scholarly journals Apple chlorotic leaf spot virus 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana

2007 ◽  
Vol 88 (1) ◽  
pp. 316-324 ◽  
Author(s):  
Hajime Yaegashi ◽  
Tsubasa Takahashi ◽  
Masamichi Isogai ◽  
Takashi Kobori ◽  
Satoshi Ohki ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Silencing Suppressor ◽  
Spot Virus ◽  
Double Stranded Rna ◽  
Chlorotic Leaf ◽  
Green Fluorescent

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.

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 Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein

2000 ◽  
Vol 81 (8) ◽  
pp. 2085-2093 ◽  
Author(s):  
Hiroshi Satoh ◽  
Hironori Matsuda ◽  
Takehiro Kawamura ◽  
Masamichi Isogai ◽  
Nobuyuki Yoshikawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Leaf Spot ◽  
Fluorescent Protein ◽  
Cell Trafficking ◽  
Leaf Epidermis ◽  
Spot Virus ◽  
Tubule Formation ◽  
Mature Leaves ◽  
Chlorotic Leaf ◽  
Green Fluorescent

The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP–GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP–GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP–GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287–457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.

scholarly journals Identification of an RNA Silencing Suppressor from a Plant Double-Stranded RNA Virus

2005 ◽  
Vol 79 (20) ◽  
pp. 13018-13027 ◽  
Author(s):  
Xuesong Cao ◽  
Peng Zhou ◽  
Xiaoming Zhang ◽  
Shifeng Zhu ◽  
Xuehua Zhong ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Rna Virus ◽  
Regulation Of Gene Expression ◽  
Potato Virus X ◽  
Dsrna Virus ◽  
Silencing Suppressor ◽  
Double Stranded Rna ◽  
Green Fluorescent

ABSTRACT RNA silencing is a mechanism which higher plants and animals have evolved to defend against viral infection in addition to regulation of gene expression for growth and development. As a counterdefense, many plant and some animal viruses studied to date encode RNA silencing suppressors (RSS) that interfere with various steps of the silencing pathway. In this study, we report the first identification of an RSS from a plant double-stranded RNA (dsRNA) virus. Pns10, encoded by S10 of Rice dwarf phytoreovirus (RDV), exhibited RSS activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c carrying GFP. The other gene segments of the RDV genome did not have such a function. Pns10 suppressed local and systemic silencing induced by sense RNA but did not interfere with local and systemic silencing induced by dsRNA. Expression of Pns10 also increased the expression of β-glucuronidase in transient assays and enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, our results establish Pns10 as an RSS encoded by a plant dsRNA virus and further suggest that Pns10 targets an upstream step of dsRNA formation in the RNA silencing pathway.

scholarly journals Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein

2005 ◽  
Vol 86 (1) ◽  
pp. 225-229 ◽  
Author(s):  
Masamichi Isogai ◽  
Nobuyuki Yoshikawa
Keyword(s):  
Leaf Spot ◽  
Rna Binding ◽  
Movement Protein ◽  
Binding Domain ◽  
Sequence Specificity ◽  
Nucleic Acid Binding ◽  
Spot Virus ◽  
Uv Crosslinking ◽  
Chlorotic Leaf ◽  
Rna Binding Domain

The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin–UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82–126 and 127–287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

scholarly journals Phenotypes and Functional Effects Caused by Various Viral RNA Silencing Suppressors in Transgenic Nicotiana benthamiana and N. tabacum

2008 ◽  
Vol 21 (2) ◽  
pp. 178-187 ◽  
Author(s):  
Shahid Aslam Siddiqui ◽  
Cecilia Sarmiento ◽  
Erkki Truve ◽  
Harry Lehto ◽  
Kirsi Lehto
Keyword(s):  
Mosaic Virus ◽  
Potato Virus ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Potato Virus X ◽  
Mottle Virus ◽  
African Cassava Mosaic Virus ◽  
Rice Yellow Mottle Virus ◽  
Green Fluorescent

RNA silencing suppressor genes derived from six virus genera were transformed into Nicotiana benthamiana and N. tabacum plants. These suppressors were P1 of Rice yellow mottle virus (RYMV), P1 of Cocksfoot mottle virus, P19 of Tomato bushy stunt virus, P25 of Potato virus X, HcPro of Potato virus Y (strain N), 2b of Cucumber mosaic virus (strain Kin), and AC2 of African cassava mosaic virus (ACMV). HcPro caused the most severe phenotypes in both Nicotiana spp. AC2 also produced severe effects in N. tabacum but a much milder phenotype in N. benthamiana, although both HcPro and AC2 affected the leaf tissues of the two Nicotiana spp. in similar ways, causing hyperplasia and hypoplasia, respectively. P1-RYMV caused high lethality in the N. benthamiana plants but only mild effects in the N. tabacum plants. Phenotypic alterations produced by the other transgenes were minor in both species. Interestingly, the suppressors had very different effects on crucifer-infecting Tobamovirus (crTMV) infections. AC2 enhanced both spread and brightness of the crTMV-green fluorescent protein (GFP) lesions, whereas 2b and both P1 suppressors enhanced spread but not brightness of these lesions. P19 promoted spread of the infection into new foci within the infiltrated leaf, whereas HcPro and P25 suppressed the spread of crTMV-GFP lesions.

scholarly journals Movement protein of Apple chlorotic leaf spot virus is genetically unstable and negatively regulated by Ribonuclease E in E. coli

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Rahul Mohan Singh ◽  
Dharam Singh ◽  
Vipin Hallan
Keyword(s):  
Leaf Spot ◽  
Movement Protein ◽  
Spot Virus ◽  
Ribonuclease E ◽  
E Coli ◽  
Chlorotic Leaf

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 Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

2010 ◽  
Vol 84 (21) ◽  
pp. 11542-11554 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rik Huismann ◽  
Rob Goldbach ◽  
Marcel Prins ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Micro Rna ◽  
Impatiens Necrotic Spot Virus ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

scholarly journals Are Tubules Generated by the 3a Protein Necessary for Cucumber Mosaic Virus Movement?

1999 ◽  
Vol 12 (11) ◽  
pp. 985-993 ◽  
Author(s):  
Tomas Canto ◽  
Peter Palukaitis
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Virus Movement ◽  
Wild Type ◽  
Gene Encoding ◽  
Green Fluorescent ◽  
Infected Tobacco

The 3a movement protein of cucumber mosaic virus (CMV), fused to the jellyfish green fluorescent protein (3a-GFP) generated surface punctate aggregates as well as tubules protruding from infected tobacco and Nicotiana benthamiana protoplasts. Fluorescent tubules also appeared on the surface of protoplasts prepared from transgenic tobacco plants expressing 3a-GFP, indicating that the 3a protein is the only viral component required for the formation of the tubules. CMV with a mutation in the gene encoding the 3a protein, M8 CMV, could infect tobacco systemically, but tubules were not detected protruding from infected protoplasts when the mutated 3a protein was fused to the GFP [(M8)3a-GFP]. This indicates that the ability of the 3a protein to generate tubules in the surface of protoplasts is not a function required for the spread of CMV in tobacco. On the other hand, the (M8)3a-GFP did not traffic through plasmodesmata interconnecting tobacco epidermal cells, in contrast to the wild-type 3a-GFP. This suggests that there may be a correlation between the ability of the 3a protein to assemble tubules in protoplasts and its ability to promote movement within certain tissues.

scholarly journals Recovery of Nicotiana benthamiana Plants from a Necrotic Response Induced by a Nepovirus Is Associated with RNA Silencing but Not with Reduced Virus Titer

2007 ◽  
Vol 81 (22) ◽  
pp. 12285-12297 ◽  
Author(s):  
Juan Jovel ◽  
Melanie Walker ◽  
Hélène Sanfaçon
Keyword(s):  
Host Defense ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Virus Titer ◽  
Defense Responses ◽  
Viral Rna ◽  
Small Interfering Rnas ◽  
Green Fluorescent

ABSTRACT Recovery of plants from virus-induced symptoms is often described as a consequence of RNA silencing, an antiviral defense mechanism. For example, recovery of Nicotiana clevelandii from a nepovirus (tomato black ring virus) is associated with a decreased viral RNA concentration and sequence-specific resistance to further virus infection. In this study, we have characterized the interaction of another nepovirus, tomato ringspot virus (ToRSV), with host defense responses during symptom induction and subsequent recovery. Early in infection, ToRSV induced a necrotic phenotype in Nicotiana benthamiana that showed characteristics typical of a hypersensitive response. RNA silencing was also activated during ToRSV infection, as evidenced by the presence of ToRSV-derived small interfering RNAs (siRNAs) that could direct degradation of ToRSV sequences introduced into sensor constructs. Surprisingly, disappearance of symptoms was not accompanied by a commensurate reduction in viral RNA levels. The stability of ToRSV RNA after recovery was also observed in N. clevelandii and Cucumis sativus and in N. benthamiana plants carrying a functional RNA-dependent RNA polymerase 1 ortholog from Medicago truncatula. In experiments with a reporter transgene (green fluorescent protein), ToRSV did not suppress the initiation or maintenance of transgene silencing, although the movement of the silencing signal was partially hindered. Our results demonstrate that although RNA silencing is active during recovery, reduction of virus titer is not required for the initiation of this phenotype. This scenario adds an unforeseen layer of complexity to the interaction of nepoviruses with the host RNA silencing machinery. The possibility that viral proteins, viral RNAs, and/or virus-derived siRNAs inactivate host defense responses is discussed.

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