scholarly journals Functional Characterization of RNA Silencing Suppressor P0 from Pea Mild Chlorosis Virus

2020 ◽  
Vol 21 (19) ◽  
pp. 7136
Author(s):  
Qian Sun ◽  
Tao Zhuo ◽  
Tianyu Zhao ◽  
Cuiji Zhou ◽  
Yuanyuan Li ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Plant Viruses ◽  
C Terminus ◽  
Systemic Rna ◽  
Green Fluorescent ◽  
Leafroll Virus ◽  
F Box Protein ◽  
Viral Suppressors

To counteract host antiviral RNA silencing, plant viruses encode numerous viral suppressors of RNA silencing (VSRs). P0 proteins have been identified as VSRs in many poleroviruses. However, their suppressor function has not been fully characterized. Here, we investigated the function of P0 from pea mild chlorosis virus (PMCV) in the suppression of local and systemic RNA silencing via green fluorescent protein (GFP) co-infiltration assays in wild-type and GFP-transgenic Nicotiana benthamiana (line 16c). Amino acid deletion analysis showed that N-terminal residues Asn 2 and Val 3, but not the C-terminus residues from 230–270 aa, were necessary for PMCV P0 (P0PM) VSR activity. P0PM acted as an F-box protein, and triple LPP mutation (62LPxx79P) at the F-box-like motif abolished its VSR activity. In addition, P0PM failed to interact with S-phase kinase-associated protein 1 (SKP1), which was consistent with previous findings of P0 from potato leafroll virus. These data further support the notion that VSR activity of P0 is independent of P0–SKP1 interaction. Furthermore, we examined the effect of P0PM on ARGONAUTE1 (AGO1) protein stability, and co-expression analysis showed that P0PM triggered AGO1 degradation. Taken together, our findings suggest that P0PM promotes degradation of AGO1 to suppress RNA silencing independent of SKP1 interaction.

scholarly journals Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures

2016 ◽  
Vol 106 (6) ◽  
pp. 653-662 ◽  
Author(s):  
Kenji Kubota ◽  
James C. K. Ng
Keyword(s):  
Rna Silencing ◽  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Suppressor Activity ◽  
Systemic Movement ◽  
Symptom Modulation ◽  
Green Fluorescent ◽  
Rna Accumulation ◽  
Local Necrosis

RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host’s antiviral defenses and in symptom modulation.

scholarly journals Cell-to-Cell, but Not Long-Distance, Spread of RNA Silencing That Is Induced in Individual Epidermal Cells

2004 ◽  
Vol 78 (6) ◽  
pp. 3149-3154 ◽  
Author(s):  
Eugene V. Ryabov ◽  
Rene van Wezel ◽  
John Walsh ◽  
Yiguo Hong
Keyword(s):  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Epidermal Cells ◽  
Turnip Crinkle Virus ◽  
Mechanical Inoculation ◽  
Long Distance ◽  
Systemic Spread ◽  
Systemic Rna ◽  
Green Fluorescent

ABSTRACT A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFPΔCP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFPΔCP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFPΔCP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFPΔCP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.

scholarly journals Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

2007 ◽  
Vol 81 (19) ◽  
pp. 10379-10388 ◽  
Author(s):  
Hannes Vogler ◽  
Rashid Akbergenov ◽  
Padubidri V. Shivaprasad ◽  
Vy Dang ◽  
Monika Fasler ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Micro Rna ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Suppressor Activity ◽  
Virus Family ◽  
Green Fluorescent

ABSTRACT Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

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 Identification of a Novel Saturable Endoplasmic Reticulum Localization Mechanism Mediated by the C-Terminus of aDictyostelium Protein Disulfide Isomerase

2000 ◽  
Vol 11 (10) ◽  
pp. 3469-3484 ◽  
Author(s):  
Jean Monnat ◽  
Eva M. Neuhaus ◽  
Marius S. Pop ◽  
David M. Ferrari ◽  
Barbara Kramer ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Disulfide Isomerase ◽  
Fluorescent Protein ◽  
Null Mutation ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
C Terminus ◽  
Complementary Action ◽  
Green Fluorescent ◽  
Protein Disulfide

Localization of soluble endoplasmic reticulum (ER) resident proteins is likely achieved by the complementary action of retrieval and retention mechanisms. Whereas the machinery involving the H/KDEL and related retrieval signals in targeting escapees back to the ER is well characterized, other mechanisms including retention are still poorly understood. We have identified a protein disulfide isomerase (Dd-PDI) lacking the HDEL retrieval signal normally found at the C terminus of ER residents in Dictyostelium discoideum. Here we demonstrate that its 57 residue C-terminal domain is necessary for intracellular retention of Dd-PDI and sufficient to localize a green fluorescent protein (GFP) chimera to the ER, especially to the nuclear envelope. Dd-PDI and GFP-PDI57 are recovered in similar cation-dependent complexes. The overexpression of GFP-PDI57 leads to disruption of endogenous PDI complexes and induces the secretion of PDI, whereas overexpression of a GFP-HDEL chimera induces the secretion of endogenous calreticulin, revealing the presence of two independent and saturable mechanisms. Finally, low-level expression of Dd-PDI but not of PDI truncated of its 57 C-terminal residues complements the otherwise lethal yeast TRG1/PDI1 null mutation, demonstrating functional disulfide isomerase activity and ER localization. Altogether, these results indicate that the PDI57 peptide contains ER localization determinants recognized by a conserved machinery present in D. discoideum and Saccharomyces cerevisiae.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

scholarly journals RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

2021 ◽  
Vol 12 ◽  
Author(s):  
Shakil Ahmad ◽  
Momana Jamil ◽  
Muhammad Fahim ◽  
Shujing Zhang ◽  
Farman Ullah ◽  
...  
Keyword(s):  
Growth Factors ◽  
Imaginal Disc ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Small Body ◽  
Functional Characterization ◽  
Oral Feeding ◽  
Developmental Defects ◽  
Melon Fly ◽  
Green Fluorescent

This study reports the first successful use of oral feeding dsRNA technique for functional characterization of imaginal disc growth factors (IDGFs) genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6) in melon fly Zeugodacus cucurbitae. Phylogenetic and domain analysis indicates that these genes had high similarity with other Tephritidae fruit flies homolog and contain only one conserved domain among these five genes, which is glyco-18 domain (glyco-hydro-18 domain). Gene expression analysis at different developmental stages revealed that these genes were expressed at larval, pupal, and adult stages. To understand their role in different developmental stages, larvae were fed dsRNA-corresponding to each of the five IDGFs, in an artificial diet. RNAi-mediated knockdown of IDGF1 shows no phenotypic effects but caused mortality (10.4%), while IDGF4_0 caused malformed pharate at the adult stage where insects failed to shed their old cuticle and remained attached with their body, highest mortality (49.2%) was recorded compared to dsRNA-green fluorescent protein (GFP) or DEPC. Silencing of IDGF3_1 and IDGF4_1 cause lethal phenotype in larvae, (17.2%) and (40%) mortality was indexed in Z. cucurbitae. IDGF6 was mainly expressed in pupae and adult stages, and its silencing caused a malformation in adult wings. The developmental defects such as malformation in wings, larval–larval lethality, pupal–adult malformation, and small body size show that IDGFs are key developmental genes in the melon fly. Our results provide a baseline for the melon fly management and understanding of IDGFs specific functions in Z. cucurbitae.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

2018 ◽  
Vol 96 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Robert Lodge ◽  
François Marceau
Keyword(s):  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Denaturation ◽  
Epifluorescence Microscopy ◽  
Terminal Sequence ◽  
Protein Ligands ◽  
C Terminus ◽  
Bona Fide ◽  
Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

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