scholarly journals An Importin-β-like Protein from Nicotiana benthamiana Interacts with the RNA Silencing Suppressor P1b of the Cucumber Vein Yellowing Virus, Modulating Its Activity

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2406
Author(s):  
Beatriz García ◽  
Leonor Bedoya ◽  
Juan Antonio García ◽  
Bernardo Rodamilans
Keyword(s):  
Viral Infection ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Expression System ◽  
Bimolecular Fluorescence Complementation ◽  
Plum Pox Virus ◽  
Type B ◽  
Relevant Role ◽  
Successful Replication ◽  
Yellowing Virus

During a plant viral infection, host–pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-β-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.

scholarly journals Rice Stripe Mosaic Virus–Encoded P4 Is a Weak Suppressor of Viral RNA Silencing and Is Required for Disease Symptom Development

2020 ◽  
Vol 33 (3) ◽  
pp. 412-422
Author(s):  
Chao Zhang ◽  
Dong Chen ◽  
Guoyi Yang ◽  
Xiyuan Yu ◽  
Jianguo Wu
Keyword(s):  
Mosaic Virus ◽  
Rna Silencing ◽  
Matrix Protein ◽  
Expression System ◽  
Potato Virus X ◽  
Bimolecular Fluorescence Complementation ◽  
Developmental Defects ◽  
The Matrix ◽  
Silencing Pathways ◽  
Viral Suppressors

Viral suppressors of RNA silencing (VSRs) are a cluster of viral proteins that have evolved to counteract eukaryotic antiviral RNA silencing pathways, thereby contributing to viral pathogenicity. In this study, we revealed that the matrix protein P4 encoded by rice stripe mosaic virus (RSMV), which is an emerging cytoplasmic rhabdovirus, is a weak RNA silencing suppressor. By conducting yeast two-hybrid, bimolecular fluorescence complementation, and subcellular colocalization assays, we proved that P4 interacts with the rice endogenous suppressor of gene silencing 3 (OsSGS3). We also determined that P4 overexpression has no effect on OsSGS3 transcription. However, P4 can promote the degradation of OsSGS3 via ubiquitination and autophagy. Additionally, a potato virus X–based expression system was used to confirm that P4 enhances the development of mosaic symptoms on Nicotiana benthamiana leaves by promoting hydrogen peroxide accumulation but not cell death. To verify whether P4 is a pathogenicity factor in host plants, we generated transgenic P4-overexpressing rice plants that exhibited disease-related developmental defects including decreased plant height and excessive tillering. Our data suggest that RSMV-encoded P4 serves as a weak VSR that inhibits antiviral RNA silencing by targeting OsSGS3.

scholarly journals The Cucumber vein yellowing virus Silencing Suppressor P1b Can Functionally Replace HCPro in Plum pox virus Infection in a Host-Specific Manner

2012 ◽  
Vol 25 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Alberto Carbonell ◽  
Gabriela Dujovny ◽  
Juan Antonio García ◽  
Adrian Valli
Keyword(s):  
Rna Silencing ◽  
Deleterious Effect ◽  
Prunus Persica ◽  
Plant Viruses ◽  
Natural Host ◽  
Plum Pox Virus ◽  
Silencing Suppressor ◽  
Specific Manner ◽  
Silencing Suppression ◽  
Yellowing Virus

Plant viruses of the genera Potyvirus and Ipomovirus (Potyviridae family) use unrelated RNA silencing suppressors (RSS) to counteract antiviral RNA silencing responses. HCPro is the RSS of Potyvirus spp., and its activity is enhanced by the upstream P1 protein. Distinctively, the ipomovirus Cucumber vein yellowing virus (CVYV) lacks HCPro but contains two P1 copies in tandem (P1aP1b), the second of which functions as RSS. Using chimeras based on the potyvirus Plum pox virus (PPV), we found that P1b can functionally replace HCPro in potyviral infections of Nicotiana plants. Interestingly, P1a, the CVYV protein homologous to potyviral P1, disrupted the silencing suppression activity of P1b and reduced the infection efficiency of PPV in Nicotiana benthamiana. Testing the influence of RSS in host specificity, we found that a P1b-expressing chimera poorly infected PPV's natural host, Prunus persica. Conversely, P1b conferred on PPV chimeras the ability to replicate locally in cucumber, CVYV's natural host. The deleterious effect of P1a on PPV infection is host dependent, because the P1aP1b-expressing PPV chimera accumulated in cucumber to higher levels than PPV expressing P1b alone. These results demonstrate that a potyvirus can use different RSS, and that particular RSS and upstream P1-like proteins contribute to defining the virus host range.

scholarly journals RNA-Dependent RNA Polymerase 1 from Nicotiana tabacum Suppresses RNA Silencing and Enhances Viral Infection in Nicotiana benthamiana

2010 ◽  
Vol 22 (4) ◽  
pp. 1358-1372 ◽  
Author(s):  
Xiao-Bao Ying ◽  
Li Dong ◽  
Hui Zhu ◽  
Cheng-Guo Duan ◽  
Quan-Sheng Du ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Rna Polymerase ◽  
Viral Infection ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Rna Dependent Rna Polymerase ◽  
Rna Polymerase 1

scholarly journals Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana.

2006 ◽  
Vol 53 (2) ◽  
pp. 289-298 ◽  
Author(s):  
Mateusz Wydro ◽  
Edward Kozubek ◽  
Przemysław Lehmann
Keyword(s):  
Gene Expression ◽  
Industrial Production ◽  
Nicotiana Benthamiana ◽  
Rna Silencing ◽  
Expression System ◽  
Transient Gene Expression ◽  
Maximum Level ◽  
Gene Expression System ◽  
Gfp Gene ◽  
Viral Suppressors

Here we report on a simple and reproducible system of Agrobacterium-mediated transient gene expression assay that utilizes infiltration of young Nicotiana benthamiana leaves. Although some of the phenomena described in this paper have been already reported by other researchers, here we have further developed them. The highest level of transient gfp gene expression was detected in the youngest leaves of N. benthamiana infiltrated with A. tumefaciens strains AGL0 and EHA105 precultured in the presence of 450-600 microM acetosyringone. Although the maximum level of transient gfp gene expression was restricted presumably by RNA silencing, it was completely suppressed in the presence of the viral protein HC-Pro. The transient expression system described here can be used to identify new viral suppressors of RNA silencing, for detailed analysis of unidentified genes and for industrial production of proteins in plants as well.

scholarly journals Heterologous RNA-silencing suppressors from both plant- and animal-infecting viruses support plum pox virus infection

2012 ◽  
Vol 93 (7) ◽  
pp. 1601-1611 ◽  
Author(s):  
Varvara I. Maliogka ◽  
María Calvo ◽  
Alberto Carbonell ◽  
Juan Antonio García ◽  
Adrian Valli
Keyword(s):  
Viral Infection ◽  
Rna Silencing ◽  
Influenza A ◽  
Plum Pox Virus ◽  
Ns1 Protein ◽  
Multifunctional Protein ◽  
Highly Sensitive ◽  
The Family ◽  
Silencing Suppression ◽  
Insight Into

HCPro, the RNA-silencing suppressor (RSS) of viruses belonging to the genus Potyvirus in the family Potyviridae, is a multifunctional protein presumably involved in all essential steps of the viral infection cycle. Recent studies have shown that plum pox potyvirus (PPV) HCPro can be replaced successfully by cucumber vein yellowing ipomovirus P1b, a sequence-unrelated RSS from a virus of the same family. In order to gain insight into the requirement of a particular RSS to establish a successful potyviral infection, we tested the ability of different heterologous RSSs from both plant- and animal-infecting viruses to substitute for HCPro. Making use of engineered PPV chimeras, we show that PPV HCPro can be replaced functionally by some, but not all, unrelated RSSs, including the NS1 protein of the mammal-infecting influenza A virus. Interestingly, the capacity of a particular RSS to replace HCPro does not correlate strictly with its RNA silencing-suppression strength. Altogether, our results suggest that not all suppression strategies are equally suitable for efficient escape of PPV from the RNA-silencing machinery. The approach followed here, based on using PPV chimeras in which an under-consideration RSS substitutes for HCPro, could further help to study the function of diverse RSSs in a ‘highly sensitive’ RNA-silencing context, such as that taking place in plant cells during the process of a viral infection.

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro

2006 ◽  
Vol 80 (20) ◽  
pp. 10055-10063 ◽  
Author(s):  
Adrian Valli ◽  
Ana Montserrat Martín-Hernández ◽  
Juan José López-Moya ◽  
Juan Antonio García
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Serine Proteinase ◽  
Plum Pox Virus ◽  
Coding Region ◽  
Long Distance ◽  
Systemic Spread ◽  
The Family ◽  
Silencing Suppression ◽  
Green Fluorescent

ABSTRACT The P1 protein of viruses of the family Potyviridae is a serine proteinase, which is highly variable in length and sequence, and its role in the virus infection cycle is not clear. One of the proposed activities of P1 is to assist HCPro, the product that viruses of the genus Potyvirus use to counteract antiviral defense mediated by RNA silencing. Indeed, an HCPro-coding region is present in all the genomes of members of the genera Potyvirus, Rymovirus, and Tritimovirus that have been sequenced. However, it was recently reported that a sequence coding for HCPro is lacking in the genome of Cucumber vein yellowing virus (CVYV), a member of the genus Ipomovirus, the fourth monopartite genus of the family. In this study, we provide further evidence that P1 enhances the activity of HCPro in members of the genus Potyvirus and show that it is duplicated in the ipomovirus CVYV. The two CVYV P1 copies are arranged in tandem, and the second copy (P1b) has RNA silencing suppression activity. CVYV P1b suppressed RNA silencing induced either by sense green fluorescent protein (GFP) mRNA or by a GFP inverted repeat RNA, indicating that CVYV P1b acts downstream of the formation of double-stranded RNA. CVYV P1b also suppressed local silencing in agroinfiltrated patches of transgenic Nicotiana benthamiana line 16c and delayed its propagation to the neighboring cells. However, neither the short-distance nor long-distance systemic spread of silencing of the GFP transgene was completely blocked by CVYV P1b. CVYV P1b and P1-HCPro from the potyvirus Plum pox virus showed very similar behaviors in all the assays carried out, suggesting that evolution has found a way to counteract RNA silencing by similar mechanisms using very different proteins in viruses of the same family.

Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection

2016 ◽  
Vol 211 ◽  
pp. 38-45 ◽  
Author(s):  
Paolo Margaria ◽  
Laura Miozzi ◽  
Marina Ciuffo ◽  
Cristina Rosa ◽  
Michael J. Axtell ◽  
...  
Keyword(s):  
Viral Infection ◽  
Solanum Lycopersicum ◽  
Small Rna ◽  
Nicotiana Benthamiana
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