scholarly journals The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity

2013 ◽  
Vol 26 (2) ◽  
pp. 168-181 ◽  
Author(s):  
Sotaro Chiba ◽  
Kamal Hleibieh ◽  
Alice Delbianco ◽  
Elodie Klein ◽  
Claudio Ratti ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Rna Silencing ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nucleolar Localization ◽  
Silencing Suppressor ◽  
Long Distance ◽  
Microscopy Imaging ◽  
Silencing Suppression ◽  
Long Distance Movement

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

scholarly journals Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families

2002 ◽  
Vol 76 (24) ◽  
pp. 12981-12991 ◽  
Author(s):  
Natalia E. Yelina ◽  
Eugene I. Savenkov ◽  
Andrey G. Solovyev ◽  
Sergey Y. Morozov ◽  
Jari P. T. Valkonen
Keyword(s):  
Transgenic Plants ◽  
Viral Infection ◽  
Symptom Severity ◽  
Rna Silencing ◽  
Systemic Infection ◽  
Stress Factors ◽  
Viral Gene ◽  
Long Distance ◽  
Silencing Suppression ◽  
Long Distance Movement

ABSTRACT RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the γb gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the γb protein may be a long-distance movement factor and have antisilencing activity. This was shown for γb proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, γb and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the γb cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus γb proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.

scholarly journals Citrus tristeza virus p23: Determinants for Nucleolar Localization and Their Influence on Suppression of RNA Silencing and Pathogenesis

2013 ◽  
Vol 26 (3) ◽  
pp. 306-318 ◽  
Author(s):  
Susana Ruiz-Ruiz ◽  
Nuria Soler ◽  
Jesús Sánchez-Navarro ◽  
Carmen Fagoaga ◽  
Carmelo López ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rna Silencing ◽  
Citrus Tristeza Virus ◽  
Ectopic Expression ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nucleolar Localization ◽  
Deletion Mutants ◽  
Tristeza Virus ◽  
Citrus Tristeza

Citrus tristeza virus (CTV) encodes a singular protein (p23, 209 amino acids) with multiple functions, including RNA silencing suppression (RSS). Confocal laser-scanning microscopy of green fluorescent protein (GFP)-p23 agroexpressed in Nicotiana benthamiana revealed its accumulation in the nucleolus, Cajal bodies, and plasmodesmata. To dissect the nucleolar localization signal (NoLS) typically associated with basic motifs, seven truncated and 10 point-mutated versions of p23 were assayed. Deletion mutants showed that regions 50 to 86 and 100 to 157 (excluding fragment 106 to 114), both with basic motifs and the first with a zinc-finger, contain the (bipartite) NoLS. Alanine substitutions delimited this signal to three cysteines of the Zn-finger and some basic amino acids. RSS activity of p23 in N. benthamiana was abolished by essentially all mutants, indicating that it involves most p23 regions. The necrotic-inducing ability of p23 when launched in N. benthamiana from Potato virus X was only retained by deletion mutant 158-209 and one substitution mutant, showing that the Zn-finger and flanking basic motifs form part of the pathogenic determinant. Ectopic expression of p23 and some deletion mutants in transgenic Mexican lime demarcated a similar determinant, suggesting that p23 affects related pathways in citrus and N. benthamiana. Both RSS activity and pathogenicity of p23 appear related to its nucleolar localization.

scholarly journals Protease Activity, Self Interaction, and Small Interfering RNA Binding of the Silencing Suppressor P1b from Cucumber Vein Yellowing Ipomovirus

2007 ◽  
Vol 82 (2) ◽  
pp. 974-986 ◽  
Author(s):  
Adrian Valli ◽  
Gabriela Dujovny ◽  
Juan Antonio García
Keyword(s):  
Zinc Finger ◽  
Rna Silencing ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Plant Viruses ◽  
Bimolecular Fluorescence Complementation ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Silencing Suppression ◽  
Interfering Rna

ABSTRACT The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.

scholarly journals Analysis of Cell-to-Cell and Long-Distance Movement of a Bean Dwarf Mosaic Geminivirus-Green Fluorescent Protein Reporter in Host and Nonhost Species: Identification of Sites of Resistance

1999 ◽  
Vol 12 (4) ◽  
pp. 345-355 ◽  
Author(s):  
H. L. Wang ◽  
M. R. Sudarshana ◽  
R. L. Gilbertson ◽  
W. J. Lucas
Keyword(s):  
Green Fluorescent Protein ◽  
Viral Infection ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Systemic Infection ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Long Distance ◽  
Green Fluorescent ◽  
Long Distance Movement

A bean dwarf mosaic geminivirus-green fluorescent protein (BDMV-GFP) reporter system was employed to analyze the viral infection process in host and nonhost species. Five classes of BDMV/host interaction were identified: (i) adapted hosts (susceptible Phaseolus vulgaris cultivars) permissive for systemic infection; (ii) adapted hosts (resistant P. vulgaris cv. Othello) displaying the development of a hypersensitive response (HR) associated with resistance to systemic infection; (iii) adapted (resistant P. vulgaris cv. Black Turtle Soup T-39) and nonadapted (Vigna unguiculata) hosts in which cell-to-cell, but not long-distance, movement was permitted; (iv) nonadapted hosts (Glycine max) in which systemic infection was coat protein-dependent; and (v) nonhosts (Cucurbita maxima, Gossypium barbadense, and Zea mays) in which the virus was confined to inoculated cells. Confocal laser scanning microscopy, fluorescence microscopy, and histochemical analyses were used to identify the cellular distribution of BDMV-GFP and the host response to viral infection. With this approach, the HR in P. vulgaris cv. Othello was visualized within cells of the epidermis, cortex, and phloem of inoculated hypocotyls. Infection studies performed with four begomoviruses and infectious BDMV/tomato mottle geminivirus pseudorecombinants revealed that the HR determinant(s) mapped to the BDMV DNA-B component.

scholarly journals Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz H. D. Panariello ◽  
Justin K. Kindler ◽  
Kenneth J. Spolnik ◽  
Ygal Ehrlich ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Root Canal ◽  
Laser Scanning ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Electromagnetic Stimulation ◽  
Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

scholarly journals RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro

2006 ◽  
Vol 80 (20) ◽  
pp. 10055-10063 ◽  
Author(s):  
Adrian Valli ◽  
Ana Montserrat Martín-Hernández ◽  
Juan José López-Moya ◽  
Juan Antonio García
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Plum Pox Virus ◽  
Coding Region ◽  
Long Distance ◽  
Systemic Spread ◽  
The Family ◽  
Silencing Suppression ◽  
Green Fluorescent

ABSTRACT The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

scholarly journals Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement protein

Virology ◽  
2008 ◽  
Vol 382 (2) ◽  
pp. 199-206 ◽  
Author(s):  
Hajime Yaegashi ◽  
Akihiro Tamura ◽  
Masamichi Isogai ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Leaf Spot ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Movement Protein ◽  
Long Distance ◽  
Spot Virus ◽  
Chlorotic Leaf ◽  
Long Distance Movement

scholarly journals Translocation of Tomato Bushy Stunt Virus P19 Protein into the Nucleus by ALY Proteins Compromises Its Silencing Suppressor Activity

2006 ◽  
Vol 80 (18) ◽  
pp. 9064-9072 ◽  
Author(s):  
Tomas Canto ◽  
Joachim F. Uhrig ◽  
Maud Swanson ◽  
Kathryn M. Wright ◽  
Stuart A. MacFarlane
Keyword(s):  
Rna Silencing ◽  
Tomato Bushy Stunt Virus ◽  
Bimolecular Fluorescence Complementation ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Long Distance ◽  
Suppressor Activity ◽  
Recognition Motif ◽  
Silencing Suppression ◽  
Suppressor Of Rna Silencing

ABSTRACT The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.

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