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1
Hoffer, Paul, Sergey Ivashuta, Olga Pontes, Alexa Vitins, Craig Pikaard, Andrew Mroczka, Nicholas Wagner, and Toni Voelker. "Posttranscriptional gene silencing in nuclei." Proceedings of the National Academy of Sciences 108, no. 1 (December 20, 2010): 409–14. http://dx.doi.org/10.1073/pnas.1009805108.
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In plants, small interfering RNAs (siRNAs) with sequence homology to transcribed regions of genes can guide the sequence-specific degradation of corresponding mRNAs, leading to posttranscriptional gene silencing (PTGS). The current consensus is that siRNA-mediated PTGS occurs primarily in the cytoplasm where target mRNAs are localized and translated into proteins. However, expression of an inverted-repeat double-stranded RNA corresponding to the soybeanFAD2-1Adesaturase intron is sufficient to silenceFAD2-1, implicating nuclear precursor mRNA (pre-mRNA) rather than cytosolic mRNA as the target of PTGS. SilencingFAD2-1using intronic or 3′-UTR sequences does not affect transcription rates of the target genes but results in the strong reduction of target transcript levels in the nucleus. Moreover, siRNAs corresponding to pre-mRNA–specific sequences accumulate in the nucleus. In Arabidopsis, we find that two enzymes involved in PTGS, Dicer-like 4 and RNA-dependent RNA polymerase 6, are localized in the nucleus. Collectively, these results demonstrate that siRNA-directed RNA degradation can take place in the nucleus, suggesting the need for a more complex view of the subcellular compartmentation of PTGS in plants.
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Kubota, Kenji, Shinya Tsuda, Atsushi Tamai, and Tetsuo Meshi. "Tomato Mosaic Virus Replication Protein Suppresses Virus-Targeted Posttranscriptional Gene Silencing." Journal of Virology 77, no. 20 (October 15, 2003): 11016–26. http://dx.doi.org/10.1128/jvi.77.20.11016-11026.2003.
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ABSTRACT Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L11 and L11A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L11A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed.
3
Qu, Feng, Tao Ren, and T. Jack Morris. "The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step." Journal of Virology 77, no. 1 (January 1, 2003): 511–22. http://dx.doi.org/10.1128/jvi.77.1.511-522.2003.
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ABSTRACT Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants.
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Havelda, Zoltán, Csaba Hornyik, Aniello Crescenzi, and József Burgyán. "In Situ Characterization of Cymbidium Ringspot Tombusvirus Infection-Induced Posttranscriptional Gene Silencing in Nicotiana benthamiana." Journal of Virology 77, no. 10 (May 15, 2003): 6082–86. http://dx.doi.org/10.1128/jvi.77.10.6082-6086.2003.
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ABSTRACT In plants, posttranscriptional gene silencing (PTGS) is an ancient and effective defense mechanism against viral infection. A number of viruses encode proteins that suppress virus-activated PTGS. The p19 protein of tombusviruses is a potent PTGS suppressor which interferes with the onset of PTGS-generated systemic signaling and is not required for viral replication or for viral movement in Nicotiana benthamiana. This unique feature of p19 suppressor allowed us to analyze the mechanism of PTGS-based host defense and its viral suppression without interfering with other viral functions. In contrast to the necrotic symptoms caused by wild-type tombusvirus, the infection of p19-defective mutant virus results in the development of a typical PTGS-associated recovery phenotype in N. benthamiana. In this report we show the effect of PTGS on the viral infection process for N. benthamiana infected with either wild-type Cymbidium Ringspot Tombusvirus (CymRSV) or a p19-defective mutant (Cym19stop). In situ analyses of different virus-derived products revealed that PTGS is not able to reduce accumulation of virus in primary infected cells regardless of the presence of p19 PTGS suppressor. We also showed that both CymRSV and Cym19stop viruses move systemically in the vasculature, with similar efficiencies. However, in contrast to the uniform accumulation of CymRSV throughout systemically infected leaves, the presence of Cym19stop virus was confined to and around the vascular bundles. These results suggest that the role of p19 is to prevent the onset of mobile signal-induced systemic PTGS ahead of the viral infection front, leading to generalized infection.
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Havelda, Zoltán, Csaba Hornyik, Anna Válóczi, and József Burgyán. "Defective Interfering RNA Hinders the Activity of a Tombusvirus-Encoded Posttranscriptional Gene Silencing Suppressor." Journal of Virology 79, no. 1 (January 1, 2005): 450–57. http://dx.doi.org/10.1128/jvi.79.1.450-457.2005.
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ABSTRACT Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.
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Dong, Xiangli, Rene van Wezel, John Stanley, and Yiguo Hong. "Functional Characterization of the Nuclear Localization Signal for a Suppressor of Posttranscriptional Gene Silencing." Journal of Virology 77, no. 12 (June 15, 2003): 7026–33. http://dx.doi.org/10.1128/jvi.77.12.7026-7033.2003.
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ABSTRACT The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17KVQHRIAKKTTRRRR31. When expressed from potato virus X, C2-RRRR31DVGG (in which the four consecutive arginine residues 28RRRR31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K17D-GFP, C2-HR21DV-GFP, and C2-KK25DI-GFP localized to nuclei and produced necrosis, but only C2-K17D-GFP suppressed PTGS. The N-terminal portions C21-31 and C217-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.
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Bazzini, A. A., H. E. Hopp, R. N. Beachy, and S. Asurmendi. "Posttranscriptional Gene Silencing Does Not Play a Significant Role in Potato virus X Coat Protein-Mediated Resistance." Phytopathology® 96, no. 11 (November 2006): 1175–78. http://dx.doi.org/10.1094/phyto-96-1175.
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The expression of a gene that encodes coat protein (CP) of Potato virus X (PVX) in transgenic tobacco plants confers a high level of CP-mediated rresistance (CP-MR) against PVX infection. To determine if posttranscriptional gene silencing (PTGS) plays a role in resistance, transgenic plants expressing PVX CP were challenged against PVX under conditions in which PTGS was suppressed by low temperatures or using viruses carrying PTGS suppressors. The data demonstrate that PTGS does not play a significant role in PVX CP-MR.
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Zhang, Xinyan, Ying Zhu, Xiaodan Liu, Xinyu Hong, Yang Xu, Ping Zhu, Yang Shen, et al. "Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis." Science 348, no. 6230 (April 2, 2015): 120–23. http://dx.doi.org/10.1126/science.aaa2618.
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Plant immunity against foreign gene invasion takes advantage of posttranscriptional gene silencing (PTGS). How plants elaborately avert inappropriate PTGS of endogenous coding genes remains unclear. We demonstrate in Arabidopsis that both 5′-3′ and 3′-5′ cytoplasmic RNA decay pathways act as repressors of transgene and endogenous PTGS. Disruption of bidirectional cytoplasmic RNA decay leads to pleiotropic developmental defects and drastic transcriptomic alterations, which are substantially rescued by PTGS mutants. Upon dysfunction of bidirectional RNA decay, a large number of 21- to 22-nucleotide endogenous small interfering RNAs are produced from coding transcripts, including multiple microRNA targets, which could interfere with their cognate gene expression and functions. This study highlights the risk of unwanted PTGS and identifies cytoplasmic RNA decay pathways as safeguards of plant transcriptome and development.
9
Pfeffer, S., P. Dunoyer, F. Heim, K. E. Richards, G. Jonard, and V. Ziegler-Graff. "P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing." Journal of Virology 76, no. 13 (July 1, 2002): 6815–24. http://dx.doi.org/10.1128/jvi.76.13.6815-6824.2002.
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ABSTRACT Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5′-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.
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Martínez de Alba, A. E., R. Flores, and C. Hernández. "Two Chloroplastic Viroids Induce the Accumulation of Small RNAs Associated with Posttranscriptional Gene Silencing." Journal of Virology 76, no. 24 (December 15, 2002): 13094–96. http://dx.doi.org/10.1128/jvi.76.24.13094-13096.2002.
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ABSTRACT In plants, posttranscriptional gene silencing (PTGS) has been reported for cytoplasmic RNAs from endogenous nuclear genes, transgenes, viruses, and, recently, for a viroid with nuclear replication and accumulation. However, phenomena of this kind have not been described for mitochondrial or chloroplastic RNAs. Here we show that viroids that replicate and accumulate in the chloroplast are also targets of PTGS and this process may control viroid titer.
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Chicas, Agustin, Emma C. Forrest, Silvia Sepich, Carlo Cogoni, and Giuseppe Macino. "Small Interfering RNAs That Trigger Posttranscriptional Gene Silencing Are Not Required for the Histone H3 Lys9 Methylation Necessary for Transgenic Tandem Repeat Stabilization in Neurospora crassa." Molecular and Cellular Biology 25, no. 9 (May 1, 2005): 3793–801. http://dx.doi.org/10.1128/mcb.25.9.3793-3801.2005.
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ABSTRACT In Neurospora crassa, the introduction of a transgene can lead to small interfering RNA (siRNA)-mediated posttranscriptional gene silencing (PTGS) of homologous genes. siRNAs can also guide locus-specific methylation of Lys9 of histone H3 (Lys9H3) in Schizosaccharomyces pombe. Here we tested the hypothesis that transgenically derived siRNAs may contemporaneously both activate the PTGS mechanism and induce chromatin modifications at the transgene and the homologous endogenous gene. We carried out chromatin immunoprecipitation using a previously characterized albino-1 (al-1) silenced strain but detected no alterations in the pattern of histone modifications at the endogenous al-1 locus, suggesting that siRNAs produced from the transgenic locus do not trigger modifications in trans of those histones tested. Instead, we found that the transgenic locus was hypermethylated at Lys9H3 in our silenced strain and remained hypermethylated in the quelling defective mutants (qde), further demonstrating that the PTGS machinery is dispensable for Lys9H3 methylation. However, we found that a mutant in the histone Lys9H3 methyltransferase dim-5 was unable to maintain PTGS, with transgenic copies being rapidly lost, resulting in reversion of the silenced phenotype. These results indicate that the defect in PTGS of the Δdim-5 strain is due to the inability to maintain the transgene in tandem, suggesting a role for DIM-5 in stabilizing such repeated sequences. We conclude that in Neurospora, siRNAs produced from the transgenic locus are used in the RNA-induced silencing complex-mediated PTGS pathway and do not communicate with an RNAi-induced initiation of transcriptional gene silencing complex to effect chromatin-based silencing.
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Sun, Di, Yanjun Li, Zeyang Ma, Xingxing Yan, Niankui Li, Baoshuan Shang, Xiaomei Hu, Kai Cui, Hisashi Koiwa, and Xiuren Zhang. "The epigenetic factor FVE orchestrates cytoplasmic SGS3-DRB4-DCL4 activities to promote transgene silencing in Arabidopsis." Science Advances 7, no. 32 (August 2021): eabf3898. http://dx.doi.org/10.1126/sciadv.abf3898.
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Posttranscriptional gene silencing (PTGS) is a regulatory mechanism to suppress undesired transcripts. Here, we identified Flowering locus VE (FVE), a well-known epigenetic component, as a new player in cytoplasmic PTGS. Loss-of-function fve mutations substantially reduced the accumulation of transgene-derived small interfering RNAs (siRNAs). FVE interacts with suppressor of gene silencing 3 (SGS3), a master component in PTGS. FVE promotes SGS3 homodimerization that is essential for its function. FVE can bind to single-stranded RNA and double-stranded RNA (dsRNA) with moderate affinities, while its truncated form FVE-8 has a significantly increased binding affinity to dsRNA. These affinities affect the association and channeling of SGS3-RNA to downstream dsRNA binding protein 4 (DRB4)/Dicer-like protein 2/4 (DCL2/4) complexes. Hence, FVE, but not FVE-8, biochemically enhances the DRB4/DCL2/4 activity in vitro. We surmise that FVE promotes production of transgene-derived siRNAs through concertedly tuning SGS3-DRB4/DCL2/4 functions. Thus, this study revealed a noncanonical role of FVE in PTGS.
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Demirer, Gozde S., Huan Zhang, Natalie S. Goh, Rebecca L. Pinals, Roger Chang, and Markita P. Landry. "Carbon nanocarriers deliver siRNA to intact plant cells for efficient gene knockdown." Science Advances 6, no. 26 (June 2020): eaaz0495. http://dx.doi.org/10.1126/sciadv.aaz0495.
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Posttranscriptional gene silencing (PTGS) is a powerful tool to understand and control plant metabolic pathways, which is central to plant biotechnology. PTGS is commonly accomplished through delivery of small interfering RNA (siRNA) into cells. Standard plant siRNA delivery methods (Agrobacterium and viruses) involve coding siRNA into DNA vectors and are only tractable for certain plant species. Here, we develop a nanotube-based platform for direct delivery of siRNA and show high silencing efficiency in intact plant cells. We demonstrate that nanotubes successfully deliver siRNA and silence endogenous genes, owing to effective intracellular delivery and nanotube-induced protection of siRNA from nuclease degradation. This study establishes that nanotubes could enable a myriad of plant biotechnology applications that rely on RNA delivery to intact cells.
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Vanitharani, Ramachandran, Padmanabhan Chellappan, Justin S. Pita, and Claude M. Fauquet. "Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing." Journal of Virology 78, no. 17 (September 1, 2004): 9487–98. http://dx.doi.org/10.1128/jvi.78.17.9487-9498.2004.
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ABSTRACT Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.
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Moore, Carolyn J., Paul W. Sutherland, Richard L. S. Forster, Richard C. Gardner, and Robin M. MacDiarmid. "Dark Green Islands in Plant Virus Infection are the Result of Posttranscriptional Gene Silencing." Molecular Plant-Microbe Interactions® 14, no. 8 (August 2001): 939–46. http://dx.doi.org/10.1094/mpmi.2001.14.8.939.
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Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.
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Zhan, Binhui, Wenyang Zhao, Shifang Li, Xiuling Yang, and Xueping Zhou. "Functional Scanning of Apple Geminivirus Proteins as Symptom Determinants and Suppressors of Posttranscriptional Gene Silencing." Viruses 10, no. 9 (September 11, 2018): 488. http://dx.doi.org/10.3390/v10090488.
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Apple geminivirus (AGV) is a recently identified geminivirus which is isolated from the apple tree in China. We carried out functional scanning of apple geminivirus proteins as symptom determinants and suppressors of posttranscriptional gene silencing (PTGS). Our results indicated that AGV V2 is an important virulence factor localized to the nucleus and cytoplasm that suppresses PTGS and induces severe symptoms of crinkling and necrosis. AGV C1 is also a virulence determinant which elicits systemic necrosis when expressed from a PVX-based vector. The AGV C4 is targeted to cytoplasm, plasma membrane, nucleus, and chloroplasts. The inoculation of PVX-C4 on N. benthamiana induced severe upward leaf curling, which implied that AGV C4 also functions as a symptom determinant, and mutation analyses suggested that the acylated residues on Gly2 and Cys8 play important roles in its subcellular localization and symptom development.
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van Wezel, Rene, Huanting Liu, Zirong Wu, John Stanley, and Yiguo Hong. "Contribution of the Zinc Finger to Zinc and DNA Binding by a Suppressor of Posttranscriptional Gene Silencing." Journal of Virology 77, no. 1 (January 1, 2003): 696–700. http://dx.doi.org/10.1128/jvi.77.1.696-700.2003.
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ABSTRACT The zinc finger C36-X1-C38-X7-C46-X6-H53 of the nuclearly localized C2 protein of Tomato yellow leaf curl virus China is involved in pathogenicity and suppression of posttranscriptional gene silencing (PTGS). Here, we demonstrate that the zinc finger is indispensable for the C2 protein to bind zinc and DNA. Mutation of cysteine residue C36, C38, or C46 reduced the zinc and DNA binding capacity of C2 protein. When expressed from potato virus X, all three mutants, C2-C36R, C2-C38N, and C2-C46I, tagged with a green fluorescent protein (GFP) were still capable of transporting GFP into but aggregated abnormally in nuclei. Our data establish that zinc- and DNA-binding activity correlates with C2-mediated pathogenesis and PTGS suppression.
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Chellappan, Padmanabhan, Ramachandran Vanitharani, and Claude M. Fauquet. "Short Interfering RNA Accumulation Correlates with Host Recovery in DNA Virus-Infected Hosts, and Gene Silencing Targets Specific Viral Sequences." Journal of Virology 78, no. 14 (July 15, 2004): 7465–77. http://dx.doi.org/10.1128/jvi.78.14.7465-7477.2004.
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ABSTRACT Viruses are both inducers and targets of posttranscriptional gene silencing (PTGS), a natural defense mechanism in plants. Here we report molecular evidence of the ability of single-stranded DNA (ssDNA) viruses to induce PTGS in infected plants irrespective of the severity of or recovery from the symptoms. Our results reveal that five distinct species of cassava-infecting geminiviruses were capable of triggering PTGS by producing two classes of virus-specific short interfering RNAs (siRNAs) of 21 to 26 nucleotides in two plant hosts, tobacco (Nicotiana benthamiana) and cassava (Manihot esculenta, Crantz). However, the efficacy of virus-induced PTGS varied depending on the intrinsic features of the virus and its interaction with the plant host. We found that symptom recovery over time in plants infected with the isolates of African cassava mosaic virus (ACMV-[CM]) or Sri Lankan cassava mosaic virus was associated with a much higher level of virus-derived siRNA accumulation compared to plants infected with viruses that do not show symptom recovery. Furthermore, we determined that the C terminus of AC1 that overlaps with the N terminus of AC2 early viral genes involved in virus replication were the primary targets for ACMV-[CM]-induced PTGS, whereas the C terminus of BC1 was targeted for the East African cassava mosaic Cameroon virus. In addition, our results reveal the possibility for double-stranded RNA formation during transcription in ssDNA viruses, which explains in part how these viruses can trigger PTGS in plants.
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Hily, Jean-Michel, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza. "Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in Nicotiana benthamiana and Prunus domestica." Journal of the American Society for Horticultural Science 132, no. 6 (November 2007): 850–58. http://dx.doi.org/10.21273/jashs.132.6.850.
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Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.
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Yu, Diqiu, Baofang Fan, Stuart A. MacFarlane, and Zhixiang Chen. "Analysis of the Involvement of an Inducible Arabidopsis RNA-Dependent RNA Polymerase in Antiviral Defense." Molecular Plant-Microbe Interactions® 16, no. 3 (March 2003): 206–16. http://dx.doi.org/10.1094/mpmi.2003.16.3.206.
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RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a to-bamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.
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Michaeli, Simon, Marion Clavel, Esther Lechner, Corrado Viotti, Jian Wu, Marieke Dubois, Thibaut Hacquard, et al. "The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1." Proceedings of the National Academy of Sciences 116, no. 45 (October 18, 2019): 22872–83. http://dx.doi.org/10.1073/pnas.1912222116.
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RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.
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Rodríguez-Negrete, Edgar A., Jimena Carrillo-Tripp, and Rafael F. Rivera-Bustamante. "RNA Silencing against Geminivirus: Complementary Action of Posttranscriptional Gene Silencing and Transcriptional Gene Silencing in Host Recovery." Journal of Virology 83, no. 3 (November 19, 2008): 1332–40. http://dx.doi.org/10.1128/jvi.01474-08.
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ABSTRACT RNA silencing in plants is a natural defense system mechanism against invading nucleic acids such as viruses. Geminiviruses, a family of plant viruses characterized by a circular, single-stranded DNA genome, are thought to be both inducers and targets of RNA silencing. Some natural geminivirus-host interactions lead to symptom remission or host recovery, a process commonly associated with RNA silencing-mediated defense. Pepper golden mosaic virus (PepGMV)-infected pepper plants show a recovery phenotype, which has been associated with the presence of virus-derived small RNAs. The results presented here suggest that PepGMV is targeted by both posttranscriptional and transcriptional gene silencing mechanisms. Two types of virus-related small interfering RNAs (siRNAs) were detected: siRNAs of 21 to 22 nucleotides (nt) in size that are related to the coding regions (Rep, TrAP, REn, and movement protein genes) and a 24-nt population primarily associated to the intergenic regions. Methylation levels of the PepGMV A intergenic and coat protein (CP) coding region were measured by a bisulfite sequencing approach. An inverse correlation was observed between the methylation status of the intergenic region and the concentration of viral DNA and symptom severity. The intergenic region also showed a methylation profile conserved in all times analyzed. The CP region, on the other hand, did not show a defined profile, and its methylation density was significantly lower than the one found on the intergenic region. The participation of both PTGS and TGS mechanisms in host recovery is discussed.
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Stenger, Drake C., Brock A. Young, Feng Qu, T. Jack Morris, and Roy French. "Wheat streak mosaic virus Lacking Helper Component-Proteinase Is Competent to Produce Disease Synergism in Double Infections with Maize chlorotic mottle virus." Phytopathology® 97, no. 10 (October 2007): 1213–21. http://dx.doi.org/10.1094/phyto-97-10-1213.
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The tritimovirus Wheat streak mosaic virus (WSMV) and the machlomovirus Maize chlorotic mottle virus (MCMV) each cause systemic chlorosis in infected maize plants. Infection of maize with both viruses produces corn lethal necrosis disease (CLND). Here, we report that complete deletion of the WSMV helper component-proteinase (HC-Pro) coding region had no effect on induction of CLND symptoms following coinoculation of maize with WSMV and MCMV. We further demonstrated that elevation of virus titers in double infections, relative to single infections, also was independent of WSMV HC-Pro. Thus, unlike potyvirus HC-Pro, WSMV HC-Pro was dispensable for disease synergism. Because disease synergism involving potyviruses requires HC-Pro-mediated suppression of posttranscriptional gene silencing (PTGS), we hypothesized that WSMV HC-Pro may not be a suppressor of PTGS. Indeed, WSMV HC-Pro did not suppress PTGS of a green fluorescent protein (GFP) transgene in an Agrobacterium-mediated coinfiltration assay in which potyvirus HC-Pro acted as a strong suppressor. Furthermore, coinfiltration with potyvirus HC-Pro, but not WSMV HC-Pro, resulted in elevated levels of the GFP target mRNA under conditions which trigger PTGS. Collectively, these results revealed significant differences in HC-Pro function among divergent genera of the family Potyviridae and suggest that the tritimovirus WSMV utilizes a gene other than HC-Pro to suppress PTGS and mediate synergistic interactions with unrelated viruses.
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Tsuda, Shinya, Kenji Kubota, Ayami Kanda, Takehiro Ohki, and Tetsuo Meshi. "Pathogenicity of Pepper mild mottle virus Is Controlled by the RNA Silencing Suppression Activity of Its Replication Protein but Not the Viral Accumulation." Phytopathology® 97, no. 4 (April 2007): 412–20. http://dx.doi.org/10.1094/phyto-97-4-0412.
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Pepper mild mottle virus (PMMoV) infects pepper plants, causing mosaic symptoms on the upper developing leaves. We investigated the relationship between a virus pathogenicity determinant domain and the appearance of mosaic symptoms. Genetically modified PMMoV mutants were constructed, which had a base substitution in the 130K replication protein gene causing an amino acid change or a truncation of the 3′ terminal pseudoknot structure. Only one substitution mutant (at amino acid residue 349) failed to cause symptoms, although its accumulation was relatively high. Conversely, the pseudoknot mutants showed the lower accumulation, but they still caused mosaic symptoms as severe as the wild-type virus. Therefore, the level of virus accumulation in a plant does not necessarily correlate with the development of mosaic symptoms. The activity to suppress posttranscriptional gene silencing (PTGS) was impaired in the asymptomatic mutant. Consequently, pathogenicity causing mosaic symptoms should be controlled by combat between host PTGS and its suppression by the 130K replication protein rather than virus accumulation.
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van Wezel, Rene, Xiangli Dong, Huanting Liu, Po Tien, John Stanley, and Yiguo Hong. "Mutation of Three Cysteine Residues in Tomato yellow leaf curl virus-China C2 Protein Causes Dysfunction in Pathogenesis and Posttranscriptional Gene—Silencing Suppression." Molecular Plant-Microbe Interactions® 15, no. 3 (March 2002): 203–8. http://dx.doi.org/10.1094/mpmi.2002.15.3.203.
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The nuclear localized C2 protein of the monopartite bego-movirus Tomato yellow leaf curl virus-China (TYLCV-C) contributes to viral pathogenicity. Here, we have investigated TYLCV-C C2 protein domains that play a role in the phenotype. Alignment of the C2 protein with 67 homologues from monopartite and bipartite begomoviruses re-vealed that a putative zinc-finger motif C36-X1-C38-X7-C46-X6-H53-X4-H58C59 and four potential phosphorylation sites(T52, S61, Y68, and S74) are highly conserved. When ex-pressed from a Potato virus X (PVX) vector, TYLCV-C C2 protein mutants C2-T52M, C2-H58S, C2-C59S, C2-S61R, and C2-S74D, like the wild-type C2 protein, induced local necrotic ringspots and systemic necrosis in Nicotiana ben-thamiana plants. Mutants C2-H53P and C2-Y68D produced irregular necrotic lesions on inoculated leaves that were distinct from the wild-type phenotype. In contrast, mutants C2-C36R, C2-C38N, and C2-C46I induced chlorosis and mosaic symptoms rather than necrosis. We demonstrate that TYLCV-C C2, like its counterpart in the bipartite begomovirus African cassava mosaic virus, mediates suppression of posttranscriptional gene silencing (PTGS). Moreover, the individual mutations C36R, C38N, and C46I abolished the ability of C2 protein to suppress PTGS. These results suggest that the three cysteine residues within the putative zinc-finger motif are essential for C2 protein to induce necrosis and to act as a suppressor of PTGS.
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Molnár, Attila, Tibor Csorba, Lóránt Lakatos, Éva Várallyay, Christophe Lacomme, and József Burgyán. "Plant Virus-Derived Small Interfering RNAs Originate Predominantly from Highly Structured Single-Stranded Viral RNAs." Journal of Virology 79, no. 12 (June 15, 2005): 7812–18. http://dx.doi.org/10.1128/jvi.79.12.7812-7818.2005.
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ABSTRACT RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.
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Qiu, Wenping, Jong-Won Park, and Herman B. Scholthof. "Tombusvirus P19-Mediated Suppression of Virus-Induced Gene Silencing Is Controlled by Genetic and Dosage Features That Influence Pathogenicity." Molecular Plant-Microbe Interactions® 15, no. 3 (March 2002): 269–80. http://dx.doi.org/10.1094/mpmi.2002.15.3.269.
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The p19 protein (P19) of Tomato bushy stunt virus (TBSV) is a pathogenicity determinant with host-dependent effects on virus spread and symptom induction. In addition, results in this study confirm that Potato virus X-mediated delivery of P19 suppresses posttranscriptional gene silencing (PTGS). To study the relevance of this activity for TBSV biology, we evaluated whether TBSV activates virus-induced gene silencing (VIGS) and if this process is suppressed by P19. TBSV vectors with the green fluorescent protein (GFP) gene, either active or inactive for P19 expression, were inoculated onto GFP-transgenic Nicotiana bentha-miana plants. In the absence of P19 expression, VIGS was activated, as evidenced by the disappearance of GFP mRNA and green fluorescence. Coexpression of GFP and P19 from the TBSV vector suppressed VIGS, except in the newly emerging leaves. The suppressor activity required a central P19 region that is also known to be essential for host-dependent virus spread and symptom induction. Defective interfering RNAs (DIs) that contained the 3′ end of the GFP gene induced silencing very effectively. The concomitant DI-instigated reduction in P19 accumulation failed to suppress this process, analogous to the known P19 dosage effects for other biological activities. In conclusion, (i) TBSV and its DIs are very effective inducers of VIGS, (ii) P19 is a strong suppressor of PTGS, (iii) P19 is a moderate suppressor of VIGS, and (iv) the suppressor activity is influenced by genetic and dosage features that are also important for P19-associated pathogenesis.
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Tenllado, F., and J. R. Dı́az-Ruı́z. "Double-Stranded RNA-Mediated Interference with Plant Virus Infection." Journal of Virology 75, no. 24 (December 15, 2001): 12288–97. http://dx.doi.org/10.1128/jvi.75.24.12288-12297.2001.
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ABSTRACT Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not single-stranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.
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Taha, Omnia, Inas Farouk, Abdelhadi Abdallah, and Naglaa A. Abdallah. "Use of Posttranscription Gene Silencing in Squash to Induce Resistance against the Egyptian Isolate of theSquash Leaf Curl Virus." International Journal of Genomics 2016 (2016): 1–9. http://dx.doi.org/10.1155/2016/6053147.
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Squash leaf curl virus(SqLCV) is a bipartite begomovirus affecting squash plants. It is transmitted by whiteflyBemisia tabacibiotype B causing severe leaf curling, vein banding, and molting ending by stunting. In this study full-length genomic clone of SqLCV Egyptian isolated and posttranscriptional gene silencing (PTGS) has been induced to develop virus resistance. The Noubaria SqLCV has more than 95% homology with Jordon, Israel, Lebanon, Palestine, and Cairo isolates. Two genes fragment from SqLCV introduced in sense and antisense orientations using pFGC5049 vector to be expressed as hairpin RNA. The first fragment was 348 bp from replication associated protein gene (Rep). The second fragment was 879 bp representing the full sequence of the movement protein gene (BC1). Using real-time PCR, a silencing record of 97% has been recorded toRep/TrAPconstruct; as a result it has prevented the appearance of viral symptoms in most tested plants up to two months after infection, while construct containing theBC1gene scored a reduction in the accumulation of viral genome expression as appearing in real-time PCR results 4.6-fold giving a silencing of 79%, which had a positive effect on symptoms development in most tested plants.
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Jackel, Jamie N., R. Cody Buchmann, Udit Singhal, and David M. Bisaro. "Analysis of Geminivirus AL2 and L2 Proteins Reveals a Novel AL2 Silencing Suppressor Activity." Journal of Virology 89, no. 6 (December 31, 2014): 3176–87. http://dx.doi.org/10.1128/jvi.02625-14.
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ABSTRACTBoth posttranscriptional and transcriptional gene silencing (PTGS and TGS, respectively) participate in defense against the DNA-containing geminiviruses. As a countermeasure, members of the genusBegomovirus(e.g.,Cabbage leaf curl virus) encode an AL2 protein that is both a transcriptional activator and a silencing suppressor. The related L2 protein ofBeet curly top virus(genusCurtovirus) lacks transcription activation activity. Previous studies showed that both AL2 and L2 suppress silencing by a mechanism that correlates with adenosine kinase (ADK) inhibition, while AL2 in addition activates transcription of cellular genes that negatively regulate silencing pathways. The goal of this study was to clarify the general means by which these viral proteins inhibit various aspects of silencing. We confirmed that AL2 inhibits systemic silencing spread by a mechanism that requires transcription activation activity. Surprisingly, we also found that reversal of PTGS and TGS by ADK inactivation depended on whether experiments were conducted in vegetative or reproductiveNicotiana benthamianaplants (i.e., before or after the vegetative-to-reproductive transition). While AL2 was able to reverse silencing in both vegetative and reproductive plants, L2 and ADK inhibition were effective only in vegetative plants. This suggests that silencing maintenance mechanisms can change during development or in response to stress. Remarkably, we also observed that AL2 lacking its transcription activation domain could reverse TGS in reproductive plants, revealing a third, previously unsuspected AL2 suppression mechanism that depends on neither ADK inactivation nor transcription activation.IMPORTANCERNA silencing in plants is a multivalent antiviral defense, and viruses respond by elaborating multiple and sometimes multifunctional proteins that inhibit various aspects of silencing. The studies described here add an additional layer of complexity to this interplay. By examining geminivirus AL2 and L2 suppressor activities, we show that L2 is unable to suppress silencing inNicotiana benthamianaplants that have undergone the vegetative-to-reproductive transition. As L2 was previously shown to be effective in matureArabidopsisplants, these results illustrate that silencing mechanisms can change during development or in response to stress in ways that may be species specific. The AL2 and L2 proteins are known to share a suppression mechanism that correlates with the ability of both proteins to inhibit ADK, while AL2 in addition can inhibit silencing by transcriptionally activating cellular genes. Here, we also provide evidence for a third AL2 suppression mechanism that depends on neither transcription activation nor ADK inactivation. In addition to revealing the remarkable versatility of AL2, this work highlights the utility of viral suppressors as probes for the analysis of silencing pathways.
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Ceniceros-Ojeda, Esther Adriana, Edgar Antonio Rodríguez-Negrete, and Rafael Francisco Rivera-Bustamante. "Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)." Journal of Virology 90, no. 8 (January 20, 2016): 3828–38. http://dx.doi.org/10.1128/jvi.02385-15.
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ABSTRACTGeminiviruses are important plant pathogens characterized by circular, single-stranded DNA (ssDNA) genomes. However, in the nuclei of infected cells, viral double-stranded DNA (dsDNA) associates with host histones to form a minichromosome. In phloem-limited geminiviruses, the characterization of viral minichromosomes is hindered by the low concentration of recovered complexes due to the small number of infected cells. Nevertheless, geminiviruses are both inducers and targets of the host posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) machinery. We have previously characterized a “recovery” phenomenon observed in pepper plants infected with pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Initial micrococcal nuclease-based assays pinpointed the presence of different viral chromatin complexes in symptomatic and recovered tissues. Using the pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction that does not disturb the basic chromatin structural integrity, as evaluated by the detection of core histones. Using this procedure, we have further characterized two populations of viral minichromosomes in PepGMV-infected plants. After further purification using sucrose gradient sedimentation, we also observed that minichromosomes isolated from symptomatic tissue showed a relaxed conformation (based on their sedimentation rate), are associated with a chromatin activation marker (H3K4me3), and present a low level of DNA methylation. The minichromosome population obtained from recovered tissue, on the other hand, sedimented as a compact structure, is associated with a chromatin-repressive marker (H3K9me2), and presents a high level of DNA methylation.IMPORTANCEViral minichromosomes have been reported in several animal and plant models. However, in the case of geminiviruses, there has been some recent discussion about the importance of this structure and the significance of the epigenetic modifications that it can undergo during the infective cycle. Major problems in this type of studies are the low concentration of these complexes in an infected plant and the asynchronicity of infected cells along the process; therefore, the complexes isolated in a given moment usually represent a mixture of cells at different infection stages. The recovery process observed in PepGMV-infected plants and the isolation procedure described here provide two distinct populations of minichromosomes that will allow a more precise characterization of the modifications of viral DNA and its host proteins associated along the infective cycle. This structure could be also an interesting model to study several processes involving plant chromatin.