scholarly journals Investigation of the Effects of P1 on Hc-Pro-Mediated Gene Silencing Suppression Through Genetics and Omics Approaches

2020 ◽  
Author(s):  
Sin-Fen Hu ◽  
Wei-Lun Wei ◽  
Syuan-Fei Hong ◽  
Ru-Ying Fang ◽  
Hsin-Yi Wu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Transgenic Arabidopsis ◽  
Rna Degradation ◽  
Interacting Proteins ◽  
Viral Suppressor ◽  
Posttranscriptional Gene Silencing ◽  
New Findings ◽  
A Subunit ◽  
Silencing Suppression ◽  
Interfering Rna

Abstract Background: Viral suppressor negatively controls posttranscriptional gene silencing (PTGS) byinhibiting microRNA (miRNA) and short-interfering RNA (siRNA)regulation in plants. The first identified viral suppressor-P1/HC-Pro is afusion protein. Upon infecting plants, theP1 protein itself gets released from HC-Pro bythe self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression.We performed proteomics to identifyP1-interacting proteins and observedwhole gene correlationsin P1/HC-Pro-mediated PTGS suppression through transcriptomic studied comparative networks.Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might workthrough the P1 binding to VERNALIZATION INDEPENDENCE 3/ SUPERKILLER8 (VIP3/SKI8),a subunit of the exosome, to interferewith the 5'-fragment of the PTGS-cleaved RNA degradation product.Second,specifically the AGO1 wasposttranslationaldegraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant).Third, transcriptomic comparative networkshighlighted critical genes inPTGS, including miRNA targets,calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response.Conclusion: Through thesetransgenic and omics approaches, we revealed an overall perspective and new findings in our understandingof the mechanism of P1/HC-Pro-mediated PTGS suppression. Many of the criticalgenes that weresignificantlyimpacted in the omics profiles will be further investigated by CRISPR-knockoutor gain-of-function to understand PTGS in plant better.

scholarly journals Investigation of the effects of P1 on HC-Pro-mediated gene silencing suppression through genetics and omics approaches

2020 ◽  
Author(s):  
Sin-Fen Hu ◽  
Wei-Lun Wei ◽  
Syuan-Fei Hong ◽  
Ru-Ying Fang ◽  
Hsin-Yi Wu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Rna Degradation ◽  
Novel Genes ◽  
Posttranscriptional Gene Silencing ◽  
Comparative Network Analysis ◽  
New Findings ◽  
A Subunit ◽  
Silencing Suppression ◽  
Viral Suppressors ◽  
Interfering Rna

Abstract Background: Posttranscriptional gene silencing (PTGS) is one of the most important mechanisms for plants during viral infection. However, viruses have also developed viral suppressors to negatively control PTGS by inhibiting microRNA (miRNA) and short-interfering RNA (siRNA) regulation in plants. The first identified viral suppressor, P1/HC-Pro, is a fusion protein. Upon infecting plants, the P1 protein itself is released from HC-Pro by the self-cleaving activity of P1. P1 has an unknown function in enhancing HC-Pro-mediated PTGS suppression. We performed proteomics to identify P1-interacting proteins. We also performed transcriptomics that were generated from Col-0 and various P1/HC-Pro-related transgenic plants to identify novel genes. The results showed several novel genes were identified through the comparative network analysis that might be involved in P1/HC-Pro-mediated PTGS suppression. Results: First, we demonstrated that P1 enhances HC-Pro function and that the mechanism might work through P1 binding to VERNALIZATION INDEPENDENCE 3/SUPERKILLER 8 (VIP3/SKI8), a subunit of the exosome, to interfere with the 5'-fragment of the PTGS-cleaved RNA degradation product. Second, specifically the AGO1 was specifically posttranslationally degraded in transgenic Arabidopsis expressing P1/HC-Pro of turnip mosaic virus (TuMV) (P1/HCTu plant). Third, the comparative network highlighted potentially critical genes in PTGS, including miRNA targets, calcium signaling, hormone (JA, ET, and ABA) signaling, and defense response. Conclusion: Through these genetic and omics approaches, we revealed an overall perspective to identify many critical genes involved in PTGS. These new findings significantly impact in our understanding of P1/HC-Pro-mediated PTGS suppression.

scholarly journals The Three Essential Motifs in P0 for Suppression of RNA Silencing Activity of Potato leafroll virus Are Required for Virus Systemic Infection

Viruses ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 170 ◽  
Author(s):  
Mamun-Or Rashid ◽  
Xiao-Yan Zhang ◽  
Ying Wang ◽  
Da-Wei Li ◽  
Jia-Lin Yu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Systemic Infection ◽  
Rna Degradation ◽  
Potato Leafroll Virus ◽  
Posttranscriptional Gene Silencing ◽  
Conserved Region ◽  
Leafroll Virus ◽  
The Impact

Higher plants exploit posttranscriptional gene silencing as a defense mechanism against virus infection by the RNA degradation system. Plant RNA viruses suppress posttranscriptional gene silencing using their encoded proteins. Three important motifs (F-box-like motif, G139/W140/G141-like motif, and C-terminal conserved region) in P0 of Potato leafroll virus (PLRV) were reported to be essential for suppression of RNA silencing activity. In this study, Agrobacterium-mediated transient experiments were carried out to screen the available amino acid substitutions in the F-box-like motif and G139/W140/G141-like motif that abolished the RNA silencing suppression activity of P0, without disturbing the P1 amino acid sequence. Subsequently, four P0 defective mutants derived from a full-length cDNA clone of PLRV (L76F and W87R substitutions in the F-box-like motif, G139RRR substitution in the G139/W140/G141-like motif, and F220R substitution in the C-terminal conserved region) were successfully generated by reverse PCR and used to investigate the impact of these substitutions on PLRV infectivity. The RT-PCR and western blot analysis revealed that these defective mutants affected virus accumulation in inoculated leaves and systemic movement in Nicotiana benthamiana as well as in its natural hosts, potato and black nightshade. These results further demonstrate that the RNA silencing suppressor of PLRV is required for PLRV accumulation and systemic infection.

scholarly journals Posttranscriptional gene silencing in nuclei

2010 ◽  
Vol 108 (1) ◽  
pp. 409-414 ◽  
Author(s):  
Paul Hoffer ◽  
Sergey Ivashuta ◽  
Olga Pontes ◽  
Alexa Vitins ◽  
Craig Pikaard ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Target Genes ◽  
Rna Degradation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Posttranscriptional Gene Silencing ◽  
Subcellular Compartmentation ◽  
Specific Degradation ◽  
Precursor Mrna ◽  
Specific Sequences

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.

scholarly journals Potyvirus-induced gene silencing: the dynamic process of systemic silencing and silencing suppression

2007 ◽  
Vol 88 (8) ◽  
pp. 2337-2346 ◽  
Author(s):  
Elin Gammelgård ◽  
Maradumane Mohan ◽  
Jari P. T. Valkonen
Keyword(s):  
Gene Silencing ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Deletion Mutants ◽  
Virus Induced Gene Silencing ◽  
Potato Virus A ◽  
Silencing Suppression ◽  
Dark Green ◽  
Green Fluorescent ◽  
Interfering Rna

Potato virus A (PVA; genus Potyvirus) was used for virus-induced gene silencing in a model system that included transgenic Nicotiana benthamiana (line 16c) expressing the gfp transgene for green fluorescent protein (GFP) and chimeric PVA (PVA–GFP) carrying gfp in the P1-encoding region. Infection of the 16c plants with PVA–GFP in five experiments resulted in a reproducible pattern of systemic gfp transgene silencing, despite the presence of the strong silencing-suppressor protein, HC-Pro, produced by the virus. PVA–GFP was also targeted by silencing, and virus-specific short interfering RNA accumulated from the length of the viral genome. Viral deletion mutants lacking the gfp insert appeared in systemically infected leaves and reversed silencing of the gfp transgene in limited areas. However, systemic gfp silencing continued in newly emerging leaves in the absence of the gfp-carrying virus, which implicated a systemic silencing signal that moved from lower leaves without interference by HC-Pro. Use of GFP as a visual marker revealed a novel, mosaic-like recovery phenotype in the top leaves. The leaf areas appearing red or purple under UV light (no GFP expression) contained little PVA and gfp mRNA, and corresponded to the dark-green islands observed under visible light. The surrounding green fluorescent tissues contained actively replicating viral deletion mutants that suppressed GFP silencing. Taken together, systemic progression of gene silencing and antiviral defence (RNA silencing) and circumvention of the silencing by the virus could be visualized and analysed in a novel manner.

scholarly journals Tomato Mosaic Virus Replication Protein Suppresses Virus-Targeted Posttranscriptional Gene Silencing

2003 ◽  
Vol 77 (20) ◽  
pp. 11016-11026 ◽  
Author(s):  
Kenji Kubota ◽  
Shinya Tsuda ◽  
Atsushi Tamai ◽  
Tetsuo Meshi
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Rna Degradation ◽  
Plant Viruses ◽  
Replication Protein ◽  
Tomato Mosaic Virus ◽  
Replicase Gene ◽  
Posttranscriptional Gene Silencing ◽  
Recombinant Viruses ◽  
Ptgs Suppressor

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.

scholarly journals Global Role for Polyadenylation-Assisted Nuclear RNA Degradation in Posttranscriptional Gene Silencing

2007 ◽  
Vol 28 (2) ◽  
pp. 656-665 ◽  
Author(s):  
Shao-Win Wang ◽  
Abigail L. Stevenson ◽  
Stephen E. Kearsey ◽  
Stephen Watt ◽  
Jürg Bähler
Keyword(s):  
Gene Expression ◽  
Gene Silencing ◽  
Global Gene Expression ◽  
Rna Degradation ◽  
Genomic Integrity ◽  
Repeat Elements ◽  
Posttranscriptional Gene Silencing ◽  
Rna Targets ◽  
Nuclear Rna ◽  
Rna Quality Control

ABSTRACT Fission yeast Cid14, a component of the TRAMP (Cid14/Trf4-Air1-Mtr4 polyadenylation) complex, polyadenylates nuclear RNA and stimulates degradation by the exosome for RNA quality control. Here, we analyze patterns of global gene expression in cells lacking the Cid14 or the Dis3/Rpr44 subunit of the nuclear exosome. We found that transcripts from many genes induced during meiosis, including key regulators, accumulated in the absence of Cid14 or Dis3. Moreover, our data suggest that additional substrates include transcripts involved in heterochromatin assembly. Mutant cells lacking Cid14 and/or Dis3 accumulate transcripts corresponding to naturally silenced repeat elements within heterochromatic domains, reflecting defects in centromeric gene silencing and derepression of subtelomeric gene expression. We also uncover roles for Cid14 and Dis3 in maintaining the genomic integrity of ribosomal DNA. Our data indicate that polyadenylation-assisted nuclear RNA turnover functions in eliminating a variety of RNA targets to control diverse processes, such as heterochromatic gene silencing, meiotic differentiation, and maintenance of genomic integrity.

scholarly journals 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

2005 ◽  
Vol 25 (9) ◽  
pp. 3793-3801 ◽  
Author(s):  
Agustin Chicas ◽  
Emma C. Forrest ◽  
Silvia Sepich ◽  
Carlo Cogoni ◽  
Giuseppe Macino
Keyword(s):  
Gene Silencing ◽  
Neurospora Crassa ◽  
Chromatin Immunoprecipitation ◽  
Histone H3 ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Endogenous Gene ◽  
Homologous Genes ◽  
Interfering Rna

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.

Post-transcriptional gene silencing in plants

2001 ◽  
Vol 114 (17) ◽  
pp. 3083-3091 ◽  
Author(s):  
Hervé Vaucheret ◽  
Christophe Béclin ◽  
Mathilde Fagard
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
Degradation Mechanism ◽  
Rna Degradation ◽  
Transcriptional Gene Silencing ◽  
Rna Helicases ◽  
C Elegans ◽  
Post Transcriptional Gene Silencing ◽  
Interfering Rna ◽  
Resistance To Viruses

Post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism that shows similarities to RNA interference (RNAi) in animals. Indeed, both involve double-stranded RNA (dsRNA), spread within the organism from a localised initiating area, correlate with the accumulation of small interfering RNA (siRNA) and require putative RNA-dependent RNA polymerases, RNA helicases and proteins of unknown functions containing PAZ and Piwi domains. However, some differences are evident. First, PTGS in plants requires at least two genes – SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) and MET1 (which encodes a DNA-methyltransferase) – that are absent in C. elegans and thus are not required for RNAi. Second, all Arabidopsis mutants that exhibit impaired PTGS are hypersusceptible to infection by the cucumovirus CMV, indicating that PTGS participates in a mechanism for plant resistance to viruses. Interestingly, many viruses have developed strategies to counteract PTGS and successfully infect plants – for example, by potentiating endogenous suppressors of PTGS. Whether viruses can counteract RNAi in animals and whether endogenous suppressors of RNAi exist in animals is still unknown.

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