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 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 Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

2020 ◽  
Vol 21 (4) ◽  
pp. 1414 ◽  
Author(s):  
Hui Li ◽  
Xiaobao Ying ◽  
Lina Shang ◽  
Bryce Redfern ◽  
Nicholas Kypraios ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Nuclear Localization Sequence ◽  
Localization Sequence ◽  
Candidatus Liberibacter ◽  
Phloem Necrosis ◽  
Liberibacter Asiaticus ◽  
Green Fluorescent

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

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 Effects of the noncoding subgenomic RNA of red clover necrotic mosaic virus in virus infection

2021 ◽  
Author(s):  
Pulkit Kanodia ◽  
W. Allen Miller
Keyword(s):  
Gene Expression ◽  
Virus Infection ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Red Clover ◽  
Plant Viruses ◽  
Viral Rna ◽  
Productive Infection ◽  
Deficient Mutant ◽  
Host Interactions

In recent years, a new class of viral noncoding subgenomic (ncsg)RNA has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant (xr) RNA structure, which blocks the progression of 5’→3’ exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate: (i) absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wildtype RCNMV or an SR1f-deficient mutant RCNMV revealed that wt RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10 to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3’ region of RCNMV RNA1 support the premature mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here we begin to address this question by investigating the noncoding subgenomic (ncsg)RNA of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well-studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wildtype RCNMV vs an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana , which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

scholarly journals ORF6 of Tobacco mosaic virus is a determinant of viral pathogenicity in Nicotiana benthamiana

2004 ◽  
Vol 85 (10) ◽  
pp. 3123-3133 ◽  
Author(s):  
Tomas Canto ◽  
Stuart A. MacFarlane ◽  
Peter Palukaitis
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Host Responses ◽  
Reading Frame ◽  
Virus Accumulation ◽  
Green Fluorescent

Tobacco mosaic virus (TMV) contains a sixth open reading frame (ORF6) that potentially encodes a 4·8 kDa protein. Elimination of ORF6 from TMV attenuated host responses in Nicotiana benthamiana without alteration in virus accumulation. Furthermore, heterologous expression of TMV ORF6 from either potato virus X (PVX) or tobacco rattle virus (TRV) vectors enhanced the virulence of both viruses in N. benthamiana, also without effects on their accumulation. By contrast, the presence or absence of TMV ORF6 had no effect on host response or virus accumulation in N. tabacum plants infected with TMV or PVX. TMV ORF6 also had no effect on the synergism between TMV and PVX in N. tabacum. However, the presence of the TMV ORF6 did have an effect on the pathogenicity of a TRV vector in N. tabacum. In three different types of assay carried out in N. benthamiana plants, expression of TMV ORF6 failed to suppress gene silencing. Expression in N. benthamiana epidermal cells of the encoded 4·8 kDa protein fused to the green fluorescent protein at either end showed, in addition to widespread cytosolic fluorescence, plasmodesmatal targeting specific to both fusion constructs. The role of the ORF6 in host responses is discussed.

scholarly journals Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

2017 ◽  
Vol 114 (7) ◽  
pp. E1282-E1290 ◽  
Author(s):  
Kiwamu Hyodo ◽  
Kenji Hashimoto ◽  
Kazuyuki Kuchitsu ◽  
Nobuhiro Suzuki ◽  
Tetsuro Okuno
Keyword(s):  
Mosaic Virus ◽  
Rna Virus ◽  
Plant Stress ◽  
Rna Replication ◽  
Plant Viruses ◽  
Brome Mosaic Virus ◽  
Viral Rna ◽  
Genome Replication ◽  
Dependent Manner ◽  
Positive Strand Rna

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant–virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of N. benthamiana (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.

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 Comparative analysis of viral RNA signatures on different RIG-I-like receptors

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Raul Y Sanchez David ◽  
Chantal Combredet ◽  
Odile Sismeiro ◽  
Marie-Agnès Dillies ◽  
Bernd Jagla ◽  
...  
Keyword(s):  
Rna Virus ◽  
Affinity Purification ◽  
Viral Rna ◽  
Coding Region ◽  
Chikv Infection ◽  
Rna Molecules ◽  
Positive Sense ◽  
Rna Ligands ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.

scholarly journals Downregulation of the NbNACa1 Gene Encoding a Movement-Protein-Interacting Protein Reduces Cell-to-Cell Movement of Brome mosaic virus in Nicotiana benthamiana

2007 ◽  
Vol 20 (6) ◽  
pp. 671-681 ◽  
Author(s):  
Masanori Kaido ◽  
Yosuke Inoue ◽  
Yoshika Takeda ◽  
Kazuhiko Sugiyama ◽  
Atsushi Takeda ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Sequence Similarity ◽  
Microprojectile Bombardment ◽  
Cell Movement ◽  
Brome Mosaic Virus ◽  
Viral Movement

The 3a movement protein (MP) plays a central role in the movement of the RNA plant virus, Brome mosaic virus (BMV). To identify host factor genes involved in viral movement, a cDNA library of Nicotiana benthamiana, a systemic host for BMV, was screened with far-Western blotting using a recombinant BMV MP as probe. One positive clone encoded a protein with sequence similarity to the α chain of nascent-polypeptide-associated complex from various organisms, which is proposed to contribute to the fidelity of translocation of newly synthesized proteins. The orthologous gene from N. benthamiana was designated NbNACa1. The binding of NbNACa1 to BMV MP was confirmed in vivo with an agroinfiltration-immunoprecipitation assay. To investigate the involvement of NbNACa1 in BMV multiplication, NbNACa1-silenced (GSNAC) transgenic N. benthamiana plants were produced. Downregulation of NbNACa1 expression reduced virus accumulation in inoculated leaves but not in protoplasts. A microprojectile bombardment assay to monitor BMV-MP-assisted viral movement demonstrated reduced virus spread in GSNAC plants. The localization to the cell wall of BMV MP fused to green fluorescent protein was delayed in GSNAC plants. From these results, we propose that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.

scholarly journals Development of Tobacco Mosaic Virus Infection Sites in Nicotiana benthamiana

1999 ◽  
Vol 12 (2) ◽  
pp. 143-152 ◽  
Author(s):  
Judit Szécsi ◽  
Xin Shun Ding ◽  
Chae Oh Lim ◽  
Mohammed Bendahmane ◽  
Moo Je Cho ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Subcellular Location ◽  
Cell Types ◽  
Adjacent Cell ◽  
In Planta ◽  
Cytoplasmic Bodies

To monitor infection of Nicotiana benthamiana by tobacco mosaic virus (TMV), leaves were inoculated with viral constructs expressing the green fluorescent protein (GFP) from jellyfish (Aequorea victoria) fused to the movement protein (MP) of TMV (MP:GFP) or as a free GFP in place of the coat protein (CP). Infection sites produced by TMV expressing the MP:GFP appeared as fluorescent rings larger in diameter and less fluorescent than fluorescent disks induced by constructs encoding free GFP. These results suggest that protein expression driven by the MP subgenomic promoter (sgp) initiates and ends earlier and is at lower level than that observed for proteins driven by the CP sgp. Similarly, analyses of cross sections through the infection sites revealed that in different cell types the accumulation of MP:GFP was regulated differently than the accumulation of free GFP. Immunocytochemistry and electron microscopy showed that near the leading edge of the fluorescent ring the MP:GFP and the viral 126 kDa and 183 kDa replicase proteins accumulated in paired cytoplasmic bodies that formed often on opposite sides of adjacent cell walls containing plasmodesmata. In the dimly fluorescent center of the rings the 126 kDa and 183 kDa proteins, but not the MP:GFP, were localized in unpaired cytoplasmic bodies containing ropelike, fibrillar structures. The paired bodies were similar to previously described viroplasms, while the unpaired bodies were similar to X-bodies. These data indicate that the accumulation of proteins expressed from different sgps of TMV has a specific spatial and temporal pattern in planta. In addition, the cytoplasmic bodies containing the 126 kDa and 183 kDa proteins are dynamic entities whose protein content and subcellular location change during infection.

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