A Conserved Carboxylesterase Inhibits Tobacco mosaic virus (TMV) Accumulation in Nicotiana benthamiana Plants

A carboxylesterase (CXE) or carboxylic-ester hydrolase is an enzyme that catalyzes carboxylic ester and water into alcohol and carboxylate. In plants, CXEs have been implicated in defense, development, and secondary metabolism. We discovered a new CXE gene in Nicotiana benthamiana that is related to virus resistance. The transcriptional level of NbCXE expression was significantly increased after Tobacco mosaic virus (TMV) infection. Transient over-expression of NbCXE inhibited TMV accumulation in N. benthamiana plants. Conversely, when the NbCXE gene was silenced with a Tobacco rattle virus (TRV)-based gene silencing system, TMV RNA accumulation was increased in NbCXE-silenced plants after infection. NbCXE protein was shown to interact with TMV coat protein (CP) in vitro. Additionally, the expressions of host defense-related genes were increased in transient NbCXE-overexpressed plants but decreased in NbCXE silenced N. benthamiana plants. In summary, our study showed that NbCXE is a novel resistance-related gene involved in host defense responses against TMV infection.

Download Full-text

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

Journal of General Virology ◽

10.1099/vir.0.80270-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 3123-3133 ◽

Cited By ~ 19

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.

Download Full-text

In Vitro- and In Vivo-Generated Defective RNAs of Satellite Panicum Mosaic Virus Define cis-Acting RNA Elements Required for Replication and Movement

Journal of Virology ◽

10.1128/jvi.74.5.2247-2254.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2247-2254 ◽

Cited By ~ 21

Author(s):

Wenping Qiu ◽

Scholthof G. Karen-Beth

Keyword(s):

Mosaic Virus ◽

De Novo ◽

Stop Codon ◽

Dependent Manner ◽

Defective Rnas ◽

Systemic Spread ◽

Rna Elements ◽

Rna Domains ◽

Rna Accumulation

ABSTRACT Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3′ untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5′ UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5′ UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential “trigger” for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

Download Full-text

Role of the 3′ tRNA-Like Structure in Tobacco Mosaic Virus Minus-Strand RNA Synthesis by the Viral RNA-Dependent RNA Polymerase In Vitro

Journal of Virology ◽

10.1128/jvi.74.24.11671-11680.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11671-11680 ◽

Cited By ~ 49

Author(s):

T. A. M. Osman ◽

C. L. Hemenway ◽

K. W. Buck

Keyword(s):

Rna Polymerase ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Central Core ◽

Minus Strand ◽

Stem Loop ◽

Polymerase Binding

ABSTRACT A template-dependent RNA polymerase has been used to determine the sequence elements in the 3′ untranslated region of tobacco mosaic virus RNA that are required for promotion of minus-strand RNA synthesis and binding to the RNA polymerase in vitro. Regions which were important for minus-strand synthesis were domain D1, which is equivalent to a tRNA acceptor arm; domain D2, which is similar to a tRNA anticodon arm; an upstream domain, D3; and a central core, C, which connects domains D1, D2, and D3 and determines their relative orientations. Mutational analysis of the 3′-terminal 4 nucleotides of domain D1 indicated the importance of the 3′-terminal CA sequence for minus-strand synthesis, with the sequence CCCA or GGCA giving the highest transcriptional efficiency. Several double-helical regions, but not their sequences, which are essential for forming pseudoknot and/or stem-loop structures in domains D1, D2, and D3 and the central core, C, were shown to be required for high template efficiency. Also important were a bulge sequence in the D2 stem-loop and, to a lesser extent, a loop sequence in a hairpin structure in domain D1. The sequence of the 3′ untranslated region upstream of domain D3 was not required for minus-strand synthesis. Template-RNA polymerase binding competition experiments showed that the highest-affinity RNA polymerase binding element region lay within a region comprising domain D2 and the central core, C, but domains D1 and D3 also bound to the RNA polymerase with lower affinity.

Download Full-text

An analysis of tobacco mosaic virus replicative structures synthesized in vitro

Plant Molecular Biology ◽

10.1007/bf00027137 ◽

1986 ◽

Vol 6 (6) ◽

pp. 455-465 ◽

Cited By ~ 15

Author(s):

Nevin Dale Young ◽

Milton Zaitlin

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus

Download Full-text

Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Journal of Virology ◽

10.1128/jvi.00540-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8329-8344 ◽

Cited By ~ 67

Author(s):

Jamie Ashby ◽

Emmanuel Boutant ◽

Mark Seemanpillai ◽

Adrian Sambade ◽

Christophe Ritzenthaler ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Cell Extract ◽

Transport Processes ◽

In Vitro Assays ◽

Protein Functions ◽

Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

Download Full-text

In vitro and in vivo aggregation of the defective PM2 tobacco mosaic virus protein

Journal of Molecular Biology ◽

10.1016/s0022-2836(66)80056-6 ◽

1966 ◽

Vol 19 (1) ◽

pp. 140-IN8 ◽

Cited By ~ 21

Author(s):

Albert Siegel ◽

G.J. Hills ◽

Roy Markham

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Virus Protein

Download Full-text

On in vitro and in vivo binding of tobacco mosaic virus to cytoplasmic membranes

Biochemie und Physiologie der Pflanzen ◽

10.1016/s0015-3796(84)80090-6 ◽

1984 ◽

Vol 179 (6) ◽

pp. 507-516 ◽

Cited By ~ 4

Author(s):

Barbara Pustowoit ◽

Wladimir Pustowoit ◽

Gottfried Schuster

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

In Vivo Binding ◽

Cytoplasmic Membranes

Download Full-text

Visualization by atomic force microscopy of tobacco mosaic virus movement protein–RNA complexes formed in vitro

Journal of General Virology ◽

10.1099/0022-1317-82-6-1503 ◽

2001 ◽

Vol 82 (6) ◽

pp. 1503-1508 ◽

Cited By ~ 45

Author(s):

O. I. Kiselyova ◽

I. V. Yaminsky ◽

E. M. Karger ◽

O. Yu. Frolova ◽

Y. L. Dorokhov ◽

...

Keyword(s):

Atomic Force Microscopy ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Structural Conversion ◽

Force Microscopy ◽

Rna Transcripts ◽

Atomic Force ◽

Rna Complexes

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive ‘beads-on-a-string’ into a ‘thick string’ form that was partly resistant to RNase. The ‘thick string’-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The ‘thick string’ complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.

Download Full-text