scholarly journals Suppression of Bamboo Mosaic Virus Accumulation by a Putative Methyltransferase in Nicotiana benthamiana

2009 ◽  
Vol 83 (11) ◽  
pp. 5796-5805 ◽  
Author(s):  
Chun-Wei Cheng ◽  
Yi-Yuong Hsiao ◽  
Hui-Chuan Wu ◽  
Chi-Mau Chuang ◽  
Jao-Shien Chen ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Rna Virus ◽  
Infectious Clone ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Virus Induced Gene Silencing ◽  
Innate Defense ◽  
Predicted Signal Peptide

ABSTRACT Bamboo mosaic virus (BaMV) is a 6.4-kb positive-sense RNA virus belonging to the genus Potexvirus of the family Flexiviridae. The 155-kDa viral replicase, the product of ORF1, comprises an N-terminal S-adenosyl-l-methionine (AdoMet)-dependent guanylyltransferase, a nucleoside triphosphatase/RNA 5′-triphosphatase, and a C-terminal RNA-dependent RNA polymerase (RdRp). To search for cellular factors potentially involved in the regulation of replication and/or transcription of BaMV, the viral RdRp domain was targeted as bait to screen against a leaf cDNA library of Nicotiana benthamiana using a yeast two-hybrid system. A putative methyltransferase (PNbMTS1) of 617 amino acid residues without an established physiological function was identified. Cotransfection of N. benthamiana protoplasts with a BaMV infectious clone and the PNbMTS1-expressing plasmid showed a PNbMTS1 dosage-dependent inhibitory effect on the accumulation of BaMV coat protein. Deletion of the N-terminal 36 amino acids, deletion of a predicted signal peptide or transmembrane segment, or mutations in the putative AdoMet-binding motifs of PNbMTS1 abolished the inhibitory effect. In contrast, suppression of PNbMTS1 by virus-induced gene silencing in N. benthamiana increased accumulation of the viral coat protein as well as the viral genomic RNA. Collectively, PNbMTS1 may function as an innate defense protein against the accumulation of BaMV through an uncharacterized mechanism.

scholarly journals RNA virus interference via CRISPR/Cas13a system in plants

2017 ◽  
Author(s):  
Rashid Aman ◽  
Zahir Ali ◽  
Haroon Butt ◽  
Ahmed Mahas ◽  
Fatima Aljedaani ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Coat Protein ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Phage Genome ◽  
Rna Viruses ◽  
Rna Virus ◽  
Turnip Mosaic Virus ◽  
Green Fluorescent

AbstractCRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants. CRISPR/Cas13a produced interference against green fluorescent protein (GFP) expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. crRNAs targeting the HC-Pro and GFP sequences exhibited better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs. Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses, and for other RNA manipulations in plants.

scholarly journals Identification of a movement protein of Mirafiori lettuce big-vein ophiovirus

2013 ◽  
Vol 94 (5) ◽  
pp. 1145-1150 ◽  
Author(s):  
Akihiro Hiraguri ◽  
Shoko Ueki ◽  
Hideki Kondo ◽  
Koji Nomiyama ◽  
Takumi Shimizu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Protein Gene ◽  
Movement Protein ◽  
Rna Virus ◽  
Infectious Clone ◽  
Microprojectile Bombardment ◽  
Tomato Mosaic Virus ◽  
Intercellular Movement ◽  
Heterologous Virus

Mirafiori lettuce big-vein virus (MiLBVV) is a member of the genus Ophiovirus, which is a segmented negative-stranded RNA virus. In microprojectile bombardment experiments to identify a movement protein (MP) gene of ophioviruses that can trans-complement intercellular movement of an MP-deficient heterologous virus, a plasmid containing an infectious clone of a tomato mosaic virus (ToMV) derivative expressing the GFP was co-bombarded with plasmids containing one of three genes from MiLBVV RNAs 1, 2 and 4 onto Nicotiana benthamiana. Intercellular movement of the movement-defective ToMV was restored by co-expression of the 55 kDa protein gene, but not with the two other genes. Transient expression in epidermal cells of N. benthamiana and onion showed that the 55 kDa protein with GFP was localized on the plasmodesmata. The 55 kDa protein encoded in the MiLBVV RNA2 can function as an MP of the virus. This report is the first to describe an ophiovirus MP.

scholarly journals Multiple aromatic amino acids are involved in potyvirus movement by forming π-stackings to maintain coat protein accumulation

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Zhi-Yong Yan ◽  
Xiao-Jie Xu ◽  
Le Fang ◽  
Chao Geng ◽  
Yan-Ping Tian ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Infectious Clone ◽  
Three Dimensional ◽  
Cell Movement ◽  
Systemic Infection ◽  
Watermelon Mosaic Virus ◽  
Protein Accumulation ◽  
Three Dimensional Model ◽  
Aromatic Residues

AbstractCoat protein (CP) is required for potyviruses to move and establish a systemic infection in plants. π-stackings formed by aromatic residues play critical roles in maintaining protein stability and functions. As we know, many aromatic residues located in the core region of potyvirus CPs are conserved. However, their roles in potyvirus infection remain largely unknown. Here, through analysis of the three-dimensional model of the tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) CP, 16 aromatic residues were predicated to form π-stackings. The results of transient expression experiments demonstrated that deletion of any of these 16 aromatic residues reduced CP accumulation. Infectivity assays showed that deletion of any of these aromatic residues in the TVBMV infectious clone abolished cell-to-cell movement and reduced replication of the virus. Substitution of Y105 and Y147 individually with non-aromatic residues alanine or glycine reduced CP accumulation, virus replication, and abolished the ability of TVBMV to move intercellularly, while substitution of these two residues individually with aromatic residues phenylalanine or tryptophan, had no or little effect on CP accumulation and TVBMV systemic movement and replication. Similar results were obtained from the CP mutants of watermelon mosaic virus (WMV, genus Potyvirus). Taken together, our results demonstrate that multiple aromatic residues in CP are involved in potyvirus movement by forming π-stackings to maintain CP accumulation.

scholarly journals Autophagy is involved in assisting the replication of Bamboo mosaic virus in Nicotiana benthamiana

2019 ◽  
Vol 70 (18) ◽  
pp. 4657-4670 ◽  
Author(s):  
Ying-Ping Huang ◽  
Ying-Wen Huang ◽  
Yung-Jen Hsiao ◽  
Siou-Cen Li ◽  
Yau-Huei Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Critical Role ◽  
Viral Rna ◽  
Autophagosome Formation ◽  
Orange Fluorescent Protein ◽  
Positive Sense ◽  
Trigger Cell Death

Abstract Autophagy plays a critical role in plants under biotic stress, including the response to pathogen infection. We investigated whether autophagy-related genes (ATGs) are involved in infection with Bamboo mosaic virus (BaMV), a single-stranded positive-sense RNA virus. Initially, we observed that BaMV infection in Nicotiana benthamiana leaves upregulated the expression of ATGs but did not trigger cell death. The induction of ATGs, which possibly triggers autophagy, increased rather than diminished BaMV accumulation in the leaves, as revealed by gene knockdown and transient expression experiments. Furthermore, the inhibitor 3-methyladenine blocked autophagosome formation and the autophagy inducer rapamycin, which negatively and positively affected BaMV accumulation, respectively. Pull-down experiments with an antibody against orange fluorescent protein (OFP)-NbATG8f, an autophagosome marker protein, showed that both plus- and minus-sense BaMV RNAs could associate with NbATG8f. Confocal microscopy revealed that ATG8f-enriched vesicles possibly derived from chloroplasts contained both the BaMV viral RNA and its replicase. Thus, BaMV infection may induce the expression of ATGs possibly via autophagy to selectively engulf a portion of viral RNA-containing chloroplast. Virus-induced vesicles enriched with ATG8f could provide an alternative site for viral RNA replication or a shelter from the host silencing mechanism.

scholarly journals Inhibitory Effect of Osthole from Cnidium monnieri on Tobacco Mosaic Virus (TMV) Infection in Nicotiana glutinosa

Molecules ◽  
2019 ◽  
Vol 25 (1) ◽  
pp. 65 ◽  
Author(s):  
Ya-Han Chen ◽  
Dong-Sheng Guo ◽  
Mei-Huan Lu ◽  
Jian-Ying Yue ◽  
Yan Liu ◽  
...  
Keyword(s):  
Coat Protein ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Plant Virus ◽  
Inhibitory Effect ◽  
Nicotiana Glutinosa ◽  
1H And 13C Nmr ◽  
Virus Activity ◽  
Viral Particles ◽  
Coumarin Compound

The coumarin compound of osthole was extracted from Cnidium monnieri and identified by LC-MS and 1H- and 13C-NMR. Osthole was tested for anti-virus activity against tobacco mosaic virus (TMV) using the half-leaf method. The results showed that stronger antiviral activity on TMV infection appeared in Nicotiana glutinosa than that of eugenol and ningnanmycin, with inhibitory, protective, and curative effects of 72.57%, 70.26%, and 61.97%, respectively. Through observation of the TMV particles, we found that osthole could directly affect the viral particles. Correspondingly, the level of coat protein detected by Western blot was significantly reduced when the concentrations of osthole increased in tested plants compared to that of the control. These results suggest that osthole has anti-TMV activity and may be used as a biological reagent to control the plant virus in the half-leaf method.

scholarly journals Sequence Variations Among 17 New Radish Isolates of Turnip mosaic virus Showing Differential Pathogenicity and Infectivity in Nicotiana benthamiana, Brassica rapa, and Raphanus sativus

2019 ◽  
Vol 109 (5) ◽  
pp. 904-912 ◽  
Author(s):  
Junsu Gong ◽  
Hye-Kyoung Ju ◽  
Ik-Hyun Kim ◽  
Eun-Young Seo ◽  
In-Sook Cho ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Chinese Cabbage ◽  
Nicotiana Benthamiana ◽  
Turnip Mosaic Virus ◽  
Amino Acid Residues ◽  
Systemic Necrosis ◽  
Infectious Clones ◽  
Sequence Variations ◽  
Distinct Sequence

Infectious clones were generated from 17 new Korean radish isolates of Turnip mosaic virus (TuMV). Phylogenetic analysis indicated that all new isolates, and three previously characterized Korean radish isolates, belong to the basal-BR group (indicating that the pathotype can infect both Brassica and Raphanus spp.). Pairwise analysis revealed genomic nucleotide and polyprotein amino acid identities of >87.9 and >95.7%, respectively. Five clones (HJY1, HJY2, KIH2, BE, and prior isolate R007) had lower sequence identities than other isolates and produced mild symptoms in Nicotiana benthamiana. These isolates formed three distinct sequence classes (HJY1/HJY2/R007, KIH2, and BE), and several differential amino acid residues (in P1, P3, 6K2, and VPg) were present only in mild isolates HJY1, HJY2, and R007. The remaining isolates all induced systemic necrosis in N. benthamiana. Four mild isolates formed a phylogenetic subclade separate from another subclade including all of the necrosis-inducing isolates plus mild isolate KIH2. Symptom severity in radish and Chinese cabbage genotypes was not correlated with pathogenicity in N. benthamiana; indeed, Chinese cabbage cultivar Norang was not infected by any isolate, whereas Chinese cabbage cultivar Chusarang was uniformly susceptible. Four isolates were unable to infect radish cultivar Iljin, but no specific amino acid residues were correlated with avirulence. These results may lead to the identification of new resistance genes against TuMV.

scholarly journals A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

2019 ◽  
Author(s):  
Mei Liu ◽  
Zhiling Liang ◽  
Miguel A. Aranda ◽  
Ni Hong ◽  
Liming Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Nicotiana Benthamiana ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Economic Significance ◽  
Potential Alternative ◽  
Low Efficiency

AbstractCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiencies. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits. Here, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69-300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged. These results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function in cucurbits.One sentence summaryA CGMMV-based vector enables gene function studies in cucurbits, an extremely low efficiency species for genetic transformation.

scholarly journals Maintenance of coat protein N-terminal net charge and not primary sequence is essential for zucchini yellow mosaic virus systemic infectivity

2004 ◽  
Vol 85 (11) ◽  
pp. 3421-3430 ◽  
Author(s):  
Boaz Kimalov ◽  
Amit Gal-On ◽  
Ran Stav ◽  
Eduard Belausov ◽  
Tzahi Arazi
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Zucchini Yellow Mosaic Virus ◽  
Cell Movement ◽  
Yellow Mosaic Virus ◽  
Virus Infectivity ◽  
Amino Acid Residues ◽  
Primary Sequence ◽  
Net Charge ◽  
Truncation Mutant

Zucchini yellow mosaic virus (ZYMV) surface exposed coat protein (CP) N-terminal domain (Nt) is 43 aa long and contains an equal number of positively and negatively charged amino acid residues (CP-Nt net charge=0). A ZYMV-AGII truncation mutant lacking the first 20 aa of its CP-Nt (AGII-CPΔ20; CP-Nt net charge=+2) was found to be systemically non-infectious even though AGII mutants harbouring larger CP-Nt deletions were previously demonstrated to be fully infectious. Nevertheless, AGII-CPΔ20 infectivity was restored by fusion to its CP-Nt two Asp residues or a negatively charged Myc peptide, both predicted to neutralize CP-Nt net positive charge. To evaluate further the significance of CP-Nt net charge for AGII infectivity, a series of CP-Nt net charge mutants was generated and analysed for systemic infectivity of squash plants. AGII-CPKKK harbouring a CP-Nt amino fusion of three Lys residues (CP-Nt net charge=+3) was not systemically infectious. Addition of up to four Asp residues to CP-Nt did not abolish virus infectivity, although certain mutants were genetically unstable and had delayed infectivity. Addition of five negatively charged residues abolished infectivity (AGII-CPDDDDD; CP-Nt net charge=−5) even though a recombinant CPDDDDD could assemble into potyviral-like particle in bacteria. Neutralization of CP-Nt net charge by fusing Asp or Lys residues recovered infectivity of AGII-CPKKK and AGII-CPDDDDD. GFP-tagging of these mutants has demonstrated that both viruses have defective cell-to-cell movement. Together, these findings suggest that maintenance of CP-Nt net charge and not primary sequence is essential for ZYMV infectivity.

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