Olive mild mosaic virus coat protein and p6 are suppressors of RNA silencing and their silencing confers resistance against OMMV

AbstractRNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress it. Here the genome of Olive mild mosaic virus (OMMV) was screened for silencing suppressors using a green fluorescent based transient suppression assay. The full OMMV cDNA and 5 different OMMV open reading frames (ORFs) were cloned into Gateway binary destination vector pK7WG2, transformed into Agrobacterium tumefaciens C58C1 and agroinfiltrated into Nicotiana benthamiana 16C plants. Among all ORFs tested, CP and p6 showed suppressor activity, with CP showing a significant higher activity when compared to p6, yet lower than that of the full OMMV. This suggests that OMMV silencing suppression results from a complementary action of both CP and p6.Such discovery led to the use of those viral suppressors in the development of OMMV resistant plants through pathogen-derived resistance (PDR) based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a highly significant reduction in viral accumulation and symptom attenuation was observed as compared to that seen when each construct was used alone, and to the respective controls, thus showing clear effectiveness against OMMV infection. Data here obtained indicate that the use of both OMMV viral suppressors as transgenes is a very efficient and promising approach to obtain plants resistant to OMMV.ImportanceOMMV silencing suppressors were determined. Among all ORFs tested, CP and p6 showed suppressor activity, with CP showing a significant higher activity when compared to p6, yet lower than that of the full OMMV, suggesting a complementary action of both CP and p6 in silencing suppression.This is the first time that a silencing suppressor was found in a necrovirus and that two independent proteins act as silencing suppressors in a member of the Tombusviridae family.When silencing of both suppressors was achieved, a highly significant reduction in viral accumulation and symptom attenuation was observed as compared to that seen when each was used alone, thus showing clear effectiveness against OMMV infection. A high percentage of resistant plants was obtained (60%), indicating that the use of both OMMV viral suppressors as transgenes is a very efficient and promising approach to obtain plants resistant to OMMV.

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Olive Mild Mosaic Virus Coat Protein and P6 Are Suppressors of RNA Silencing, and Their Silencing Confers Resistance against OMMV

Viruses ◽

10.3390/v10080416 ◽

2018 ◽

Vol 10 (8) ◽

pp. 416 ◽

Cited By ~ 2

Author(s):

Carla Varanda ◽

Patrick Materatski ◽

Maria Campos ◽

Maria Clara ◽

Gustavo Nolasco ◽

...

Keyword(s):

Mosaic Virus ◽

Rna Silencing ◽

Defense Mechanism ◽

Plant Viruses ◽

Open Reading Frames ◽

Mild Mosaic ◽

Suppressor Activity ◽

Mild Mosaic Virus ◽

Virus Coat ◽

Viral Suppressors

RNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. In this study, the genome of the Olive mild mosaic virus (OMMV) was screened for silencing suppressors. The full OMMV cDNA and 5 OMMV open reading frames (ORFs) were cloned into the Gateway binary vector pK7WG2, transformed into Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana 16C plants. CP and p6 showed suppressor activity, with CP showing significantly higher activity than p6, yet activity that was lower than the full OMMV, suggesting a complementary action of CP and p6. These viral suppressors were then used to induce OMMV resistance in plants based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants, which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a significant reduction in viral accumulation and symptom attenuation was observed as compared to those of the controls, as well as to when each construct was used alone, proving them to be effective against OMMV infection. This is the first time that a silencing suppressor was found in a necrovirus, and that two independent proteins act as silencing suppressors in a virus member of the Tombusviridae family.

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Identification of Divergent Isolates of Banana Mild Mosaic Virus and Development of a New Diagnostic Primer to Improve Detection

Pathogens ◽

10.3390/pathogens9121045 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1045

Author(s):

Marwa Hanafi ◽

Rachid Tahzima ◽

Sofiene Ben Kaab ◽

Lucie Tamisier ◽

Nicolas Roux ◽

...

Keyword(s):

Mosaic Virus ◽

High Throughput Sequencing ◽

Plant Viruses ◽

Mild Mosaic ◽

Genome Variability ◽

Molecular Tests ◽

Pcr Products ◽

Reverse Primer ◽

Mild Mosaic Virus ◽

Germplasm Accessions

Banana mild mosaic virus (BanMMV) (Betaflexiviridae, Quinvirinae, unassigned species) is a filamentous virus belonging to the Betaflexiviridae family. It infects Musa spp. with a very wide geographic distribution. The genome variability of plant viruses, including the members of the Betaflexiviridae family, makes their molecular detection by specific primers particularly challenging. During routine indexing of the Musa germplasm accessions, a discrepancy was observed between electron microscopy and immunocapture (IC) reverse transcription (RT) polymerase chain reaction (PCR) test results for one asymptomatic accession. Filamentous viral particles were observed while molecular tests failed to amplify any fragment. The accession underwent high-throughput sequencing and two complete genomes of BanMMV with 75.3% of identity were assembled. Based on these sequences and on the 54 coat protein sequences available from GenBank, a new forward primer, named BanMMV CP9, compatible with Poty1, an oligodT reverse primer already used in diagnostics, was designed. A retrospective analysis of 110 different germplasm accessions from diverse origins was conducted, comparing BanMMCP2 and BanMMV CP9 primers. Of these 110 accessions, 16 tested positive with both BanMMCP2 and BanMMV CP9, 3 were positive with only BanMMCP2 and 2 tested positive with only BanMMV CP9. Otherwise, 89 were negative with the two primers and free of flexuous virions. Sanger sequencing was performed from purified PCR products in order to confirm the amplification of the BanMMV sequence for the five accessions with contrasting results. It is highly recommended to use the two primers successively to improve the inclusiveness of the protocol.

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Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors

Journal of General Virology ◽

10.1099/vir.0.006098-0 ◽

2009 ◽

Vol 90 (2) ◽

pp. 519-525 ◽

Cited By ~ 25

Author(s):

Sandra Martínez-Turiño ◽

Carmen Hernández

Keyword(s):

Coat Protein ◽

Potato Virus ◽

Rna Silencing ◽

Potato Virus X ◽

Plant Viruses ◽

Small Interfering Rnas ◽

Silencing Suppression ◽

Viral Suppressors

Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.

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The Potyvirus Silencing Suppressor Protein VPg Mediates Degradation of SGS3 via Ubiquitination and Autophagy Pathways

Journal of Virology ◽

10.1128/jvi.01478-16 ◽

2016 ◽

Vol 91 (1) ◽

Cited By ~ 57

Author(s):

Xiaofei Cheng ◽

Aiming Wang

Keyword(s):

Mosaic Virus ◽

Rna Silencing ◽

Plant Viruses ◽

Turnip Mosaic Virus ◽

Host Protein ◽

Ubiquitin Proteasome ◽

Cellular Pathways ◽

Viral Suppressors ◽

Interfering Rna ◽

Innate Antiviral Immunity

ABSTRACT RNA silencing is an innate antiviral immunity response of plants and animals. To counteract this host immune response, viruses have evolved an effective strategy to protect themselves by the expression of viral suppressors of RNA silencing (VSRs). Most potyviruses encode two VSRs, helper component-proteinase (HC-Pro) and viral genome-linked protein (VPg). The molecular biology of the former has been well characterized, whereas how VPg exerts its function in the suppression of RNA silencing is yet to be understood. In this study, we show that infection by Turnip mosaic virus (TuMV) causes reduced levels of suppressor of gene silencing 3 (SGS3), a key component of the RNA silencing pathway that functions in double-stranded RNA synthesis for virus-derived small interfering RNA (vsiRNA) production. We also demonstrate that among 11 TuMV-encoded viral proteins, VPg is the only one that interacts with SGS3. We furthermore present evidence that the expression of VPg alone, independent of viral infection, is sufficient to induce the degradation of SGS3 and its intimate partner RNA-dependent RNA polymerase 6 (RDR6). Moreover, we discover that the VPg-mediated degradation of SGS3 occurs via both the 20S ubiquitin-proteasome and autophagy pathways. Taken together, our data suggest a role for VPg-mediated degradation of SGS3 in suppression of silencing by VPg. IMPORTANCE Potyviruses represent the largest group of known plant viruses and cause significant losses of many agriculturally important crops in the world. In order to establish infection, potyviruses must overcome the host antiviral silencing response. A viral protein called VPg has been shown to play a role in this process, but how it works is unclear. In this paper, we found that the VPg protein of Turnip mosaic virus (TuMV), which is a potyvirus, interacts with a host protein named SGS3, a key protein in the RNA silencing pathway. Moreover, this interaction leads to the degradation of SGS3 and its interacting and functional partner RDR6, which is another essential component of the RNA silencing pathway. We also identified the cellular pathways that are recruited for the VPg-mediated degradation of SGS3. Therefore, this work reveals a possible mechanism by which VPg sabotages host antiviral RNA silencing to promote virus infection.

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The cysteine-rich proteins of beet necrotic yellow vein virus and tobacco rattle virus contribute to efficient suppression of silencing in roots

Journal of General Virology ◽

10.1099/vir.0.043513-0 ◽

2012 ◽

Vol 93 (8) ◽

pp. 1841-1850 ◽

Cited By ~ 25

Author(s):

Ida Bagus Andika ◽

Hideki Kondo ◽

Masamichi Nishiguchi ◽

Tetsuo Tamada

Keyword(s):

Mosaic Virus ◽

Potato Virus ◽

Rna Silencing ◽

Tobacco Rattle Virus ◽

Potato Virus X ◽

Plant Viruses ◽

Yellow Vein ◽

Negative Strand ◽

Silencing Suppression ◽

Leaves And Roots

Many plant viruses encode proteins that suppress RNA silencing, but little is known about the activity of silencing suppressors in roots. This study examined differences in the silencing suppression activity of different viruses in leaves and roots of Nicotiana benthamiana plants. Infection by tobacco mosaic virus, potato virus Y and cucumber mosaic virus but not potato virus X (PVX) resulted in strong silencing suppression activity of a transgene in both leaves and roots, whereas infection by beet necrotic yellow vein virus (BNYVV) and tobacco rattle virus (TRV) showed transgene silencing suppression in roots but not in leaves. For most viruses tested, viral negative-strand RNA accumulated at a very low level in roots, compared with considerable levels of positive-strand genomic RNA. Co-inoculation of leaves with PVX and either BNYVV or TRV produced an increase in PVX negative-strand RNA and subgenomic RNA (sgRNA) accumulation in roots. The cysteine-rich proteins (CRPs) BNYVV p14 and TRV 16K showed weak silencing suppression activity in leaves. However, when either of these CRPs was expressed from a PVX vector, there was an enhancement of PVX negative-strand RNA and sgRNA accumulation in roots compared with PVX alone. Such enhancement of PVX sgRNAs was also observed by expression of CRPs of other viruses and the well-known suppressors HC-Pro and p19 but not of the potato mop-top virus p8 CRP. These results indicate that BNYVV- and TRV-encoded CRPs suppress RNA silencing more efficiently in roots than in leaves.

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Variability in the level of RNA silencing suppression caused by triple gene block protein 1 (TGBp1) from various potexviruses during infection

Journal of General Virology ◽

10.1099/vir.0.008243-0 ◽

2009 ◽

Vol 90 (4) ◽

pp. 1014-1024 ◽

Cited By ~ 51

Author(s):

Hiroko Senshu ◽

Johji Ozeki ◽

Ken Komatsu ◽

Masayoshi Hashimoto ◽

Kouji Hatada ◽

...

Keyword(s):

Virus Infection ◽

Rna Silencing ◽

Triple Gene Block ◽

Plant Viruses ◽

Virus Species ◽

Suppressor Activity ◽

Gene Block ◽

Gfp Fluorescence ◽

Silencing Suppression ◽

Triple Gene Block Protein

RNA silencing is an important defence mechanism against virus infection, and many plant viruses encode RNA silencing suppressors as a counter defence. In this study, we analysed the RNA silencing suppression ability of multiple virus species of the genus Potexvirus. Nicotiana benthamiana plants exhibiting RNA silencing of a green fluorescent protein (GFP) transgene showed reversal of GFP fluorescence when systemically infected with potexviruses. However, the degree of GFP fluorescence varied among potexviruses. Agrobacterium-mediated transient expression assay in N. benthamiana leaves demonstrated that the triple gene block protein 1 (TGBp1) encoded by these potexviruses has drastically different levels of silencing suppressor activity, and these differences were directly related to variations in the silencing suppression ability during virus infection. These results suggest that suppressor activities differ even among homologous proteins encoded by viruses of the same genus, and that TGBp1 contributes to the variation in the level of RNA silencing suppression by potexviruses. Moreover, we investigated the effect of TGBp1 encoded by Plantago asiatica mosaic virus (PlAMV), which exhibited a strong suppressor activity, on the accumulation of microRNA, virus genomic RNA and virus-derived small interfering RNAs.

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