Tobacco mosaic virus 126-kDa Protein Increases the Susceptibility of Nicotiana tabacum to Other Viruses and Its Dosage Affects Virus-Induced Gene Silencing

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein–green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

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Satellite Panicum Mosaic Virus Capsid Protein Elicits Symptoms on a Nonhost Plant and Interferes with a Suppressor of Virus-Induced Gene Silencing

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2004.17.3.263 ◽

2004 ◽

Vol 17 (3) ◽

pp. 263-271 ◽

Cited By ~ 20

Author(s):

Wenping Qiu ◽

Karen-Beth G. Scholthof

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Capsid Protein ◽

Fluorescent Protein ◽

Potato Virus X ◽

Helper Virus ◽

Gene Vector ◽

Virus Induced Gene Silencing ◽

Suppressor Activity ◽

Pathogenicity Factor

The capsid protein (CP) of satellite panicum mosaic virus (SPMV) has been implicated as a pathogenicity factor, inducing severe chlorosis on millet plants co-infected with SPMV and its helper virus, Panicum mosaic virus (PMV). In this study, we tested the effects of SPMV CP on Nicotiana benthamiana, a plant that does not support PMV+SPMV infections. SPMV CP expressed from a Potato virus X (PVX) gene vector elicited necrotic lesions on N. benthamiana. Pathogenicity factors often have the additional feature of acting as suppressors of gene silencing; therefore, several assays were developed to test if SPMV CP could act in such a capacity. The results showed that SPMV CP failed to act as a suppressor of posttranscriptional gene silencing when such tests were performed with transgenic N. benthamiana plants silenced for green fluorescent protein (GFP) expression by agroinfiltration or plant virus vectors. However SPMV CP expressed from the PVX gene vector did interfere with suppressor activity associated with PVX p25. This included a rebounded level of GFP silencing along the vascular tissues, including the veins on upper noninoculated leaves. Therefore, the roles of the SPMV CP now include encapsidation of the SPMV RNA, activity as a pathogenicity factor in both host and nonhost plants, and the enigmatic feature of interfering with suppression of gene silencing.

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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.

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Tombusvirus P19-Mediated Suppression of Virus-Induced Gene Silencing Is Controlled by Genetic and Dosage Features That Influence Pathogenicity

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.3.269 ◽

2002 ◽

Vol 15 (3) ◽

pp. 269-280 ◽

Cited By ~ 112

Author(s):

Wenping Qiu ◽

Jong-Won Park ◽

Herman B. Scholthof

Keyword(s):

Gene Silencing ◽

Fluorescent Protein ◽

Biological Activities ◽

Potato Virus X ◽

Virus Spread ◽

Virus Induced Gene Silencing ◽

Suppressor Activity ◽

Gfp Gene ◽

Pathogenicity Determinant ◽

Green Fluorescent

The p19 protein (P19) of Tomato bushy stunt virus (TBSV) is a pathogenicity determinant with host-dependent effects on virus spread and symptom induction. In addition, results in this study confirm that Potato virus X-mediated delivery of P19 suppresses posttranscriptional gene silencing (PTGS). To study the relevance of this activity for TBSV biology, we evaluated whether TBSV activates virus-induced gene silencing (VIGS) and if this process is suppressed by P19. TBSV vectors with the green fluorescent protein (GFP) gene, either active or inactive for P19 expression, were inoculated onto GFP-transgenic Nicotiana bentha-miana plants. In the absence of P19 expression, VIGS was activated, as evidenced by the disappearance of GFP mRNA and green fluorescence. Coexpression of GFP and P19 from the TBSV vector suppressed VIGS, except in the newly emerging leaves. The suppressor activity required a central P19 region that is also known to be essential for host-dependent virus spread and symptom induction. Defective interfering RNAs (DIs) that contained the 3′ end of the GFP gene induced silencing very effectively. The concomitant DI-instigated reduction in P19 accumulation failed to suppress this process, analogous to the known P19 dosage effects for other biological activities. In conclusion, (i) TBSV and its DIs are very effective inducers of VIGS, (ii) P19 is a strong suppressor of PTGS, (iii) P19 is a moderate suppressor of VIGS, and (iv) the suppressor activity is influenced by genetic and dosage features that are also important for P19-associated pathogenesis.

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Involvement of the chloroplast gene ferredoxin 1 in multiple responses of Nicotiana benthamiana to Potato virus X infection

Journal of Experimental Botany ◽

10.1093/jxb/erz565 ◽

2019 ◽

Vol 71 (6) ◽

pp. 2142-2156 ◽

Cited By ~ 2

Author(s):

Xue Yang ◽

Yuwen Lu ◽

Fang Wang ◽

Ying Chen ◽

Yanzhen Tian ◽

...

Keyword(s):

Transgenic Plants ◽

Mosaic Virus ◽

Potato Virus ◽

Tobacco Rattle Virus ◽

Potato Virus X ◽

Tomato Mosaic Virus ◽

Pcr Analysis ◽

Coat Proteins ◽

Virus Induced Gene Silencing ◽

Callose Deposition

Abstract The chloroplast protein ferredoxin 1 (FD1), with roles in the chloroplast electron transport chain, is known to interact with the coat proteins (CPs) of Tomato mosaic virus and Cucumber mosaic virus. However, our understanding of the roles of FD1 in virus infection remains limited. Here, we report that the Potato virus X (PVX) p25 protein interacts with FD1, whose mRNA and protein levels are reduced by PVX infection or by transient expression of p25. Silencing of FD1 by Tobacco rattle virus-based virus-induced gene silencing (VIGS) promoted the local and systemic infection of plants by PVX. Use of a drop-and-see (DANS) assay and callose staining revealed that the permeability of plasmodesmata (PDs) was increased in FD1-silenced plants together with a consistently reduced level of PD callose deposition. After FD1 silencing, quantitative reverse transcription–real-time PCR (qRT–PCR) analysis and LC-MS revealed these plants to have a low accumulation of the phytohormones abscisic acid (ABA) and salicylic acid (SA), which contributed to the decreased callose deposition at PDs. Overexpression of FD1 in transgenic plants manifested resistance to PVX infection, but the contents of ABA and SA, and the PD callose deposition were not increased in transgenic plants. Overexpression of FD1 interfered with the RNA silencing suppressor function of p25. These results demonstrate that interfering with FD1 function causes abnormal plant hormone-mediated antiviral processes and thus enhances PVX infection.

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Agrobacterium-Mediated Gene Transient Overexpression and Tobacco Rattle Virus (TRV)-Based Gene Silencing in Cassava

International Journal of Molecular Sciences ◽

10.3390/ijms20163976 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3976 ◽

Cited By ~ 6

Author(s):

Hongqiu Zeng ◽

Yanwei Xie ◽

Guoyin Liu ◽

Yunxie Wei ◽

Wei Hu ◽

...

Keyword(s):

Gene Silencing ◽

Functional Genomics ◽

Transient Expression ◽

Fluorescent Protein ◽

Tobacco Rattle Virus ◽

Mosaic Disease ◽

African Cassava Mosaic Virus ◽

Cassava Mosaic Disease ◽

Virus Induced Gene Silencing ◽

Transient Expression Assay

Agrobacterium-mediated transient expression and virus-induced gene silencing (VIGS) are very useful in functional genomics in plants. However, whether these methods are effective in cassava (Manihot esculenta), one of the most important tropical crops, remains elusive. In this study, we used green fluorescent protein (GFP) and β-glucuronidase (GUS) as reporter genes in a transient expression assay. GFP or GUS could be detected in the infiltrated leaves at 2 days postinfiltration (dpi) and were evidenced by visual GFP and GUS assays, reverse-transcription PCR, and Western blot. In addition, phytoene desaturase (PDS) was used to show the silencing effect in a VIGS system. Both Agrobacterium GV3101 and AGL-1 with tobacco rattle virus (TRV)-MePDS-infiltrated distal leaves showed an albino phenotype at 20 dpi; in particular, the AGL-1-infiltrated plants showed an obvious albino area in the most distal leaves. Moreover, the silencing effect was validated by molecular identification. Notably, compared with the obvious cassava mosaic disease symptom infiltrated by African-cassava-mosaic-virus-based VIGS systems in previous studies, TRV-based VIGS-system-infiltrated cassava plants did not show obvious virus-induced disease symptoms, suggesting a significant advantage. Taken together, these methods could promote functional genomics in cassava.

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Virus-induced gene silencing in the perennial woody Paeonia ostii

PeerJ ◽

10.7717/peerj.7001 ◽

2019 ◽

Vol 7 ◽

pp. e7001

Author(s):

Lihang Xie ◽

Qingyu Zhang ◽

Daoyang Sun ◽

Weizong Yang ◽

Jiayuan Hu ◽

...

Keyword(s):

Gene Silencing ◽

Fluorescent Protein ◽

Molecular Genetic ◽

Functional Characterization ◽

Tobacco Rattle Virus ◽

Vacuum Infiltration ◽

Genomic Research ◽

Virus Induced Gene Silencing ◽

Tree Peony ◽

Medicinal Value

Tree peony is a perennial deciduous shrub with great ornamental and medicinal value. A limitation of its current functional genomic research is the lack of effective molecular genetic tools. Here, the first application of a Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) in the tree peony species Paeonia ostii is presented. Two different approaches, leaf syringe-infiltration and seedling vacuum-infiltration, were utilized for Agrobacterium-mediated inoculation. The vacuum-infiltration was shown to result in a more complete Agrobacterium penetration than syringe-infiltration, and thereby determined as an appropriate inoculation method. The silencing of reporter gene PoPDS encoding phytoene desaturase was achieved in TRV-PoPDS-infected triennial tree peony plantlets, with a typical photobleaching phenotype shown in uppermost newly-sprouted leaves. The endogenous PoPDS transcripts were remarkably down-regulated in VIGS photobleached leaves. Moreover, the green fluorescent protein (GFP) fluorescence was detected in leaves and roots of plants inoculated with TRV-GFP, suggesting the capability of TRV to silence genes in various tissues. Taken together, the data demonstrated that the TRV-based VIGS technique could be adapted for high-throughput functional characterization of genes in tree peony.

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Relation of Replication of Tobacco Mosaic Virus and Potato Virus X to Age in Detached Leaves of Nicotiana tabacum

Journal of Phytopathology ◽

10.1111/j.1439-0434.1981.tb03316.x ◽

1981 ◽

Vol 101 (1) ◽

pp. 16-21

Author(s):

A. V. Reunov ◽

G. D. Reunova ◽

L. A. Lapshina ◽

V. G. Reifman

Keyword(s):

Nicotiana Tabacum ◽

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Potato Virus ◽

Potato Virus X ◽

Detached Leaves

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Characterization of a Brome mosaic virus Strain and Its Use as a Vector for Gene Silencing in Monocotyledonous Hosts

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-19-1229 ◽

2006 ◽

Vol 19 (11) ◽

pp. 1229-1239 ◽

Cited By ~ 133

Author(s):

Xin Shun Ding ◽

William L. Schneider ◽

Srinivasa Rao Chaluvadi ◽

M. A. Rouf Mian ◽

Richard S. Nelson

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Gene Function ◽

Festuca Arundinacea ◽

Systemic Infection ◽

Brome Mosaic Virus ◽

Expression Vectors ◽

Virus Induced Gene Silencing ◽

Dicotyledonous Plants ◽

Virus Expression

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMVA/G (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

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