3 encodes two proteins, the cell-tocell movement protein (protein 3a) and the coat protein (CP) (protein 3b). ...
Article
We present here the first complete sequence of cucumber mosaic virus (CMV) from RNA sequencing of a dwarf periwinkle (Vinca minor) and a Wisteria sinensis in Iran by de novo assembly and annotation of contigs. The tripartite genome of CMV-Ir-VM and Ir-WS consists of a 3391-nucleotide (nt) RNA1, a 3038-nt RNA2, and a 2197-nt RNA3 segment. The sequence comparisons and phylogenetic analyses revealed that all three RNA segments of these two isolates belonged to the CMV subgroup II. RNA1, RNA2, and RNA3 of these two isolates, respectively shared 97.61–99.06%, 97.83–98.88%, and 97.19–98.86% nucleotide identities to those of CMV subgroup II isolates. In genetic analysis, the 2b gene revealed more genetic variability (π = 0.2614) compared with other regions. Our results indicated that purifying selection pressure was the major force acting upon the CMV genome, although some positively selected sites were identified for all encoded proteins.
... The systemic movement of the silencing signal takes days and typically moves from leaves (photosynthetic source) to roots and growing points (sucrose sinks) (Voinnet et al., 1998); this fl ow is characteristic of phloem (discussed in (De Schepper et al., 2013;Turgeon and Wolf, 2009)). Furthermore, phloem transport block by cadmium inhibits systemic silencing (Beclin et al., 1998;Ghoshroy et al., 1998). Later, it was also shown that phloem fl ow strongly infl uences the systemic spread of silencing in Nicotiana benthamiana and that the direction of systemic spread of silencing from inducer to sensor can be manipulated by altering sink/source relations in the plant (Tournier et al., 2006). ...
Chapter
RNA silencing denotes sequence-specific repression mediated by small RNAs. Plants have arguably the most complex RNA silencing among eukaryots because of existence of many paralogs of key protein factors, which form in an intricate network of primary and secondary small RNAs, which mediate transcriptional and post-transcriptional effects, which target endogenous protein-coding gene expression, serve as a form of innate immunity targeting viruses, and protect genome integrity by repressing retrotransposons. The complexity of RNA silencing was introduced in an accompanying review. Here, I will focus on mobility of small RNAs in plants, which allows for silencing effects occurring at a different place than where the silencing was initiated. I will discuss different types of mobility of different classes of small RNAs across plant tissues and their biological implications. NOTE: This is a chapter from an open source book. The full text is available directly through the DOI link above.
... RNA silencing, also referred to as RNA interference (RNAi), is a gene silencing phenomenon that results in inhibition of gene expression or protein synthesis induced by the presence of double-stranded RNA (dsRNA). Before the discovery of RNA silencing pathways, RNA-mediated gene silencing was reported as co-suppression in Petunia hybrida (Napoli et al. 1990), post-transcriptional gene silencing (PTGS) Post-transcriptional gene silencing (PTGS) in tobacco (Lindbo et al. 1993;Béclin et al. 1998) and quelling in the fungus Neurospora crassa (Romano and Macino 1992). The phenomenon of RNA silencing was probably first reported as early as 1928, where tobacco plants infected with tobacco ringspot virus showed symptoms only on the initially infected leaves (Wingard 1928). ...
Chapter
Over recent years, many crops have benefited from the application of genetic transformation approaches to improve important agronomic and horticultural traits. The discovery of RNA-mediated gene silencing (RNA silencing) has allowed the application of precise approaches to inhibit plant pathogens and to alter plant metabolism and development. RNA silencing is initiated by double-stranded RNA (dsRNA) leading to sequence homology-dependent translational inhibition of a target mRNA or transcriptional repression of a target gene. RNA silencing was first described in plants as post-transcriptional gene silencing (PTGS), virus cross-protection and co-suppression. Activated by the presence of short dsRNA inside the cell, RNA silencing is a form of negative gene regulation lending itself towards the generation of loss-of-function genetic changes. Applications include reducing gene expression of a pathogen, such as a virus; reducing the expression of an endogenous plant gene to alter biosynthesis, such as that of an undesired allergen, toxin or flower or fruit pigment; reducing the expression of an endogenous plant gene to alter nutritional qualities, such as altered starch content and reducing the expression of endogenous genes to alter plant development. Alteration of plant phenotypes and inhibiting plant pathogens through the direct application of dsRNA to crop plants has provided a rapid and efficient method that is feasible for some situations and has the advantage of avoiding the steps in plant transformation and regeneration. In this chapter, we review examples that outline the technology for the application of dsRNA in crops, and discuss the role this has played in crop improvement.
... It was recently shown that certain virus-encoded proteins (e.g. potyvirus HC-Pro, cucumovirus protein 2b, potexvirus protein p25, tombusvirus protein p19, tospovirus protein NSs ) inhibit host-activated PTGS (ANANDALAKSHIMI et al. 1998;BECLIN et al. 1998;VOINNET et al. 2000;CARRINGTON et al. 2001). ...
Article
Among natural resistance mechanisms to plant pathogens, cultivar resistance has been extensively used in plant breeding to introduce what can be defined as “conventional” resistance to a number of them, including viruses. The necessity of overcoming the constraints of genetic incompatibility, so as to widen the range of possibile use of genetic control of infectious agents, has propitiated the utilization of biotechnological procedures, whereby “non conventional” or transgenic resistance was developed. Transgenic resistance to plant viruses encompasses the identification, cloning and tranferring into the recipient host of single viral genes, which gives rise to what is known as “pathogen-derived resistance” (PDR). Of the hypothesized mechanisms underlying expression of PDR, post-transcriptional gene silencing has been most extensively investigated in recent years. Despite of the success that virus-resistant cropping of transgenic plants begins to enjoy, in Europe there is still a widespread sentiment against agricultural biotechnologies and the use of genetically modified plants in particular. Yet, experimental evidence is accumulating that, in the case of PDR, the feared risks associated with genetic trasformation are minimal, if not negligible
... Interestingly, TEV stimulates systemic accumulation of CMV in a nonreciprocal manner in Nicotiana spp, a phenomenon that likely involves suppression of gene silencing responses by TEV-encoded HC-Pro (Pruss et al., 1997;Anandalakshmi et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998). CMV also encodes a suppressor of gene silencing (the 2b protein), but it may function to inhibit a component of the silencing pathway that differs from that suppressed by HC-Pro (Beclin et al., 1998;Brigneti et al., 1998). The CMV 2b protein may be relatively effective in suppressing gene silencing in Arabidopsis, whereas TEV-encoded HC-Pro may be relatively effective in Nicotiana spp. ...
Article
Arabidopsis plants have a system to specifically restrict the long-distance movement of tobacco etch potyvirus (TEV) without involving either hypersensitive cell death or systemic acquired resistance. At least two dominant genes, RTM1 and RTM2, are necessary for this restriction. Through a series of coinfection experiments with heterologous viruses, the RTM1/RTM2–mediated restriction was shown to be highly specific for TEV. The RTM2 gene was isolated by a map-based cloning strategy. Isolation of RTM2 was confirmed by transgenic complementation and sequence analysis of wild-type and mutant alleles. The RTM2 gene product is a multidomain protein containing an N-terminal region with high similarity to plant small heat shock proteins (HSPs). Phylogenetic analysis revealed that the RTM2 small HSP–like domain is evolutionarily distinct from each of the five known classes of plant small HSPs. Unlike most other plant genes encoding small HSPs, expression of the RTM2 gene was not induced by high temperature and did not contribute to thermotolerance of seedlings. The RTM2 gene product was also shown to contain a large C-terminal region with multiple repeating sequences.
Article
Many important horticultural and field crops are susceptible to virus infections or may possess a degree of resistance to some viruses, but become infected by others. Plant viruses enter cells through the presence of wounds, and replicate intracellularly small genomes that encode genes required for replication, cell-to-cell movement and encapsidation. There are numerous evidences from specific virus-host interactions to require the involvement of host factors and steps during viral replication cycle. However, viruses should deal with host defense responses either by general or specific mechanisms, targeting viral components or genome itself. On the other hand, the host plants have also adapted to defend themselves against viral attack by operating different lines of resistance responses. The defense-related interactions provide new insights into the complex molecular strategies for hosts for defense and counter-defense employed by viruses.
Article
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Gene silencing and RNA-mediated virus resistance are two remarkable and potentially useful phenomena that occur in higher plants. However, in light of recent research it seems likely that the post-transcriptional type of gene silencing and RNA-mediated virus resistance are actually manifestations of the same phenomenon. The occurrence of both post-transcriptional gene silencing (PTGS) and RNA-mediated virus resistance require sequence homology between a transgene and an endogenous gene or a transgene and an infecting virus, respectively. Furthermore, both processes are characterized by high transcription rates of homologous, silenced (trans)genes but low steady-state levels of their transcripts (in case of virus-infected plants, low steady-state transcript levels of the silenced transgene and homologous viral RNA which eventually leads to virus resistance). Therefore, PTGS is a potential tool for creating virus-resistant transgenic plants that express a sequence homologous to the invading virus. It is very unlikely, though, that PTGS in plants has evolved solely for the purpose of transgene suppression so it is perhaps not surprising that some natural virus defense systems have been found to resemble gene silencing. In addition, although plants may combat virus infections by gene silencing, there is recent evidence that some plant viruses can fight back by suppressing the plant's ability to carry out the silencing process. The advantages and disadvantages of the commercial use of PTGS for creating virus-resistant plants (RNA-mediated virus resistance) is also discussed.
Conference Paper
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Transgenic clones C2, C3, C4, C5, C6, PT3 and PT5 of Prunus domestica L. transformed with the Plum pox virus coat protein gene (PPV-CP) were evaluated for Sharka resistance under high infection pressure in field natural conditions in Romania. Transgenic clone C5, recently named “HoneySweet”, showed high resistance to PPV. None of the C5 trees becames naturally infected by aphids for more than ten years. Known to develop the post-transcriptional gene silencing (PTGS) mechanism, we assessed the effect of heterologous viruses on the efficacy and stability of PTGS displayed by the C5 plum against PPV. In this way, C5 trees were graft-inoculated with different combinations of Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) and PPV-D strain. The potential for suppression of the silencing mechanism mediated by these ilarviruses was evaluated in orchard and nursery. Both trials showed that, the engineered resistance to PPV in C5 transgenic plums was stable and was not suppressed by the presence of the assayed heterologous viruses over a three-year experimental period.
Article
The nitrogen compounds nitrate and ammonium are the minerals plants need in high quantities and which limit their growth in temperate zones. The nitrate assimilation pathway starts with nitrate uptake followed by nitrate reduction resulting in ammonium which is fixed into the amino acids glutamine and glutamate in most plants. This article concentrates on nitrate uptake and nitrate reduction with respect to higher and lower plants. The physiology and the progress in molecular approaches of both processes are considered. For nitrate uptake the well established uptake systems are discussed and special attention is drawn on nitrate sensing and the distinct nitrate carriers with special consideration of the great number of carriers identified in Arabidopsis. Knowledge particularly on nitrate sensing is rare, but it seems to be the first step in a signal transduction chain triggered by nitrate. Therefore further work should consider this topic more frequently. For nitrate reductase the focus is on the post translational modifications as regulatory tool for nitrate assimilation, on the intersections of carbon and nitrogen metabolism and on the molecular approaches. Few remarks on how environmental conditions affect nitrate assimilation are also included. Further progress is needed to understand the transduction of positive and negative signals from the environment affecting the expression of genes coding for the nitrate assimilating pathway.
Article
In animals, double-stranded short interfering RNA (siRNA) and single-stranded microRNA (miRNA) regulate gene expression by targeting homologous mRNA for cleavage or by interfering with their translation, respectively [1–3]. siRNAs are processed from injected or transgene-derived, long, perfect double-stranded RNA (dsRNA), while miRNAs are processed from short, imperfect dsRNA precursors transcribed from endogenous intergenic regions [4–9]. In plants, both siRNAs and miRNAs activate cleavage of homologous RNA targets [10–12], but little is known about the genes controlling their production or action. The SGS2/SDE1 protein contributes to produce transgene siRNA [10], while DCL1 and HEN1 contribute to endogenous miRNA accumulation [8, 9]. Here, we show that: i) SGS2, SGS3 [13], AGO1 [14, 15], and HEN1 contribute to produce transgene siRNA involved in sense posttranscriptional gene silencing (S-PTGS); ii) HEN1, but not SGS2, SGS3, or AGO1, contributes to the accumulation of the endogenous miR171 miRNA and to the cleavage of Scarecrow target mRNA by miR171[11]; iii) SGS2, SGS3, AGO1, and HEN1 contribute to resistance against cucumber mosaic virus [13, 15], but not to siRNA and IR-PTGS triggered by hairpin transgenes directly producing perfect dsRNA [16]; and iv) the actions of HEN1 in miRNA/development and siRNA/S-PTGS can be uncoupled by single-point mutations at different positions in the protein.
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Одним з найважливiших та найменш контрольованих чинникiв, що зменшують врожайнiсть рослин, є фiтовiруси. До найпоширенiших вiрусних патогенiв, якi уражують сiльськогосподарськi рослини та мають насiннєвий шлях передачi, належить вiрус огiркової мозаїки. Насiннєвий шлях передачi для цього вiрусу є доволi iстотним, тому потрiбно особливу увагу придiлити обстеженню насiння, ще до його висiвання. Одним iз типових агротехнiчних прийомiв отримання здорового врожаю є використання неiнфiкованого насiннєвого матерiалу, адже вiруси посiдають чiльне мiсце серед патогенiв, якi можуть передаватися насiнням, iнфiкуючи зародковi тканини або насiннєвi покриви. Для знезараження насiння вiд вiрусiв iснує низка хiмiо- та термотерапевтичних методiв, ефективнiсть яких, однак, значно залежить як вiд насiння (виду та рослини), так i вiд виду вiрусу. Для багатьох вiрусiв, особливо з широким спектром сприйнятливих до них рослин, проблема пошуку хiмiчних сполук з антивiрусними властивостями залишається актуальною. Вчасна дiагностика вiрусних iнфекцiй дасть можливiсть в свою чергу провести вчас у обробку iнфiкованого насiння, а отже, зменшити втрати врожаю. Нами була проведена перевiрка антифiтовiрусної дiї вiдносно ВОМ препарату «Деконекс-50 ФФ», що призначений для дезiнфекцiї рiзноманiтних матерiалiв та виробiв медичного призначення, поверхонь в примiщеннях, санiтарно-технiчного обладнання. «Деконекс-50 ФФ» — дезинфiкуючий засiб, що є прозорим рiдким концентратом i мiстить 0,5 % глутарового альдегiду, 7,5 % — додецилдиметиламонiю та 12 % — глiоксаля як дiючої речовини, а також 4 % — неiоногенних ПАР i 1 % сумiшi ефiрних масел. Препарат належить до 4-го класу мало небезпечних речовин. Як уже зазначалось, для дослiдження антифiтовiрусної дiї препарату «Деконекс 50 ФФ» було обрано систему «вiрус огiркової мозаїки — рослини Cucumis sativus». Пошук препаратiв вiдносно цього вiрусу був зумовлений як надзвичайною шкодочиннiстю цього патогену щодо овочевих культур, так i широким спектром сприйнятливих до нього рослин та високою генетичною мiнливiстю вiрусу. До того ж впродовж останнiх трьох рокiв ми регулярно виявляли ВОМ у високих титрах на рослинах родини Cucurbitaceae. Аналiз рослин огiрка сорту «Мiг», отриманих з iнфiкованого ВОМ насiння, обробленого та не обробленого препаратом «Деконекс-50 ФФ», здiйснювали на 30-й день вирощування. Внаслiдок передпосiвної обробки препаратом «Деконекс-50 ФФ» iнфiкованого ВОМ насiння за результатами IФА було встановлено пропорцiйне дозозалежне зниження вiрусного навантаження. Перевiрено, що оброблене насiння Деконексом в концентрацiях 0,5 % та 1 % призводить до зниження вмiсту антигенiв ВОМ у рослин. Дослiдження «Деконексу-50 ФФ» показало, що при обробцi препаратом рiзної концентрацiї знижується вiрусне навантаження щодо ВОМ, який передається насiнням у покривних тканинах. Таким чином, дослiджений препарат може бути рекомендований для знезараження ВОМ у контамiнованому насiннi огiркiв та для профiлактичної обробки насiння овочевих культур. При цьому концентрацiя 0,5 % знизила вмiст вiрусу на 26 %, а концентрацiя 1 % — на 51 %. Нами було проведено перевiрку антифiтовiрусної дiї препарату «Деконекс-50 ФФ» вiдносно ВОМ. Внаслiдок передпосiвної обробки препаратом «Деконекс-50 ФФ» iнфiкованого ВОМ насiння за результатами IФА було встановлено пропорцiйне дозозалежне зниження вiрусу. Таким чином, дослiджений препарат може бути рекомендований для знезараження насiння огiркiв вiд ВОМ та для розробки методiв профiлактики овочевих культур.
Chapter
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RNA silencing in plants has become one of the most well studied biological phenomena in last couple of decades. Subsequently it has proved to be an important regulator of physiological processes including growth and development as well as defense. In plants, one of the primary roles of RNA silencing is to control virus infection. To counter this measure of defense by plants, viruses have developed strategies to suppress the RNA silencing phenomena. In addition to suppression activity, these proteins perform some vital functions required for their multiplication and pathogenicity. Since the first report of suppressor proteins came in the year 1997, a number of such proteins have been reported and studied till now. In this chapter we detail the plant viral suppressors and their working mechanism along with the assays used to identify them. The last segment discusses the applications of viral suppressors as important biological tools.
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This report is the outcome of an EFSA procurement aiming at investigating and summarising the state of knowledge on (I) the mode-of-action of dsRNA and miRNA pathways, (II) the potential for nontarget gene regulation by dsRNA-derived siRNAs or miRNAs, (III) the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing. The report is based on a comprehensive and systematic literature search, starting with the identification and retrieval of~190,000 publications related to the research area and further filtered down with keywords to produce focused collections used for subsequent screening of titles and abstracts. The report is comprised of an (I) Introduction to the field of small RNAs, (II) a Data and Methodologies section containing strategies used for literature search and study selection, and (III) the Results of the literature review organized according to the three main procurement tasks. The outcome of the first task reviews dsRNA and miRNA pathways in mammals (including humans), birds, fish, arthropods, annelids, molluscs, nematodes, and plants. Eight taxon-dedicated chapters are based on ~1,400 cumulative references chosen from ~10,000 inspected titles and abstracts. We review conserved and divergent aspects of small RNA pathways and dsRNA responses in animals and plants including structure and function of key proteins as well as four basic mechanisms: genome-encoded posttranscriptional regulations (miRNA), degradation of RNAs by short interfering RNA pools generated from long dsRNA (RNAi), transcriptional silencing, and sequence-independent responses to dsRNA. The outcome of the second task focuses on base pairing between small RNAs and their target RNAs and predictability of biological effects of small RNAs in animals and plants. The outcome of the last task reviews methodology, siRNA pools, and movement of small RNAs in plants. Potential transfer of small RNAs between species and circulating miRNAs in mammals is described in the final chapter. full text: http://onlinelibrary.wiley.com/doi/10.2903/sp.efsa.2017.EN-1246/pdf
Article
The 2b protein of Cucumber mosaic virus (CMV) subgroup IB strain IA (IA2b) suppresses the sense transgene-induced post-transcriptional gene silencing (S-PTGS) but not the inverted repeat-induced post-transcriptional gene silencing (IR-PTGS) of a tobacco microsome-localized α-linolenate synthase gene (NtFAD3). In contrast, the 2b protein of CMV subgroup IB strain SD (SD2b) has been reported to suppress IR-PTGS. We overexpressed the SD2b gene in tobacco, and this transgenic line was crossed with the transgenic plants showing S-PTGS and IR-PTGS of the NtFAD3 gene. The phenotype of offspring showed that SD2b inhibited S-PTGS but not IR-PTGS. Next, we determined the suppressor activity of IA2b and SD2b proteins in a transient IR-PTGS assay. The transient expression of firefly luciferase (LUC) gene was efficiently decreased by IR-PTGS. Co-infiltration of SD2b and IA2b gene partially suppressed IR-PTGS of the LUC gene in the wild-type plants; however, these 2b proteins did not suppress IR-PTGS in the RNA-dependent RNA polymerase6 (RDR6) knockdown plants. It has been reported that RDR6-dependent secondary small interfering RNA synthesis does not occur in the IR-PTGS, when targeting endogenous genes such as NtFAD3, but is efficiently induced in the IR-PTGS of the reporter transgenes. These results indicate that the SD2b and IA2b preferentially suppress the RDR6-dependent silencing pathway but do not suppress the RDR6-independent IR-PTGS pathway.
Article
Cucumber mosaic virus coat protein (CMV CP) plays a key role in cell-to-cell movement in host organisms. 1,4-Pentadien-3-one derivatives have excellent antiviral activities. In this study, we cloned, expressed and purified a CP recombinant protein. Then, we studied the binding interactions of CMV CP and 1, 4-pentadien-3-one derivatives N1–N20. Microscale thermophoresis experiments showed that N12 and N16 bound to CMV CP with dissociation constants of 0.071 and 0.11 μM, respectively. Docking and site-directed mutagenesis studies provided further insights into the interactions of N12 and N16 with Ile210, Thr69 and Ser213of CMV CP. Thus, these CMV CP residues may be important binding sites for the 1,4-pentadien-3-one derivatives N12 and N16. The data are important for designing and synthesizing new pentadienone derivatives.
Chapter
Plants have evolved mechanisms to limit viral infections and genomic damage that can occur by the invasion, proliferation, and expression of viruses and mobile genetic elements such as retroelements and transposons (1). Up to 95% of a plant’s genome is comprised of repetitive elements. The mechanisms involved with limiting expression of this “junk” deoxyribonucleic acid (DNA) have significantly hindered progress in agricultural biotechnology because DNA carrying genes of interest is often subjected to the same protective surveillance mechanisms and their expression shut down. This phenomenon, known as gene silencing, can occur immediately following integration of transgenes or over several generations. Gene silencing can affect some or all plants derived from a transgenic event, and expression can be partially or fully turned off.
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Chilli pepper is an important vegetable and spice crop grown worldwide. Chilli is susceptible to various pathogens, among them mosaic disease caused by Cucumber mosaic virus (CMV) is a major constraint for its production. Roving survey was carried out for mosaic disease assessment in chilli at 35 locations comprising five districts of south eastern Karnataka, which was later confirmed for the presence of different viruses in random samples by DAC-ELISA. Results revealed the prevalence of the disease caused by CMV up to 43.00% based on visual assessment. However, only in 64 samples out of 140 infected chilli samples showed CMV infection in DAC-ELISA and revealed the mixed infection of viruses. Mechanical sap inoculation of CMV-Ko isolate induced symptoms on chilli plants, which were similar to the symptoms observed in field. Complete genome sequence of CMV-Ko (RNA1, RNA2 and RNA3) isolate was amplified, cloned and sequenced. Sequence analysis revealed that it shared 83.7-99.1% nucleotide (nt) identity with CMV subgroup IB isolates infecting different crops in India. Recombination analysis of CMV-Ko genome showed that, RNA1 and RNA2 had recombinant origin and not RNA3. Host range studies for CMV-Ko isolate showed its potential of infecting nine host plants out of 21 used for transmission. Fifty advanced chilli lines were screened against CMV-Ko isolate and 27 immune lines to CMV were identified, which can be utilized for management of disease caused by CMV in chilli. Supplementary information: The online version contains supplementary material available at 10.1007/s13337-021-00713-3.
Article
Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.
Article
Post-transcriptional gene silencing (PTGS) in plants and quelling in fungi are transgene-induced silencing phenomena, resulting from the degradation of transgene RNAs and homologous endogenous RNAs. PTGS shows similarities with RNAi in animals, a phenomenon induced by injection of double-stranded RNA (dsRNA) or introduction of transgenes expressing dsRNA. First, PTGS and RNAi both involve dsRNA. Second, they can be dissected into three steps: localized initiation, propagation of a sequence-specific systemic signal, maintenance in silenced tissues. Finally, they both correlate with the accumulation of 25nt sense and anti-sense RNAs. Genetic dissection and cloning of genes regulating PTGS, quelling and RNAi confirmed the links between these three phenomena. Indeed, all three involve a putative RNA-dependent-RNA polymerase and a protein similar to the translation initiator factor eIF2C. However some differences can be noticed. In particular, PTGS in plants requires two genes, SGS3 (encoding a protein of unknown function) and MET1 (encoding a DNA-methyltransferase), which are not required for RNAi. Indeed, the genomes of C. elegans and Drosophila (two organisms undergoing RNAi) lack both methylation and orthologs of the SGS3 gene). Several experiments revealed that PTGS is a general mechanism of virus resistance. In particular, we showed that Arabidopsis mutants impaired in PTGS are hypersensitive to infection by the virus CMV. However, many viruses have developed strategies to counteract PTGS and therefore succeed to infect plants. Because viruses may act as targets, inducers or inhibitors of PTGS, the success and the extent of virus infection therefore depends on the competition between plant PTGS defenses and virus counteracting effects.
Article
Research in the past decade has shed light on the existence of an RNA-dependent defence mechanism, posttranscriptional gene silencing (PTGS), that occurs in plants, animals, and fungi. PTGS was found to be responsible for seemingly unrelated biological phenomena. For example, in transgenic plants, overexpression of endogenous genes sometimes produces unexpected results: co-suppression of both the transgene and the homologous endogenous gene(s), while virus-derived transgenes in plants can be poorly expressed and yet provide excellent virus resistance (RNA-mediated virus resistance). In addition, there is recent evidence that some plant viruses are capable of inhibiting PTGS by suppressing the plant's ability to carry out the silencing process, which eventually results in breaking this type of virus resistance. At present, the molecular mechanism of PTGS is being intensively studied and research evidence points to the central role of double-stranded (ds) RNAs homologous to the target gene. The host organism treats these dsRNAs as foreign and specifically degrades them along with any other homologous RNA. The purpose of this review is to summarize recent, relevant information on research results concerning the molecular mechanism of PTGS and related plant-virus interactions. In addition, the practical application of PTGS to create virus-resistant plants (RNA-mediated virus resistance) and, in support of functional genomics, to determine the role of cloned plant genes (virus-induced gene silencing, VIGS) is also discussed.
Article
Introduction Posttranscriptional gene silencing (PTGS) is a natural, universal mechanism that degrades both cellular and viral mRNA in a homology-dependant manner in diverse eukaryotes, and has now become a major area of research and development in both plants and animals. It was first discovered in plants (Napoli et al., 1990), while a mechanistically similar phenomenon is known to occur in a wide range of organisms, including Caenorhabditis elegans, Drosophila melanogaster and mammals termed RNA-interference (RNAi) (Fire et al., 1998, Hammond et al., 2000) and in Neurospora crassa termed quelling (Cogoni and Macino, 1997). Transgenes and viruses can induce PTGS in plants, and it is now recognized as a natural defense mechanism against virus infection (Hamilton and Baulcombe, 1999). Recent studies at the molecular level revealed that all of these phenomena are considered as manifestations of a general RNA-targeting pathway (Vance and Vaucheret, 2001). The mechanism by which a virus infection triggers PTGS in plants is not fully understood, but it is evident that dsRNA is a strong inducer of PTGS (Waterhouse et al., 2001). Such dsRNA molecules are produced during RNA virus replication using their own RNA-dependent RNA polymerase (RdRP), or alternatively host RdRPs convert any “aberrant” ssRNA in the cell, from viral origin or cell origin, into dsRNA (Dalmay et al., 2000; Ahlquist, 2002). The biology and biochemistry of RNAi was discussed in detail in Section I of this book.
Article
The breeding of crop plants with resistance to plant viruses is an important objectiveof modern agriculture. Although conventional breeding has proven effective insome cases, in many economically important crop-virus combinations naturalresistance is not available. With the development of biotechnology tools, pathogenderivedresistance (PDR) has become the method of choice to engineer resistance toplant viruses. In the majority of highly resistant transgenic lines, PDR was found tobe operating at the RNA level and induces homology-dependent RNA silencing, anatural defence mechanism of the plant that causes sequence-specific degradationof the viral RNA. Widely exploited by plant pathologists, RNA silencing representsa highly efficient mean to interfere with viral RNA accumulation. However, mixedviral infections are common in nature and must be considered in terms of stabilityand durability of virus resistance. Many virus genomes encode specific proteinsthat promote synergistic interactions between plant viruses by suppressing RNAsilencing. These silencing suppressors could potentially affect the engineered virusresistance, especially in the context of mixed infections in which only one of thetwo virus genomes is targeted by the resistance. In this chapter, the molecularmechanisms of RNA silencing-derived virus resistance will be reviewed and theadvantages of these strategies will be briefly outlined. The discovery of plant virussuppressors of the RNA silencing machinery will be discussed with special emphasison the potyvirus HC-Pro protein. Finally, case studies in which the durability ofRNA silencing-derived resistance was tested in the context of mixed infections willbe reviewed. In particular, recent studies demonstrating the stability of engineeredresistance to Plum pox virus in transgenic Prunus lines under conditions of mixedinfections will be discussed.
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The RNA-2 of a carrot isolate from the English serotype of tomato black ring nepovirus (TBRV-ED) has been sequenced. It is 4618 nucleotides long and contains one open reading frame encoding a polypeptide of 1344 amino acids. The 5' non-coding region contains three repetitions of a stem-loop structure also conserved in TBRV-Scottish and grapevine chrome mosaic nepovirus (GCMV). The coat protein domain was mapped to the carboxy-terminal one-third of the polyprotein. Sequence comparisons indicate that TBRV-ED RNA-2 probably arose by an RNA recombination event that resulted in the exchange of the putative movement protein gene between TBRV and GCMV.
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We recently identified a new cucumovirus-specific gene (2b) which is encoded by RNA 2 of the cucumber mosaic cucumovirus (CMV) tripartite RNA genome and whose coding sequence overlaps the C-terminal 69 codons of ORF 2a encoding the RNA polymerase protein. We have now found that although a CMV mutant lacking ORF 2b accumulated in the inoculated cotyledons of cucumber plants, it was unable to spread systemically, demonstrating involvement of 2b in long distance movement. The same mutant infected tobacco systemically with a much reduced virulence and delayed appearance of symptoms, indicating that 2b may contribute to long distance movement in this host. Deletion of the overlapping C-terminal part of ORF 2a did not change infectivity of the mutant in either host species, ruling out 2a mutation as the reason for the change of phenotype. Further infectivity studies with mutants containing partial deletions in ORF 2b further supported the conclusion that 2b encodes a host-specific long distance movement function. Sequence analysis revealed that 2b may represent a novel naturally occurring hybrid gene important to the evolutionary formation of the cucumovirus group and that it could provide a genetic basis for the wide host range of these viruses.
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cDNA clones of cucumber mosaic virus (CMV) RNA 3 were modified to express the jellyfish green fluorescent protein (GFP) in place of the 3a movement protein (MP) or coat protein (CP), as fusions to the N (GFP-3a) or C (3a-GFP) terminus of the MP or from a separate open reading frame as part of tricistronic RNAs 3. CMV RNA transcripts containing the individual modified RNAs 3 were unable to infect either Nicotiana tabacum or Nicotiana benthamiana systemically. Infection, as measured by confocal microscopy of GFP fluorescence, generally was limited to one to three epidermal cells at each inoculation site. Limited cell-to-cell movement, but not systemic movement, could be detected by complementation involving expression of MP and CP from two different RNA 3 constructs, each also expressing GFP. Infection involving RNA 3 expressing the GFP-3a fusion showed bright granules of variable size distributed predominantly and nonuniformly throughout the cytoplasm and, to a lesser extent, associated with the cell wall in single fluorescent cells, while infections expressing the 3a-GFP fusion showed bright, punctate fluorescence associated only with the cell wall. Infected cells expressing either 3a-GFP or free GFP showed a halo of less bright, fluorescent, neighboring cells, indicating limited movement of GFP. The initially infected cells also allowed movement of 10-kDa fluorescent dextran to the neighboring halo cells, while infection did not spread, suggesting different requirements for movement of either MP or dextran versus RNA.
Frequencies, timing, and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants
  • J.-C Palauqui
  • T Elmayan
  • F Dorlhac De Borne
  •  Cre  Te
  • P Charles
  • C Vaucheret
Palauqui, J.-C., Elmayan, T., Dorlhac de Borne, F., Cre  te Â, P., Charles, C., and Vaucheret, H. (1996). Frequencies, timing, and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants. Plant Physiol. 112, 1447± 1456.