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 The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step

2003 ◽  
Vol 77 (1) ◽  
pp. 511-522 ◽  
Author(s):  
Feng Qu ◽  
Tao Ren ◽  
T. Jack Morris
Keyword(s):  
Coat Protein ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Rna Degradation ◽  
Plant Viruses ◽  
Early Initiation ◽  
Turnip Crinkle Virus ◽  
Small Interfering Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Initiation Step

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.

scholarly journals Host-Dependent Requirement for the Potato leafroll virus 17-kDa Protein in Virus Movement

2002 ◽  
Vol 15 (10) ◽  
pp. 1086-1094 ◽  
Author(s):  
Lawrence Lee ◽  
Peter Palukaitis ◽  
Stewart M. Gray
Keyword(s):  
Amino Acids ◽  
Solanum Tuberosum ◽  
Nicotiana Benthamiana ◽  
Systemic Infection ◽  
Potato Leafroll Virus ◽  
Virus Movement ◽  
Wild Type ◽  
Mature Leaves ◽  
Leafroll Virus ◽  
Virus Distribution

The requirement for the 17-kDa protein (P17) of Potato leafroll virus (PLRV) in virus movement was investigated in four plant species: potato (Solanum tuberosum), Physalis floridana, Nicotiana benthamiana, and N. clevelandii. Two PLRV P17 mutants were characterized, one that does not translate the P17 and another that expresses a P17 missing the first four amino acids. The P17 mutants were able to replicate and accumulate in agroinoculated leaves of potato and P. floridana, but they were unable to move into vascular tissues and initiate a systemic infection in these plants. In contrast, the P17 mutants were able to spread systemically from inoculated leaves in both Nicotiana spp., although the efficiency of infection was reduced relative to wild-type PLRV. Examination of virus distribution in N. benthamiana plants using tissue immunoblotting techniques revealed that the wild-type PLRV and P17 mutants followed a similar movement pathway out of the inoculated leaves. Virus first moved upward to the apical tissues and then downward. The P17 mutants, however, infected fewer phloem-associated cells, were slower than wild-type PLRV in moving out of the inoculated tissue and into apical tissues, and were unable to infect any mature leaves present on the plant at the time of inoculation.

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 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 Expression dynamics of ARGONAUTE proteins during meiosis in Arabidopsis

2021 ◽  
Author(s):  
Cecilia Oliver ◽  
German Martinez
Keyword(s):  
Genetic Diversity ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Potential Role ◽  
Specific Activity ◽  
Male Meiosis ◽  
Transcriptional Gene Silencing ◽  
Posttranscriptional Gene Silencing ◽  
Argonaute Proteins ◽  
Silencing Pathways

Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing (AGO1, 2 and 5) in the cytoplasm or the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.

scholarly journals The passage of Potato leafroll virus through Myzus persicae gut membrane regulates transmission efficiency

2001 ◽  
Vol 82 (1) ◽  
pp. 17-23 ◽  
Author(s):  
J. Rouzé-Jouan ◽  
L. Terradot ◽  
F. Pasquer ◽  
S. Tanguy ◽  
D. Giblot Ducray-Bourdin
Keyword(s):  
Amino Acid ◽  
Coat Protein ◽  
Myzus Persicae ◽  
Virus Transmission ◽  
Transmission Efficiency ◽  
Potato Leafroll Virus ◽  
Transmission Process ◽  
Leafroll Virus ◽  
Readthrough Protein

Potato leafroll virus (PLRV) is transmitted by aphids in a persistent manner. Although virus circulation within the aphid leading to transmission has been well characterized, the mechanisms involved in virus recognition at aphid membranes are still poorly understood. One isolate in our collection (PLRV-14.2) has been shown to be non- or only poorly transmitted by some clones of aphids belonging to the Myzus persicae complex. To determine where the transmission process was blocked within the aphid, three virus transmission procedures were used. PLRV-14.2 could not be transmitted, or was only very poorly transmitted, after acquisition from infected plants or from purified preparations. In contrast, it could be transmitted with more than 70% efficiency when microinjected. Therefore, it is concluded that the gut membrane was a barrier regulating passage of PLRV particles from the gut lumen into the haemocoel of M. persicae. Comparison of coat protein (CP) and readthrough protein (RTP) sequences between poorly and readily transmissible isolates showed that PLRV-14.2 differed from other PLRV isolates by amino acid changes in both of these proteins. It is hypothesized that at least some of the changes found in CP and/or RTP reduced virus recognition by aphid gut receptors, resulting in reduced acquisition and subsequent transmission of PLRV-14.2.

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 A Surface Loop of the Potato Leafroll Virus Coat Protein Is Involved in Virion Assembly, Systemic Movement, and Aphid Transmission

2005 ◽  
Vol 79 (2) ◽  
pp. 1207-1214 ◽  
Author(s):  
Lawrence Lee ◽  
Igor B. Kaplan ◽  
Daniel R. Ripoll ◽  
Delin Liang ◽  
Peter Palukaitis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Virus Assembly ◽  
Systemic Infection ◽  
Aphid Transmission ◽  
Potato Leafroll Virus ◽  
Wild Type Virus ◽  
Virion Assembly ◽  
Aphid Vectors ◽  
Leafroll Virus

ABSTRACT Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.

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.

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