Article

Expression of the Rice Yellow Mottle Virus P1 Proteinin Vitroandin Vivoand Its Involvement in Virus Spread

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Abstract

Rice yellow mottle sobemovirus (RYMV) is responsible for the yellow mottle disease on rice in Africa. The expression and function of the protein P1 (17.8 kDa) encoded by the first open reading frame (ORF) of RYMV was investigated. Using an antibody raised against purified P1, two proteins with apparent molecular masses of 18 and 19 kDa were identified inin vitrotranslation reactions of transcripts of the full-length cDNA of RYMV. Likewise, gene products with similar molecular mass were detected in inoculated and systemically infected rice leaves and in infected rice protoplasts. A mutant from which ORF1 nucleotides 88 to 547 were deleted and a frameshift mutant that resulted in truncation of 83 amino acids from the C terminus of P1 were incapable of replicating in protoplasts. In contrast, a mutant that does not express P1 due to a mutation at the initiation codon replicated efficiently in protoplasts but at a reduced level (about 0.5- to 2-fold less) compared to replication of wild-type RNA. None of these mutants caused systemic infection in rice plants. Transgenic rice plants that express P1 complemented the initiation codon mutant, but not the deletion mutants, and produced systemic infection. These experiments demonstrate that P1 of RYMV is dispensible for virus replication, although nucleotide deletions or additions in ORF1 are apparently lethal for virus replication. Furthermore, P1 of RYMV is required for the infection of plants and is important for virus spread.

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... The genome of sobemoviruses consists of a 4-4,5 kb polycistronic positive single-stranded RNA, in which open reading frame 1 (ORF1) encodes P1 protein that has been characterized as suppressor of posttranscriptional gene silencing (Fusaro et al., 2012;Sarmiento et al., 2007;Siré et al., 2008). Furthermore, even though P1 does not seem to be needed for replication of sobemoviruses, it is necessary for systemic infection (Bonneau et al., 1998;Meier et al., 2006;Sivakumaran et al., 1998) and has therefore been suggested to serve as viral movement protein (MP). P1 of Sesbania mosaic sobemovirus (SeMV) interacts with the genomebound VPg and P10 in vitro indicating there is active transport of viral RNA complex facilitated by P1 acting as a MP and supported by hydrolysis of ATP by P10 (Chowdhury and Savithri, 2011). ...
... Sobemovirus P1 is needed for systemic infection, but not for replication, as previously described in several studies. For example, Southern bean mosaic virus (SBMV) P1 initiation codon mutants can replicate in host protoplasts (Sivakumaran et al., 1998) and RYMV P1 initiation codon mutant replicates in rice protoplasts, but not in rice plants whereas P1 deletion mutant fails to replicate at all (Bonneau et al., 1998). Transgenic rice plants expressing exogenous P1 complement the systemic movement of the RYMV P1 initiation codon mutant (Bonneau et al., 1998). ...
... For example, Southern bean mosaic virus (SBMV) P1 initiation codon mutants can replicate in host protoplasts (Sivakumaran et al., 1998) and RYMV P1 initiation codon mutant replicates in rice protoplasts, but not in rice plants whereas P1 deletion mutant fails to replicate at all (Bonneau et al., 1998). Transgenic rice plants expressing exogenous P1 complement the systemic movement of the RYMV P1 initiation codon mutant (Bonneau et al., 1998). Controversially, in this study we demonstrate that mutant RYMV without P1, can still replicate in rice plants. ...
Article
Sobemovirus P1 protein, characterized previously as a suppressor of posttranscriptional gene silencing, is required for systemic virus spread and infection in plants. Mutations in the ORF1 initiation codon do not affect viral replication indicating P1 is not necessary for this process. Wild type, recombinant and P1 deletion mutants of Cocksfoot mottle virus and Rice yellow mottle virus were used to infect oat, rice, wheat, barley, Arabidopsis thaliana and Nicotiana benthamiana plants. Wild type RYMV, RYMV without P1 and RYMV with CfMV P1 were detected in inoculated leaves of all tested plant species. We found that RYMV does not need P1 for replication and for local movement neither in host nor non-host species tested in this study. However, it is crucial for successful systemic spread of the virus in its host plant rice. Moreover, adding CfMV P1 into RYMV genome did not help it to overcome restriction to the inoculated leaf.
... The genomic organization is related to the Cocksfoot mottle virus (CfMV), and includes 5 ORFs (Ling et al, 2013;Sõmera et al, 2015). The most emblematic multifunctional protein in RYMV is the P1 protein encoded by ORF1 of the genome, this protein is a small Cysteine-rich protein of 18 kDa, which is required for virus movement (Bonneau et al, 1998;Siré et al, 2008), suspected in viral replication (Bonneau et al., 1998), and described as a suppressor of RNA silencing directed against exogenous genes in plant reporter systems (Voinnet et al, 1999;Siré et al, 2008;Lacombe et al, 2010;Fusaro et al, 2012). The P1 protein presents the highest diversity among the other viral proteins with 17,8% of amino acid sequence divergence (Siré et al, 2008;Sérémé et al, 2014). ...
... The genomic organization is related to the Cocksfoot mottle virus (CfMV), and includes 5 ORFs (Ling et al, 2013;Sõmera et al, 2015). The most emblematic multifunctional protein in RYMV is the P1 protein encoded by ORF1 of the genome, this protein is a small Cysteine-rich protein of 18 kDa, which is required for virus movement (Bonneau et al, 1998;Siré et al, 2008), suspected in viral replication (Bonneau et al., 1998), and described as a suppressor of RNA silencing directed against exogenous genes in plant reporter systems (Voinnet et al, 1999;Siré et al, 2008;Lacombe et al, 2010;Fusaro et al, 2012). The P1 protein presents the highest diversity among the other viral proteins with 17,8% of amino acid sequence divergence (Siré et al, 2008;Sérémé et al, 2014). ...
... To our knowledge, we here report the first 3D structure revealing ZnF features within a plant VSR protein, with very few similarities with other proteins in the databases. P1 protein is essential for RYMV systemic spread in rice tissues (Bonneau et al, 1998;Siré et al, 2008) and also plays an important role in viral replication despite being not strictly essential in this early step of RYMV infection (Bonneau et al, 1998;Nummert et al, 2017). Indeed, a mutation of P1 translation initiation codon rendering P1 undetectable in infected rice tissues almost abolished RYMV particles accumulation in infected and systemic leaves. ...
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The Rice Yellow Mottle sobemovirus (RYMV) belongs to the most damaging pathogens devastating rice fields in Africa. P1, a key protein for RYMV, was reported as a potent RNAi suppressor counteracting RNA silencing in plant reporter systems. Here we describe the complete 3D structure and dynamics of P1. Its N-terminal region contains ZnF1, a structural CCCC-type zinc finger strongly affine to zinc and a prominent short helix, rendering this region poorly amenable to structural changes. P1 C-terminal region contains ZnF2, an atypical HCHC-type ZnF that does not belong to any existing class of Zn finger proteins. ZnF2 appeared much less affine to zinc and more sensitive to oxidizing environments than ZnF1, and may serve as a sensor of plant redox status. We also identified key residues essential for RYMV infectivity and spread in rice tissues through their participation in P1 oligomerization and folding. Altogether, our results provide the first complete structure of a rice antiviral silencing suppressor and highlight P1 structural properties that may serve RYMV functions to infect and invade the host plant.
... This protein is involved in viral accumulation and posttranscriptional gene silencing by suppressing the mechanisms of host RNA silencing Lacombe et al., 2010). P1 is dispensable for the local movement and replication of RYMV but is essential for systemic infection (Brugidou et al., 1995;Bonneau et al., 1998;Nummert et al., 2017). At the 5' terminus of ORF1 is a viral genome-linked protein (VPg) instead of a cap while the 3' end of the viral genome is not polyadenylated (Hull, 1977;Yassi et al., 1994). ...
... ORF3 encodes the viral coat protein (CP) translated from the sub genomic RNA. The coat protein is responsible for cell-to-cell movement, virus packaging, and stability (Yassi et al., 1994;Bonneau et al., 1998;Opalka et al., 1998). ...
Article
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Rice is the main food crop for people in low- and lower-middle-income countries in Asia and sub-Saharan Africa (SSA). Since 1982, there has been a significant increase in the demand for rice in SSA, and its growing importance is reflected in the national strategic food security plans of several countries in the region. However, several abiotic and biotic factors undermine efforts to meet this demand. Rice yellow mottle virus (RYMV) caused by Solemoviridae is a major biotic factor affecting rice production and continues to be an important pathogen in SSA. To date, six pathogenic strains have been reported. RYMV infects rice plants through wounds and rice feeding vectors. Once inside the plant cells, viral genome-linked protein is required to bind to the rice translation initiation factor [eIF(iso)4G1] for a compatible interaction. The development of resistant cultivars that can interrupt this interaction is the most effective method to manage this disease. Three resistance genes are recognized to limit RYMV virulence in rice, some of which have nonsynonymous single mutations or short deletions in the core domain of eIF(iso)4G1 that impair viral host interaction. However, deployment of these resistance genes using conventional methods has proved slow and tedious. Molecular approaches are expected to be an alternative to facilitate gene introgression and/or pyramiding and rapid deployment of these resistance genes into elite cultivars. In this review, we summarize the knowledge on molecular genetics of RYMV-rice interaction, with emphasis on host plant resistance. In addition, we provide strategies for sustainable utilization of the novel resistant sources. This knowledge is expected to guide breeding programs in the development and deployment of RYMV resistant rice varieties.
... The CP has been shown in various virus species to be important for vector-virus interactions151617. Mutation studies with the P1 proteins of the sobemoviruses Rice yellow mottle virus (RYMV), Cocksfoot mottle virus (CoMV) and Southern cowpea mosaic virus (SCPMV) have shown that P1 is required for cell to cell movement and systemic translocation of these viruses, despite no sequence similarity to one another or to other known P1 movement proteins181920. The VTMoV-P1 ORF is annotated on the basis of its genomic location, which is similar to that of P1 genes of other sobemoviruses. Sequences from regions containing the CP and P1 ORFs of two isolates of VTMoV were determined after 24 months of continuous passage by mechanical or mirid transmission. ...
... The sequence of the VTMoV–P1 protein showed no significant similarity to any two-or three-dimensional structures of previously characterized proteins (data not shown) and the function of this protein is yet to be determined. Nevertheless , mutation studies with the P1 genes (present at a similar genomic location to the VTMoV P1 gene) of other sobemoviruses, RYMV, SCPMV, and CoMV, show that each of these proteins enable cell to cell movement and systemic spread of these viruses181920, despite a lack of sequence similarity. Therefore, diversifying selective pressure for the P1 protein may be consistent with adaptation of the virus towards achieving efficient movement through the experimental host (N. ...
Article
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Genomic mutation in plant viruses of cultivated plants is known to be influenced by virus, host and vector, but the factors influencing mutation in viruses of native plants in natural ecosystems are rarely studied. We have tested the effect of mode of transmission on mutation in Velvet tobacco mottle virus (VTMoV), a mirid-vectored sobemovirus associated with Nicotiana velutina, an Australian native xerophyte growing in a region isolated from anthropogenic influences. Two variants of VTMoV (K1 and R17) were passaged monthly in the alternative experimental plant host, N. clevelandii, for 2 years, either by mechanical inoculation or by transmission with the mirid Cyrtopeltis nicotianae. Sequence variations were scored after 24 passages in regions of the genome containing the open reading frames (ORFs) for the P1 and coat protein (CP). The mean mutation rate was 6.83 × 10−4 nt/site year, but a higher overall rate was observed for the K1 (satellite −) than the R17 (satellite +) variant. The P1 ORF showed a higher frequency of non-synonymous mutations than the CP. No clear association was found between either mutation site or mutation rate and the mode of transmission, indicating that obligatory mirid transmission had not exerted a specific bottle-neck effect on sequence variation during the experimental time frame. Failure to detect any sequence motifs linked to vector transmission suggests that a specific capsid-stylet interaction is not required for transmission by mirids.
... No. AY004291) is conserved among SeMV, SCPMV and SBMV-Ark, the functional significance of this motif is not known. Recent reports demonstrate that the ORF1 is translationally active in SCPMV, RYMV and CfMV [4, 23, 27]. The involvement of the ORF1 product in virus spread has been recently demonstrated by Bonneau et al. [4]. ...
... Recent reports demonstrate that the ORF1 is translationally active in SCPMV, RYMV and CfMV [4, 23, 27]. The involvement of the ORF1 product in virus spread has been recently demonstrated by Bonneau et al. [4]. It has also been demonstrated by mutational analysis of full-length cDNA clone of SCPMV that the cell to cell movement of the virus requires ORF1, ORF3 and CP gene products [24]. ...
Article
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The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5' non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M(r) 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326-337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a--1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via--1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters.
... mutation in the ORF1 initiation codon of RYMV ORF1, replicates efficiently in rice protoplasts, but is unable to systemically infect rice plants. In transgenic plants that express P1 in trans, the defective mutant virus recovered its ability for systemic infection (Bonneau et al., 1998). These results indicated that P1 is the RYMV MP. ...
... But the complexity of the unusual genome organizations and replication strategies for many rice viruses have so far prevented the development of infectious clone systems. To date, only two infectious clone systems of RTBV and RYMV have been developed Bonneau et al., 1998). Lack of reverse genetic systems for the rice viruses has made it impossible to use standard mutagenesis methods for functional studies of their encoded proteins that are involved in viral cell-to-cell movement. ...
Article
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To adapt to plants as hosts, plant viruses have evolutionally needed the capacity to modify the host plasmodesmata (PD) that connect adjacent cells. Plant viruses have acquired one or more genes that encode movement proteins (MPs), which facilitate the cell-to-cell movement of infectious virus entities through PD to adjacent cells. Because of the diversity in their genome organization and in their coding sequences, rice viruses may each have a distinct cell-to-cell movement strategy. The complexity of their unusual genome organizations and replication strategies has so far hampered reverse genetic research on their genome in efforts to investigate virally encoded proteins that are involved in viral movement. However, the MP of a particular virus can complement defects in cell-to-cell movement of other distantly related or even unrelated viruses. Trans-complementation experiments using a combination of a movement-defective virus and viral proteins of interest to identify MPs of several rice viruses have recently been successful. In this article, we reviewed recent research that has advanced our understanding of cell-to-cell movement of rice viruses.
... The sgRNA has been detected both in sobemovirus particles and in infected tissues [65,75,[78][79][80][81][82]. CfMV has also been reported to encapsulate at least five different viral defective interfering RNA molecules (DI RNA) corresponding to 35-40 nt of the 5 terminus linked to 850-950 nt of the 3 terminus [83]. ...
... The expression of P1 of RYMV, SCPMV and CfMV is required for systemic infection [49,81,97]. According to this, P1 was suspected to serve as viral movement protein (MP). ...
Article
Full-text available
The genus Sobemovirus, unassigned to any family, consists of viruses with single-stranded plus-oriented single-component RNA genomes and small icosahedral particles. Currently, 14 species within the genus have been recognized by the International Committee on Taxonomy of Viruses (ICTV) but several new species are to be recognized in the near future. Sobemovirus genomes are compact with a conserved structure of open reading frames and with short untranslated regions. Several sobemoviruses are important pathogens. Moreover, over the last decade sobemoviruses have become important model systems to study plant virus evolution. In the current review we give an overview of the structure and expression of sobemovirus genomes, processing and functions of individual proteins, particle structure, pathology and phylogenesis of sobemoviruses as well as of satellite RNAs present together with these viruses. Based on a phylogenetic analysis we propose that a new family Sobemoviridae should be recognized including the genera Sobemovirus and Polemovirus. Finally, we outline the future perspectives and needs for the research focusing on sobemoviruses.
... The SYCMV genome size is approximately 4.2 kb including 5 -and 3 -untranslated regions (UTRs; 77 nt and 122 nt, respectively) and four putative open reading frames (ORFs), ORF1 (78-566 nt), polyprotein ORF2a/b (524-2248 nt and1852-3417 nt) and coat protein (3227-4030 nt) (Nam et al., 2012). SYCMV ORF1 is considered to be required for virus cell-to-cell movement in systemic infection and to act as a viral suppressor of RNA silencing (VSR), such as those of Cocksfoot mottle virus (CfMV) and Rice yellow mottle virus (RYMV) (Bonneau et al., 1998;Meier et al., 2006;Sarmiento et al., 2007). SYCMV ORF2, which is divided into ORF2a and ORF2b, encodes polyproteins including serine protease (Pro), viral protein genome-linked (VPg), and RNA-dependent RNA polymerase (RdRp). ...
... In this study, SYCMV-derived vectors were developed for VIGS and heterologous protein expression in host legume plants (Fig. 1). SYCMV ORF1 (P1 protein) is considered to be required for the suppression of viral RNA silencing and for virus cell-to-cell movement in systemic infection, similar to other sobemoviruses such as CfMV and RYMV (Bonneau et al., 1998;Meier et al., 2006;Sarmiento et al., 2007). The P1 protein of SYCMV might act as VSR, similar to proteins such as P19 of genus Tombusvirus, P25 of genus Potexvirus, helper component proteinase of genus Potyvirus, and 2b of genus Cucumovirus (Siddiqui et al., 2008). ...
Article
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A new vector using Soybean yellow common mosaic virus (SYCMV) was constructed for gene function study or heterologous protein expression in soybeans. The in vitro transcript with a 5' cap analog m7GpppG from an SYCMV full-length infectious vector driven by a T7 promoter infected soybeans (pSYCMVT7-full). The symptoms observed in the soybeans infected with either the sap from SYCMV-infected leaves or pSYCMVT7-full were indistinguishable, suggesting that the vector exhibits equivalent biological activity as the virus itself. To utilize the vector further, a DNA-based vector driven by the Cauliflower mosaic virus (CaMV) 35S promoter was constructed. The complete sequence of the SYCMV genome was inserted into a binary vector flanked by a CaMV 35S promoter at the 5' terminus of the SYCMV genome and a cis-cleaving ribozyme sequence followed by a nopaline synthase terminator at the 3' terminus of the SYCMV genome (pSYCMV-full). The SYCMV-derived vector was tested for use as a virus-induced gene silencing (VIGS) vector for the functional analysis of soybean genes. VIGS constructs containing either a fragment of the Phytoene desaturase (PDS) gene (pSYCMV-PDS1) or a fragment of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RbcS) gene (pSYCMV-RbcS2) were constructed. Plants infiltrated with each vector using the Agrobacterium-mediated inoculation method exhibited distinct symptoms, such as photo-bleaching in plants infiltrated with pSYCMV-PDS1 and yellow or pale green coloring in plants infiltrated with pSYCMV-RbcS2. In addition, down-regulation of the transcripts of the two target genes was confirmed via northern blot analysis. Particle bombardment and direct plasmid DNA rubbing were also confirmed as alternative inoculation methods. To determine if the SYCMV vector can be used for the expression of heterologous proteins in soybean plants, the vector encoding amino acids 135-160 of VP1 of Foot-and-mouth disease virus (FMDV) serotype O1 Campos (O1C) was constructed (pSYCMV-FMDV). Plants infiltrated with pSYCMV-FMDV were only detected via western blotting using the O1C antibody. Based on these results, we propose that the SYCMV-derived vector can be used for gene function study or expression of useful heterologous proteins in soybeans.
... The sgRNA has been detected both in sobemovirus particles and in infected tissues [65,75,[78][79][80][81][82]. CfMV has also been reported to encapsulate at least five different viral defective interfering RNA molecules (DI RNA) corresponding to 35-40 nt of the 5 terminus linked to 850-950 nt of the 3 terminus [83]. ...
... The expression of P1 of RYMV, SCPMV and CfMV is required for systemic infection [49,81,97]. According to this, P1 was suspected to serve as viral movement protein (MP). ...
Chapter
The genus Sobemovirus, unassigned to any family, consists of viruses with single-stranded plus-oriented single-component RNA genomes and small icosahedral particles. Currently, 14 species within the genus have been recognized by the International Committee on Taxonomy of Viruses (ICTV) but several new species are to be recognized in the near future. Sobemovirus genomes are compact with a conserved structure of open reading frames and with short untranslated regions. Several sobemoviruses are important pathogens. Moreover, over the last decade sobemoviruses have become important model systems to study plant virus evolution. In the current review we give an overview of the structure and expression of sobemovirus genomes, processing and functions of individual proteins, particle structure, pathology and phylogenesis of sobemoviruses as well as of satellite RNAs present together with these viruses. Based on a phylogenetic analysis we propose that a new family Sobemoviridae should be recognized including the genera Sobemovirus and Polemovirus. Finally, we outline the future perspectives and needs for the research focusing on sobemoviruses.
... That P1 could bind zinc atoms was initially suggested by Bonneau et al., 12 but had not been demonstrated until now, due to the difficulty in obtaining soluble protein for biochemical analyses. The two key modifications of the procedure used by Bonneau et al. were first to extract and purify rP1 in the presence of DTT and second to supply ZnSO 4 during bacterial growth all along the rP1 biosynthesis process. ...
... The two key modifications of the procedure used by Bonneau et al. were first to extract and purify rP1 in the presence of DTT and second to supply ZnSO 4 during bacterial growth all along the rP1 biosynthesis process. 12 These modifications allowed the production of high amounts of soluble rP1 and provided a first indication of the key structural role of zinc in P1. By MS, we next established that P1 binds two zinc atoms. ...
... Rice yellow mottle virus (RYMV), of the Sobemovirus genus (Truve & Fargette, 2011), has a high natural molecular diversity (Pinel-Galzi et al., 2009). Its first ORF encodes a zinc finger protein with redox-dependent flexibility (Gillet et al., 2013), named P1, which has been described as a multifunctional protein that participates in virus spread and replication (Bonneau et al., 1998). In addition, P1 proteins originating from different RYMV isolates displayed a wide range of ability to suppress RNA silencing, from strong to weak RNA silencing suppression, as in the case of the Tanzanian Tz3 (P1Tz3) and Madagascar Mg1 (P1Mg1) isolates, respectively (Voinnet et al., 1999;Siré et al., 2008). ...
Article
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RNA silencing is a eukaryotic mechanism for RNA-based gene regulation that plays an essential role in diverse biological processes, such as defence against viral infections. The P1 of rice yellow mottle virus (RYMV) is a movement protein and displays RNA silencing suppression activity with variable efficiency, depending on the origin of the isolates. In this study, the positive selection pressure acting on the P1 protein gene was assessed. A site-by-site analysis of the d(N)/d(S) ratio was performed and 18 positively selected sites were identified. Four of these were mutated, and the ability to suppress RNA silencing was evaluated for the resulting mutants in a transient expression assay. All mutations affected quantitatively RNA silencing suppression, one caused a significant decrease in the activity and three significantly increased it. This work demonstrates, for what is to the best of our knowledge the first time, that the RYMV gene encoding the P1 RNA silencing suppressor is under adaptive evolution.
... The complete genome sequences of RYMV (Ngon A Yassi et al., 1994) and SCPMV ( Wu et al., 1987) reveal four open reading frames designated ORFs 1 to 4, predicted to encode at least four proteins. ORF1 of RYMV encodes a protein of 17.8 kDa that is involved in virus movement ( Bonneau et al., 1998). The functions of the ORF1 and ORF3 proteins remain unknown. ...
Article
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Rice yellow mottle virus (RYMV) and southern bean mosaic virus, cowpea strain (SCPMV) are members of the Sobemovirus genus of RNA-containing viruses. We used electron cryo-microscopy (cryo-EM) and icosahedral image analysis to examine the native structures of these two viruses at 25 Å resolution. Both viruses have a single tightly packed capsid layer with 180 subunits assembled on a T=3 icosahedral lattice. Distinctive crown-like pentamers emanate from the 12 5-fold axes of symmetry. The exterior face of SCPMV displays deep valleys along the 2-fold axes and protrusions at the quasi-3-fold axes. While having a similar topography, the surface of RYMV is comparatively smooth. Two concentric shells of density reside beneath the capsid layer of RYMV and SCPMV, which we interpret as ordered regions of genomic RNA. In the presence of divalent cations, SCPMV particles swell and fracture, whereas the expanded form of RYMV is stable. We previously proposed that the cell-to-cell movement of RYMV in xylem involves chelation of Ca2+ from pit membranes of infected cells, thereby stabilizing the capsid shells and allowing a pathway for spread of RYMV through destabilized membranes. In the context of this model, we propose that the expanded form of RYMV is an intermediate in the in vivo assembly of virions.
... Sobemoviruses encode 3 open reading frames (Fig. 1a). The 59 proximal ORF (ORF 1) codes for the movement protein (MP) which is involved in cell to cell movement of the virus and is a suppressor of post transcriptional gene silencing [4,5,6,7,8,9]. The 39 proximal ORF (ORF 3) is translated into coat protein (CP) from a subgenomic RNA (sgRNA) generated during replication (Fig. 1a). ...
Article
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Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.
... The 5Ј-terminal ORF1 encodes a protein (P1) that has been shown to be involved in the cell-to-cell movement of SCPMV . The P1 protein of rice yellow mottle sobemovirus (RYMV) is also involved in movement (Bonneau et al., 1998). The internal ORF2 encodes the P2 polyprotein of about 105 kDa (Wu et al., 1987;Othman and Hull, 1995;Lee and Anderson, 1998). ...
Article
Vigna unguiculata (cowpea) and Phaseolus vulgaris (common bean) are permissive hosts for southern cowpea mosaic virus (SCPMV) and southern bean mosaic virus (SBMV), respectively. Neither of these two sobemoviruses systemically infects the permissive host of the other. Although bean cells are permissive for SCPMV RNA synthesis, they do not support the assembly of this virus. Thus, the host range restriction of SCPMV in bean may occur at the level of movement and may involve the inability of SCPMV to assemble in this host. In this study, it was demonstrated that SCPMV accumulates in an encapsidated form in the inoculated and systemic leaves of bean plants following coinoculation with SBMV. No evidence was observed that the SCPMV that accumulated in coinoculated bean plants had an altered host range relative to wild-type SCPMV. These results suggested that SBMV complemented the host range restriction of SCPMV in bean. Additional experiments demonstrated that cowpea protoplasts are permissive for SBMV RNA synthesis and assembly. It was concluded from these results that the host range restriction of SBMV in cowpea occurs at the level of movement. In mixed infections of cowpea with SCPMV and SBMV, the latter was recovered from the inoculated but not the systemic leaves. Its recovery from the inoculated leaves, however, was not dependent on the presence of SCPMV in the inoculum. From these results, it was concluded that SCPMV did not complement the host range restriction of SBMV in cowpea.
... Except for ORFx that has been characterized recently (Ling et al., 2013), functions of the four others are well known. ORF1 encodes for P1 protein that has been initially described to be involved in virus movement (Bonneau et al., 1998) and then in RNA silencing suppression (Voinnet et al., 1999, Siré et al., 2008Lacombe et al., 2010). ORF2a and 2b encode for polyproteins that are processed to produce the RNA dependent RNA Polymerase (RdRP), the VPg and a serine protease. ...
... BYV causes pathogenicity by inhibiting micro-RNA pathway [51], but expression of P21 protein of BYV did not cause pathogenicity in the present report as well. The P1 protein of rice yellow mottle virus (RYMV) and 2b protein of peanut stunt virus (PSV) are also thought to be determinants of pathogenicity [2,30,41,60]. In contrast, ALSV vectors expressing P1 or 2b did not show any symptoms and did not increase accumulation of virus. ...
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Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs) from eight plant viruses: P19 of carnation Italian ring spot virus (tombusvirus), 2b of peanut stunt virus (cucumovirus), NSs of tomato spotted wilt virus (tospovirus), HC-Pro of bean yellow mosaic virus (potyvirus), γb of barley stripe mosaic virus (hordeivirus), P15 of peanut clump virus (pecluvirus), P1 of rice yellow mottle virus (sobemovirus), or P21 of beet yellows virus (closterovirus). These vectors were inoculated to Nicotiana benthamiana to investigate the effects of RSSs on the virulence and accumulation of ALSV. Among the vectors, ALSV expressing NSs (ALSV-NSs) developed severe mosaic symptoms in newly developed leaves followed by plant death. Infection of ALSV-γb induced characteristic concentric ringspot symptoms on leaves, and plants infected with ALSV-HC-Pro showed mosaic and dwarf symptoms. Infection of the other five ALSV vectors did not show symptoms. ELISA and immunoblot assay indicated that virus titer increased in leaves infected with ALSV-NSs, γb, HC-Pro, or P19. RT-qPCR indicated that the amount of ALSV in plants infected with ALSV-NSs was increased by approximately 45 times compared with that of wtALSV without expression of any RSS. When ALSV-P19, NSs, or HC-Pro was inoculated to Cucumis sativus plants, none of these ALSV vectors induced symptoms, but accumulation of ALSV in plants infected with ALSV-NSs was increased, suggesting that functions of RSSs on virulence and accumulation of ALSV depend on host species.
... Le RYMV appartient au genre des Sobemovirus (Hull and Fargette, 2005). Bonneau et al., 1998; Lacombe et al., 2010; Siré et al., 2008). Cette protéine a également des points communs avec les protéines doigt-de-zinc (Gillet et al., 2013). ...
Thesis
Le virus de la panachure jaune (RYMV) est une contrainte majeure pour la riziculture en Afrique. Deux gènes contrôlant des résistances récessives ont précédemment été décrits : RYMV1, qui code pour eIF(iso)4G1, un facteur d’initiation de la traduction et RYMV2, qui code pour CPR5-1, un probable composant du pore nucléaire impliqué dans la régulation des mécanismes de défense. Cependant, la capacité du virus à contourner ces résistances justifie la caractérisation de sources de résistance originales, présentes dans les espèces de riz africain Oryza glaberrima et Oryza barthii. Trois approches complémentaires ont été mises en oeuvre afin d’identifier les facteurs génétiques contrôlant ces résistances. Une approche de cartographie génétique dans des populations bi-parentales a permis l’identification du gène RYMV3, contrôlant la résistance de l’accession Tog5307 et sans doute également de l’accession Tog5672. Il s’agit de la première résistance dominante identifiée dans le pathosystème riz/RYMV. RYMV3 a été cartographié dans un intervalle de 15 kb où deux gènes sont annotés, dont un gène NB-LRR. Des comparaisons de séquences entre accessions résistantes et accessions sensibles suggèrent que le polymorphisme responsable de la résistance est une mutation ponctuelle dans le domaine LRR du gène NB-LRR. Les deux autres approches ont reposé sur l’exploitation de données de séquençage Illumina de 163 accessions O. glaberrima et 84 accessions O. barthii. Les accessions O. glaberrima ont été phénotypées à la fois pour la résistance élevée et pour la résistance partielle au RYMV, et une partie des accessions O. barthii a été évaluée pour la résistance élevée. L’analyse de la variabilité allélique aux trois gènes majeurs de résistance a permis l’identification d’un probable nouvel allèle de résistance à RYMV1 et de six à RYMV2. Ces allèles sont actuellement en cours de validation. D’autre part, une approche de génétique d’association réalisée sur 125 accessions O. glaberrima a mis en évidence deux QTL de résistance partielle sur les chromosomes 6 et 11, dont l’un colocalise, en première approche, avec le gène RYMV3. Ce travail a ainsi permis l’identification d’un gène majeur, de deux QTL et de nouveaux allèles de résistance qui contribuent à une meilleure compréhension des interactions riz/RYMV et sont utilisables en sélection pour améliorer la durabilité des variétés résistantes.
... ORFs 1, 2a and 2b are translated from the genomic RNA while ORF3, which encodes the coat protein, is translated from a subgenomic RNA. ORF1 encodes P1, a protein of around 12 to 24 kDa (depending on species) that appears to play a role in systemic silencing (Voinnet et al., 1999;Sarmiento et al., 2007;Siré et al., 2008;Lacombe et al., 2010) and virus movement (Bonneau et al., 1998;Sivakumaran et al., 1998;Meier et al., 2006;Chowdhury and Savithri, 2011). The P1 amino acid sequences from different sobemovirus species are generally highly divergent . ...
Article
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The sobemoviruses have one of the smallest of all known RNA virus genomes. ORF1 encodes P1 which plays a role in suppression of silencing and virus movement, ORFs 2a and 2b encode the replicational polyproteins P2a and P2ab, and ORF3 encodes the coat protein. Translation of ORF2a from the genomic RNA is dependent on a leaky scanning mechanism. We report the presence of an additional ORF (ORFx), conserved in all sobemoviruses. ORFx overlaps the 5' end of ORF2a in the +2 reading frame and also extends some distance upstream of ORF2a. ORFx lacks an AUG initiation codon and its expression is predicted to depend on low level initiation at near-cognate non-AUG codons, such as CUG, by a proportion of the ribosomes that are scanning the region between the ORF1 and ORF2a initiation codons. Mutations that disrupt translation of ORFx in turnip rosette virus prevent the establishment of infection.
... RYMV's RNA consists of 4450 nucleotides harbouring four open reading frames (ORFs) (Ngon et al. 1994;Yassi et al. 1994) with ORFs 2, 3, and 4 overlapping. The ORF 1 extends from nucleotide 80 to 553 and is responsible for coding protein with 157 amino acids and a size of 17.8 K (Bonneau et al. 1998); ORF 2 begins from nucleotide 608 to 3607, coding for a polyprotein containing 999 amino acids and a size of 110.7 K; ORF 3 is found in the ORF2 extending from nucleotide 3447 to 4166 and codes for a 26 K protein (Yassi et al. 1994). The ORF 1 plays a vital role in the infection of host plants. ...
Article
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Rice yellow mottle genus Sobemovirus is an important disease of rice worldwide. Although the virus attacks both upland and lowland rice cultivars, the latter are worse infected. The disease is spread by insect vectors belonging to the families Chrysomelidae, Coccinelidae, and Tettigonidae. It is also transmitted by domestic cattle (Bos spp.), donkeys (Asinus spp.), grass rats (Arvicanthis niloticus), weed species of the family Poaceae, debris, irrigation water and through mechanical inoculation. Six strains of the virus have been reported so far. Rice yellow mottle virus (RYMV) incidence ranges between 5 and 100% and yield losses vary from 20 to 100%. Effective management includes the use of resistant cultivars, control of insect vectors, good sanitation and integrated pest and production management. This paper reviews the current status of the virus, with emphasis on its geographical distribution, characteristics, strains, symptomatology, diagnosis, transmission, host range and management strategies.
... The function of the P1 protein has been investigated through expression of wild type and mutant P1 in vitro and in vivo. P1 is required for infection of the plant and for virus spread (16) and also in suppressing virus-induced gene silencing (VIGS) (69). The CP gene is required for full infectivity in rice plants since it plays a role in cell-to-cell, long distance movement and systemic infection in O. sativa. ...
Article
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Three cultivars of Oryza sativa (IR64, Azucena, and Gigante) and four cultivars of O, glaberrima (Tog5681, Tog5673, CG14, and SG329) were evaluated for their resistance to two isolates of rice yellow mottle virus (RYMV) by enzyme-linked immunosorbent assay (ELISA) and symptomatology. Cultivars Tog5681 and Gigante were highly resistant, and no symptoms were observed when either virus isolate was inoculated at 10 or 20 days postgermination and assayed by ELISA at 7, 14, 22, 35, 50, or 64 days postinoculation. Azucena showed a partial resistance, whereas the other cultivars were susceptible. Symptom appearance was associated with increase in ELISA absorbance in the systemically infected leaves. The best discrimination among the cultivars occurred when the plants were inoculated at 10 days postgermination. Crosses were made between the highly resistant (Gigante and Tog5681) and the susceptible (IR64) cultivars to determine the genetic basis of resistance to RYMV. Evaluation of F1 hybrids and interspecific progenies, as well as the segregation of resistance in F2 and F3 lines of the IR64 x Gigante cross, provided results consistent with the presence of a single recessive resistance gene common to Tog5681 and Gigante.
... Rice yellow mottle virus (RYMV), of the Sobemovirus genus (Truve & Fargette, 2011), has a high natural molecular diversity (Pinel-Galzi et al., 2009). Its first ORF encodes a zinc finger protein with redox-dependent flexibility (Gillet et al., 2013), named P1, which has been described as a multifunctional protein that participates in virus spread and replication (Bonneau et al., 1998). In addition, P1 proteins originating from different RYMV isolates displayed a wide range of ability to suppress RNA silencing, from strong to weak RNA silencing suppression, as in the case of the Tanzanian Tz3 (P1Tz3) and Madagascar Mg1 (P1Mg1) isolates, respectively (Voinnet et al., 1999;Siré et al., 2008). ...
Article
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RNA silencing is an eukaryotic mechanism for RNA-based gene regulation that plays an essential role in diverse biological processes such as defence against viral infections. The P1 of rice yellow mottle virus (RYMV) is a movement protein and displays RNA silencing suppression activity with variable efficiency, depending on the origin of the isolates. In this study, the positive selection pressure acting on the P1 protein gene was assessed. A site-by-site analysis of the dN/dS ratio was performed and 18 positively selected sites have been identified. Four of them were mutated, and the ability to suppress RNA silencing was evaluated for the resulting mutants in a transient expression assay. All mutations affected quantitatively RNA silencing suppression, one caused a significant decrease in the activity, and three significantly increased it. This work demonstrates, for the first time, that the RYMV gene encoding P1 RNA silencing suppressor is under adaptive evolution.
... Not much is known about the molecular aspects of cell-tocell movement in sobemoviruses and the ancillary proteins have not been identified. Earlier studies have shown that the protein encoded by ORF1 of Rice yellow mottle virus (RYMV) [11], Cocksfoot mottle virus (CfMV) [12] and Southern cowpea mosaic virus (SCPMV) [13] is essential for cell to cell movement. Further, ORF1 encoded product of RYMV is implicated as a RNA silencing suppressor [14,15]. ...
Article
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Sesbania mosaic virus (SeMV) is a single strand positive-sense RNA plant virus that belongs to the genus Sobemovirus. The mechanism of cell-to-cell movement in sobemoviruses has not been well studied. With a view to identify the viral encoded ancillary proteins of SeMV that may assist in cell-to-cell movement of the virus, all the proteins encoded by SeMV genome were cloned into yeast Matchmaker system 3 and interaction studies were performed. Two proteins namely, viral protein genome linked (VPg) and a 10-kDa protein (P10) c v gft encoded by OFR 2a, were identified as possible interacting partners in addition to the viral coat protein (CP). Further characterization of these interactions revealed that the movement protein (MP) recognizes cognate RNA through interaction with VPg, which is covalently linked to the 5' end of the RNA. Analysis of the deletion mutants delineated the domains of MP involved in the interaction with VPg and P10. This study implicates for the first time that VPg might play an important role in specific recognition of viral genome by MP in SeMV and shed light on the possible role of P10 in the viral movement.
... The genome is composed of a single-stranded, positive-sense RNA, of about 4451±1 nucleotides (depending on the strain), and contains four open reading frames (ORF1, ORF2a&b, ORF3 and ORF4) (Kouassi et al., 2005). The P1 protein coded by ORF1 is involved in the plant-infection process, virus spread in the plant (Bonneau et al., 1998) and has been described as a suppressor of virus-induced gene-silencing (VIGS; Voinnet et al., 1999). The polyprotein encoded by ORF2a and ORF2b contains a protease, the genome-linked viral protein (VPg) and an RNA-dependent RNA polymerase, which are involved in virus replication. ...
Chapter
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This chapter focuses on the application of molecular tools for the improvement of resistance to Rice yellow mottle virus (RYMV), which is endemic to Africa and can cause up to 80% yield loss in some rice cropping systems. The identification of the first major resistance gene (RYMV1) is also presented.
... -proximal ORF 1 encodes movement protein (MP). It involves in cell to cell movement of virus and acts as a suppressor of RNA silencing (Bonneau et al., 1998). ...
Thesis
Declaration This is to certify that the thesis titled proteomics and RNA-based vaccination strategies using model and re submitted by R. R. D. Naga Charan Konakalla for the award of degree of Doctor of Philosophy in Virology is a record of the work done by him during the period of 2014 2018 under my supervision and that it has not previously formed the basis for the award of any degree or diploma or associateship or fellowship. Declaration This is to declare that the thesis entitled and RNA-based vaccination strategies using model and regional important submitted by me for the award of Doctor of Philosophy in Virology is the record of research work done by me during the period from 2014 2018 under the supervision of Prof. M. Hema and this work done has not previously formed the basis for any award / degree / diploma / associate ship / fellowship.
... Rice yellow mottle virus is a single-stranded RNA species of the genus Sobemovirus with five open reading frames (ORF) (Truve and Fargette, 2011;Ling et al., 2013). ORF1, located at the 5 end of the genome, encodes a small protein involved in virus movement (Bonneau et al., 1998) and in gene silencing suppression (Siré et al., 2008;Lacombe et al., 2010). ORF2, which encodes the central polyprotein, has two overlapping ORFs. ...
... Like other sobemoviral P1 proteins, the RoMoV P1 is very divergent and does not share significant similarity with any known proteins. Despite being very divergent, the P1 proteins of many sobemoviruses have been demonstrated to be involved in virus spread [10][11][12] and RNAi suppression [13][14][15][16]. A PSI-BLAST (http://blast.ncbi.nlm.nih.gov/) ...
Article
Once considered a tentative member of the genus Sobemovirus, rottboellia yellow mottle virus (RoMoV) was excluded from the latest species list of the ICTV after the discovery of imperata yellow mottle virus (IYMV), which resembles RoMoV in host range and geographic origin. Here, sequence analysis of the complete genome of RoMoV suggested that it should be considered a distinct species within the genus Sobemovirus. It has the highest sequence identity (55 %) to ryegrass mottle virus (RGMoV), whereas its sequence identity to IYMV is lower (44 %). In a phylogenetic tree, RoMoV clusters together with RGMoV and artemisia virus A (ArtVA), a dicot-infecting sobemovirus.
... Rice yellow mottle virus (RYMV) P1 of RYMV is required for systemic virus spread and movement [32]. ...
Article
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We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24.
... The ORF spanning nt 421-684 in SBMV encodes the essential sobemovirus protein Px [11], which may be analogous to barnavirus P1 based on the coding organizations of these viruses. The other 5′-proximal ORF in SBMV, spanning nt 93-533, has been reported to encode a movement protein (P1 [3]), which is not expected to be present in the fungal barnaviruses sequence. As a result, the consensus sequence for the apparent new barnavirus reported here (GenBank MG686618) is 4206 nt long, appears to be coding complete for ORFs 1-4 ( Fig. 1), and was newly assembled from a total of 821 individual reads (reads per position: mean, 19; range, 2-35). ...
Article
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Because so few viruses in the family Barnaviridae have been reported, we searched for more of them in public sequence databases. Here, we report the complete coding sequence of Colobanthus quitensis associated barnavirus 1, mined from a transcriptome of the Antarctic pearlwort Colobanthus quitensis. The 4.2-kb plus-strand sequence of this virus encompasses four main open reading frames (ORFs), as expected for barnaviruses, including ORFs for a protease-containing polyprotein, an RNA-dependent RNA polymerase whose translation appears to rely on − 1 ribosomal frameshifting, and a capsid protein that is likely to be translated from a subgenomic RNA. The possible derivation of this virus from a fungus associated with C. quitensis is discussed. Electronic supplementary material The online version of this article (10.1007/s00705-018-3794-x) contains supplementary material, which is available to authorized users.
... For example, rice yellow mottle virus is responsible for the most rapidly spreading disease of rice in Africa [12]. Previous studies on sobemoviruses have shown that the protein coded by ORF1 of rice yellow mottle virus [14] and Southern cowpea mosaic virus [15] is essential for cell-to-cell movement. More recently, it was reported that the ORF1-encoded protein of Cocksfoot mottle virus (CfMV), designated P1, is essential for systemic spread of the virus [16]. ...
Article
Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N-terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement. Structured digital abstract
... The genome is composed of a single-stranded, positive-sense RNA, of about 4451±1 nucleotides (depending on the strain), and contains four open reading frames (ORF1, ORF2a&b, ORF3 and ORF4) (Kouassi et al., 2005). The P1 protein coded by ORF1 is involved in the plant-infection process, virus spread in the plant (Bonneau et al., 1998) and has been described as a suppressor of virus-induced gene-silencing (VIGS; Voinnet et al., 1999). The polyprotein encoded by ORF2a and ORF2b contains a protease, the genome-linked viral protein (VPg) and an RNA-dependent RNA polymerase, which are involved in virus replication. ...
... ! F"! à la vie cellulaire de l'hôte en interférant aussi avec le silencing Siré et al, 2008 ;Lacombe et al, 2010) et en se comportant comme un facteur de pathogénicité (Bonneau et al, 1998 ;Siddiqui et al, 2008 ...
Thesis
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RYMV phytovirus genome codes for five proteins. Together they transform cellular activity allowing virus multiplication and spreading. Among of them, P1 is a multifunctional and small cystein rich protein. It is involve in virus replication and intercellular movement and in PTGS regulation, an antiviral pathway. If functions knowledge’s about P1 are acquired, little is known about how its molecular mechanism orchestrates. My phd works gives some comprehensive element by a biochemical approach. P1 is a polymorphic protein with a redox dependent conformational change. Reduction of P1 was accompanied by the binding of two zinc atoms while its oxidation provoked zinc release and disulfide bond formation. Furthermore, the oxidation state of P1 was directly related to its oligomerization state in vitro. In a pathosystem, a viral activity detection is fallowed by oxidative stress that induced host defenses pathways. We suppose that, in planta, P1 could act as a rheostat sensing redox imbalances in the host cell. This redox dependant structural polymorphism could contribute to explain the P1’s multifunctionality and how these functions could be regulated. This property is also a way for the virus to generate multiple functions from the same genetic information. Key words : RYMV-P1, PTGS, redox, structural polymorphism, multifunctionality, zinc-binding domain
... Although all sobemoviruses possess P1, the size varies from 11.7 to 24.3 kDa, and there is no sequence homology within the genus or to any protein in the GenBank database [14]. Despite this, the P1 proteins of both CoMW and RYMV have been shown to function in both systemic movement [15,16] and as a suppressor of gene silencing [17,18]. ...
Article
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Velvet tobacco mottle virus (VTMoV) infects the native Australian plant Nicotiana velutina, which is endemic to central Australia. This virus is included in the genus Sobemovirus based on virion morphology and serological relationships. We report here the full genome sequence of VTMoV, attained using a genome-walking strategy with both degenerate and specific primers. This sequence confirms that VTMoV is a sobemovirus, with the same open reading frame (ORF) organisation as other described sobemoviruses. The VTMoV sequence is closest to those sobemoviruses isolated from monocotyledonous plants, although the narrow host range of VTMoV is limited to dicotyledonous plants.
... The function of the translation product of ORF3 is still unknown, whereas ORF4 encodes the coat protein (Yassi et al., 1994). P1 is required for viral replication and cell-to-cell movement (Bonneau et al., 1998 ). Overall, it acts as a nonautonomous cell silencing suppressor (Voinnet et al., 1999) of post-transcriptional gene silencing. ...
Article
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Ectopically expressed rice yellow mottle virus P1 fusion proteins were found to be cleaved in planta and in Escherichia coli. Cleavage takes place in the absence of bacterial protease activity, indicating that the P1 fusion is autocatalytically processed independently of host factors. N-terminal sequencing of the C-terminal cleavage product of transiently expressed P1/GFP (green fluorescence protein) in Nicotiana benthamiana showed that the cleavage site is located between the first two amino acids (aa) downstream of the P1 sequence. Mutagenesis experiments revealed that a phenylalanine to valine substitution at position 157 of the P1 aa sequence impairs proper cleavage, which is nearly unaffected by replacement of phenylalanine with tyrosine. Deletion of methionine(159) (first GFP aa residue) appeared to not affect P1/GFP cleavage. N-terminal P1-tagging with GFP turned out to impair autocleavage, whereas a small His-tag could not fully prevent cleavage. Additionally, a modified P1/GFP carrying an N-terminal deletion of 81 aa was not cleaved. These findings indicate that this region is involved in the proteolysis mechanism and that large N-terminal fusion partners might affect correct folding of the P1 necessary for self-catalysis.
... The function of the P1 protein has been investigated through expression of wild type and mutant P1 in vitro and in vivo. P1 is required for infection of the plant and for virus spread (16) and also in suppressing virus-induced gene silencing (VIGS) (69). The CP gene is required for full infectivity in rice plants since it plays a role in cell-to-cell, long distance movement and systemic infection in O. sativa. ...
... ORF1 (45-686 nt), which is located at the 5' end of the genome, encodes a P1-like protein. P1 is involved in the cell-to-cell and systemic movement of the virus [7]. ORF2, has two overlapping ORFs, encodes the putative central polyproteins. ...
Article
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The Potyviridae family, named after its type member, Potato virus Y (PVY), is the largest of the 65 plant virus groups and families currently recognized. The coding region for P1 peptidase is located at the very beginning of the viral genome of the family Potyviridae. Until recently P1 was thought of as serine peptidase with RNA-binding activity and with possible influence in cell-to-cell viral spreading. This N-terminal protein, among all of the potyviruses, is the most divergent protein: varying in length and in its amino acid sequence. Nevertheless, P1 peptidase in many ways is still a mysterious viral protein. In this review, we would like to offer a comprehensive overview, discussing the proteomic, biochemical and phylogenetic views of the P1 protein.
Article
Cocksfoot mottle virus (CfMV) localization in oat plants was analyzed during three weeks post infection by immunohistochemical staining to follow its spread through different tissues. In early stages of infection, the virus was first detectable in phloem parenchyma and bundle sheath cells of inoculated leaves. Bundle sheath and phloem parenchyma were also the cell types where the virus was first detected in stems and systemic leaves of infected plants. In later stages of infection, CfMV spread also into the mesophyll surrounding vascular bundles and was seldom detected in xylem parenchyma of inoculated leaves. In systemic leaves, CfMV was not detected from xylem. Moreover, sometimes it was found from phloem only. In straw and roots, CfMV was detected both from phloem and xylem. According to our observations, CfMV predominantly moves through phloem, which makes the systemic movement of CfMV different from that of another monocot-infecting sobemovirus, Rice yellow mottle virus (RYMV).
Article
The complete nucleotide sequence of a new member of the unassigned genus Sobemovirus, isolated from raspberry and bramble plants in north east Scotland and given the name Rubus chlorotic mottle virus (RuCMV), was obtained. The virus has a single, positive-strand RNA genome of 3983 nucleotides and, in common with other sobemoviruses, contains four open reading frames (ORFs) encoding, from 5′ to 3′, the P1 protein that is likely to be a suppressor of RNA silencing, ORF2a that has homology to serine-proteases, ORF2b that is the probable RNA dependent RNA polymerase, and ORF3 that is the coat protein. ORF2b protein is potentially expressed as a fusion with ORF2a protein by a −1 frameshift at the heptanucleotide sequence UUUAAAC. Phylogenetic analyses showed that RuCMV is a distinct virus not closely related to any of the other sequenced sobemoviruses. Based on the obtained sequence a full-length cDNA copy of RuCMV was cloned and in vitro transcripts derived from this clone were shown to be fully infectious.
Article
The distribution of Rice yellow mottle virus(RYMV) was compared in the partially resistant upland rice Oryza sativa japonica cv. Azucena and in the susceptible O. sativa indica cv. IR64, which are the two parents of a doubled haploid population studied for several traits, including genetic determinants of resistance to RYMV infection. Symptom expression, distribution and accumulation of viral coat protein and nucleic acid were studied in inoculated leaves, systemically infected leaves and apices, and in leaf host tissues. Initially, the resistance was apparent as delayed virus detection and multiplication which led to a lower virus content in inoculated leaves and in systemically infected organs. Later, the resistance was less apparent as virus titers became progressively similar in the two cultivars. Then, tolerance was observed, as symptom expression was less pronounced in cv. Azucena than in IR64, despite similar virus contents. In host tissues, partial resistance was associated with the delayed detection of virus in the bundle sheaths (mestomes) of Azucena, and, later, the mestome invasion paralleled the decline in resistance. The mature leaves of both cultivars always escaped infection. Partial virus resistance resulted from the expression of quantitative trait locus on chromosome 12, whereas tolerance is a characteristic of O. japonica, including the expression of QTL1. An impaired cell to cell movement through the mestome, possibly reinforced by slow vascular movement, are proposed to explain the partial resistance.
Article
Two quantitative trait loci (QTL12 and QTL7) acting in epistatis and associated with Rice yellow mottle virus (RYMV) infection were introgressed from the traditional Oryza sativa japonica cultivar Azucena into the improved indica cultivar IR64. The response to RYMV infection was followed over time in this nearly isogenic line (NIL). In leaves, the virus coat protein content was detected by ELISA and RNA by RT-PCR. In tissues, the coat protein was localised by immuno-fluorescence labelling and RNA by in situ hybridisation. We found (i) that Azucena combined a tolerance—characterised by mild symptoms despite generalised distribution and large accumulation of the virus—and a partial resistance, (ii)that partial resistance under the control of QTL12 and QTL7, dissociated in the NIL from tolerance and any other morphological or physiological Azucena traits, was expressed similarly in a japonica and in an indica genetic background, (iii) that partial resistance was transient and consisted in a 1-week delay in virus accumulation and symptom expression; passed this delay, partial resistance brokedown, and (iv) a similar delay in virus detection was observed at tissue levels, in the vascular bundle-sheath layers. Impaired virus movement caused by the absence or mutation of a QTL12-encoded plant factor necessary for virus transport through vascular bundle sheaths is proposed to explain this partial resistance.
Article
Rice yellow mottle virus (RYMV) accumulation in protoplasts and whole plants was investigated in two highly resistant cultivars, Tog5681 (Oryza glaberrima) and Gigante (Oryza sativa). Three susceptible cultivars, i.e. one O. glaberrima Tog5673 and two O. sativa (IR64, Ac. 2428), and a partially resistant cultivar (Azucena) were used as control. After inoculation, accumulation of coat protein (CP) and viral RNA were monitored on protoplasts, inoculated leaves, sheaths of inoculated leaves and newly infected leaves by serological and Northern blot analysis. Viral RNA accumulated to a similar extent in protoplasts from all cultivars studied. In contrast, three distinct in planta behaviors were noted. In susceptible plants (IR64, Tog5673 and Ac. 2428), there was high CP and RNA accumulation at 5 d.p.i. in whole plants, suggesting that cell to cell and vascular movements occurred before 5 d.p.i. in inoculated leaves. The second behavior concerned Azucena, which showed a delay (around 7 d.p.i.) of viral accumulation in inoculated leaves. The third behavior involved the highly resistant cultivars Tog5681 and Gigante. CP and viral RNA were not detected in these cultivars. The comparison of viral accumulation in protoplasts and plants suggested that resistance of the highly resistant cultivars Tog5681 (O. glaberrima) and Gigante (O. sativa) was not due to the inhibition of virus replication but rather to the failure of cell to cell movement.
Article
Functional analysis for gene silencing suppressor of P14 geneof Beet necrotic yellow vein virus and S6 gene ofRice black streak dwarf virus was carried out by agro-infiltration with recombinant vectors ofPotato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing ofgfp transgenicNicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, thegfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially thegfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.
Article
The genome of Ryegrass mottle virus (RGMoV) comprises 4210 nucleotides. The genomic RNA contains four open reading frames (ORFs). The largest ORF 2 encodes a polyprotein of 947 amino acids (103.6 kDa), which codes for a serine protease and an RNA-dependent RNA polymerase. The viral coat protein is encoded on ORF 4 present at the 3′-proximal region. Other ORFs 1 and 3 encode the predicted 14.6 kDa and 19.8 kDa proteins of unknown function. The consensus signal for frameshifting, heptanucleotide UUUAAAC and a stem-loop structure just downstream is in front of the AUG codon of ORF 3. Analysis of the in vitro translation products of RGMoV RNA suggests that the 68 kDa protein may represent a fusion protein of ORF 2-ORF 3 produced by frameshifting. The protease region of the polyprotein and coat protein have a low similarity with that of the sobemoviruses (approximately 25% amino acid identity), while the RNA-dependent RNA polymerase region has particularly strong similarity (54 to 60% of more than 350 amino acid residues). The sequence similarities of RGMoV to the sobemoviruses, together with the characteristic genome organization indicate that RGMoV is a new species of the genus Sobemovirus.
Article
Our previous studies have hypothesised that a complementary epistasis between a QTL located on chromosome 12 and a QTL located on chromosome 7 was one of the major genetic factors controlling partial resistance to Rice yellow mottle virus (RYMV). We report research undertaken to verify this hypothesis and to introgress the resistant allele of these two QTLs from an upland resistant japonica variety, Azucena, into a lowland susceptible indica variety IR64. Three cycles of molecular marker-assisted back cross breeding were performed using RFLP and microsatellite markers. Resistance to RYMV was evaluated in F2 and F3 offspring of the BC1 and BC2 generations. Marker-assisted introgression (MAI) was very efficient: in the selected BC3 progeny the proportion of the recipient genome was close to 95% for the ten non-carrier chromosomes, and the length of the donor chromosome segment surrounding the two QTLs was less than 20 cM. The relevancy of the complementary epistasis genetic model proposed previously was confirmed experimentally: in BC1 and BC2 generations only F3 lines having the allele of the resistant parent on QTL12 and QTL7 show partial resistance to RYMV. Comparison of our experimental process of MAI with the recommendations of analytic and simulation studies pointed out the methodological flexibility of MAI. Our results also confirmed the widely admitted, but rarely verified, assumption that QTL-alleles detected in segregating populations could be treated as units of Mendelian inheritance and that the incorporation of these alleles into elite lines would result in an enhanced performance. The next step will be the design of tools for the routine use of molecular markers in breeding for partial resistance to RYMV and the development of material for the analysis of resistance mechanisms and the structure of a virus resistance gene in rice.
Chapter
Rice (Oryza sp.) is one of the most important food crops in the tropical areas of the world. Viral infections are serious constraints in rice production in certain parts of the world. There are about 16 viruses reported to date, which cause significant yield loss to rice. They belong to different geographical regions, show genome variability, and have widely different transmission characteristics and symptom development. Although the use of conventional/natural genetic resistance in plants is always considered the most appropriate strategy against the pathogen, in case of rice-virus pathology such examples are very rare. Since the last three decades, the concepts of pathogen-derived resistance and RNA interference have proved to be effective to develop virus-resistant transgenic rice plants. The present chapter collates the various transgenic approaches used to provide broad-spectrum transgenic resistance in rice against viruses.
Chapter
Molecular taxonomy of plant viruses is now nearing completion (Mayo and Pringle, 1998; Pringle, 1998) primarily because of the evolutionary relationships that emerged from comparative studies on nucleotide and protein sequences and RNA-dependent RNA polymerase gene of various plant viruses (Koonin, 1991; Koonin and Dolja 1993; Zanotto et al., 1996). Four distinct supergroups of plus-sense RNA plant viruses are now recognized. Based on similarity of the basic genome features between different groups of animal and plant viruses, two distinct supergroups of plus-RNA plant viruses initially delimited were plant “picorna-like” and plant “alpha-like” supergroups. The two more supergroups of plus-RNA plant viruses recognized recently are the `sobemo-like“ and ”carmo-like“ supergroups.
Article
Functional analysis for gene silencing suppressor of P14 gene of Beet necrotic yellow vein virus and S6 gene of Rice black streak dwarf virus was carried out by agro- infiltration with recombinant vectors of Potato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing of gfp transgenic Nicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, the gfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially the gfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.
Chapter
The vast majority of plant virus groups contain an RNA genome, most frequently of positive polarity. This chapter provides an updated overview of genome of such plant viruses stressing the functions of coding and non coding regions of genome in virus amplification. Three concise tables accompany the text. In each table, the viruses are presented in alphabetical order by genera as officially recognized in the Plant Virus Classification of the International Committee on Taxonomy of Viruses (1996). Table 1 presents general features of RNA genomes and their associated RNAs like satellite RNA (sat-RNA) and defective interfering RNA (DI). Table 2 shows further major characteristics of viruses with a single-strand RNA (ssRNA) genome. Table 3 classifies the viruses in supergroups where these have been established, outlines gene arrangements (also consult chapter 3), and gives the main translation strategies (chapter 4) used by plant RNA viruses. Recent key references as well as certain other references, not mentioned in the review by Zaccomer et al. (1995), are provided. A few virus groups are not presented, due to lack of sufficient sequence data concerning them. These are the fabaviruses, betacryptoviruses and cytorhabdoviruses. Virus-related RNA molecules (DI and sat-RNA) and viroids are not discussed here.
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The frequency and spectrum of infections with pathogens harbouring resistance to antibiotics and other drugs has dramatically increased over the last years. One of the main causes is the extensive use of antibiotics and other drugs in human and veterinary medicine. Parasites, such as Eimeria causing coccidiosis in chicken and pathogenic bacteria like Salmonellae and Campylobacter are examples of pathogens that acquired resistance. Furthermore, continuous use of drugs in diets of animals kept for human consumption increases the risk of residues in food, that possibly affect human health. These drawbacks of antimicrobial drugs have led to a demand for alternative treatments. In this thesis an alternative approach for prevention of coccidiosis in chicken is described, based on immune intervention by passively administered, plant produced, secretory IgA.As a first step, Eimeria binding IgA fragments were selected using the phage display technique. The phage display system was adapted to be used for the display of chicken Fab fragments. A newly constructed vector, named pChick3, allows straightforward cloning of chicken variable antibody domains in frame with the constant domains of the chicken light chain and the first constant domain of the IgA heavy chain. In a following step, new plant expression vectors were designed and constructed. Ten antibodies, selected from the chicken phage antibody library were then transferred to this vector system and subsequently expressed in planta as full size IgA. Upon expression of the ten selected anti- Eimeria antibodies, differences up to 500-fold in yield were observed. Several factors on translational or protein level could cause the observed differences: e.g. processing, stability, assembly and silencing. Two were tested (silencing, chain compatibility i.e. assembly) and both have an influence on the levels of expression. An explanation may be found in the combination of several factors. These observations lead to the conclusion that an extra in planta selection step is inevitable for successful integration of phage display and plant expression systems. Finally, the structure of the chicken polymeric immunoglobulin receptor was elucidated. In a fashion similar to its mammalian counterpart, this receptor transports IgA to the gut lumen forming secretory IgA. This complex is highly stable, and IgA is protected against degradation by proteases or pH-fluctuation, which makes secretory IgA the most suitable form for passive immunization. Interestingly, the chicken SC comprises only four immunoglobulin-like domains compared to five found in mammals. Thus, an integrated system for both selection and expression of immunoglobulins was developed and with the final achievement of the production of Eimeria -specific secretory IgA in plants, the prerequisites for chicken passive immune therapy were fulfilled.
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Accumulation of the cowpea strain of southern bean mosaic sobemovirus (SBMV-C) in bean, a nonpermissive host, was facilitated by coinfection with sunn-hemp mosaic (SHMV), a tobamovirus. The rate of spread in bean of SBMV-C in the presence of SHMV was compared with the rate of spread of the bean strain of SBMV (SBMV-B) coinoculated with SHMV (...)
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SUMMARY The optimum conditions for infection of turnip protoplasts with turnip rosette virus (TRosV) were o.z to 2 × lO 5 protoplasts/ml, 5 to IO/~g virus/rot, 1/~g poly-L- ornithine/mI in o'6 M-mannitol, buffered with 5 to xo mM-tris at pH 7"o to 8.6. Under these conditions, 7o% of protoplasts became infected, as indicated by staining with fluorescent antibody. The proportion of protoplasts infected was determined by factors such as pH and buffer ions and by the concentrations of protoplasts, virus and poly-L-ornithine during inoculation. The period of pre- inoculation incubation of virus and poly-L-ornithine was also critical, but mannitol concentration and the inoculation period and temperature had little effect on percent- age protoplasts infected. Time course studies showed a single step multiplication of TRosV; the virus was detected on sucrose gradients at 24 h after inoculation and at 6o h reached its maximum concentration, 3 × I06 virions per infected protoplast.
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The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.
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The genome of rice yellow mottle virus (RYMV) is a single-stranded positive-sense RNA that is not polyadenylated, and has an M(r) of 1.4 x 10(6). We present here the 4550 nucleotide (nt) sequence of RYMV RNA, and its predicted genomic organization. The RYMV genomic RNA contains four open reading frames (ORFs). The first (nt 80 to 553) encodes a protein containing 157 amino acids with a predicted M(r) of 17.8K. No function has yet been attributed to this product. ORF2 (nt 608 to 3607) encodes a polyprotein of 999 amino acids, with a predicted M(r) of 110.7K. The first 134 amino acids of ORF2 are predicted to be the genome-linked protein, VPg, followed by the viral protease, the helicase and the RNA-dependent RNA polymerase. ORF3 is within the boundaries of ORF2 and is predicted to encode a polypeptide with 126 amino acids and an M(r) of 13.7K. No function has yet been attributed to this protein. ORF4 (nt 3447 to 4166), which overlaps the 3' terminus of ORF2, encodes a 26K protein. This polypeptide has been identified as the RYMV coat protein. The data presented here confirm that RYMV belongs to the sobemovirus group and thus is a member of the picorna-like family of plant viruses.
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Sunn-hemp mosaic tobamovirus (SHMV) facilitated the spread of the cowpea strain of southern bean sobemovirus (SBMV-C) only in inoculated leaves of common bean (Phaseolus vulgaris L. cv. Bountiful), a resistant host for SBMV-C. Tissue prints of bean primary leaves doubly inoculated with SHMV and SBMV-C, developed by Western blotting, showed the presence of the SBMV-C capsid antigen in the mesophyll and epidermis, but no antigen was detected in the conducting bundles. Typical SBMV-C virions were not seen in electron micrographs of immunogold-labelled mesophyll cells; instead, specifically labelled, amorphous protein clumps were found in the vacuole. Particles of smaller diameter than that of typical SBMV-C virions were specifically trapped by SBMV antibodies following immunosorbent electron microscopy of extracts from doubly infected leaves. SBMV-C coat protein from infected Vigna unguiculata L. (cowpea) and bean plants showed no difference in its mobility following electrophoresis in denaturing SDS-polyacrylamide gels. Lack of efficient assembly of SBMV-C virions does not impede cell-to-cell movement of the virus in doubly infected leaves of bean, yet it is probably an important factor in determining the inability of SBMV-C to move into and/or through the vascular system of this host.
Article
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RNA isolated from southern bean mosaic virions contains, in small amount, a subgenomic RNA (molecular weight, 0.38 x 10(6)) that serves in vitro as an mRNA for southern bean mosaic virus coat protein. The RNA has a 5'-linked protein indistinguishable from the protein linked to the 5' end of full-length genomic RNA. Its base sequence, determined to 91 bases from the 3' end, is identical to the 3'-terminal sequence of the genomic RNA. The results suggest that the coat protein messenger sequence exists as a "silent" cistron near the 3' end of the genomic RNA.
Article
The tobacco etch potyvirus (TEV) polyprotein is proteolytically professed by three viral proteinases (NIa, HC-Pro, and P1), While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly, To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (Delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants, The Delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The Delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus, This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both Delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host, In transgenic protoplasts, genome amplification of the Delta P1 mutant relative to parental virus was stimulated five- to sixfold, In transgenic plants, the level of accumulation of the Delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.
Chapter
Hull (1977a) proposed a new group of plant viruses based upon southern bean mosaic virus (SBMV). This group was accepted by the International Committee on Taxonomy of Viruses (ICTV) (Matthews, 1982) under the name Sobemovirus (sigla for southern bean mosaic virus) group. Although the ICTV only accepted two viruses as full members and four as possible members, for the purposes of this chapter I will consider ten viruses as being full members and six as being possible members (Table I). LTSV, SCMV, and VTMoV have been suggested as comprising another possible group (the velvet tobacco mottle virus group) (Matthews, 1982), but it is now generally accepted (see Francki et al.,1985a) that these viruses have close affinities to Sobemoviruses. The possible members show some similarities to Sobemoviruses but as more information on them becomes available, at least some of them may be grouped elsewhere.
Article
Synthesis of turnip rosette virus (TRosV)-induced RNA was examined in turnip protoplasts inoculated in vitro. TRosV RNA (mol. wt. 1.4 × 106; approx. 4.3 kb) was first detected 10 to 20 h post-inoculation by 32P labelling. When actinomycin D was added to protoplast cultures 6 h or less post-inoculation, labelling of virus RNA was less than in untreated cultures. Synthesis of TRosV RNA in infected protoplasts appeared to involve a double-stranded (ds)RNA; a replicative form (RF) of the virus RNA was isolated and characterized. The RF, mol. wt. 2.8 × 106 (approx. 4.3 kbp), was not degraded by treatment with RNase or DNase, contained 50% sequences complementary to TRosV RNA (as determined by hybridization) and was denatured to singlestranded (ss)RNAs of mol. wt. (× 10-6) of 1.4, 0.7, 0.3 and 0.09 (4.3, 2.1, 0.9 and 0.27 kb respectively). Virus infection apparently induced the synthesis of a ssRNA of mol. wt. 0.3 × 106 which could be resolved from other RNA species in nucleic acid extracts from infected protoplasts. A minor dsRNA of 1.4 × 106 mol. wt. (2.1 kbp) was also apparently induced by virus infection. These virus-induced small RNA species may represent subgenomic messengers or their RF.
Article
SUMMARY Soybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-L-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 9o % of the protoplasts were infected by CPMV when the inoculation medium contained o'4 M-sorbitol, o'5/~g virus/ml, 2"5/zg PkO/ml, IO raM-potassium phosphate buffer, pH 6.3, and o'5 mM-CaCI~, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaC12 was omitted. Inoculation at temperatures below l o °C also decreased infection substantially. With SBMV a maximum of 3o to 35 % of the protoplasts were infected when o'4 M-sorbitol, 2 to 2"5/~g virus/ml, 2 #g PLO/ml, ro mm-tris-HC1 buffer, pH 8.o, I mM-MgSO4 and 0. 5 mM-CaC12 were pre- sent in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.
Article
Rice yellow mottle virus(RYMV)is the causal pathogen of yellow mottle of rice in the area around Kisumu near Lake Victoria in Kenya. Affected plants show a yellow or orange discolouration of the leaves, reduced tillering and stunting of the plants, and sterility of the flowers. The results of this study gave the following cryptogram forRYMVR/1:1.4/23:S/S:S/Cl.RYMVis easily mechanically transmissible. Experimentally a limited number of plants belonging to the family Gramineae only were found to be a host for the virus. Rice was the only host found naturally infected withRYMV.Beetles belonging to the subfamilies Criocerinae, Cryptocephalinae, Galerucinae, Halticinae and Hispinae of the family Chrysomelidae, and the long-homed grasshopper Conocephalus merumontanus Sjöstedt (Tettigoniidae) also transmitted the virus. Chaetocnema pulla Chapuis (Halticinae) proved to carry the virus in the field.RYMVis a stable virus with a particle diameter of about 25 nm and sediments as a single component. The base composition of theRNAis similar, but not identical to that of cocksfoot mottle virus. Serologically no relationship nor any other affinity was established betweenRYMVand a number of other isometric plant viruses of comparable size.Infected rice is considered to be the main source of the virus in the rice fields. Control of the disease must be sought in cultivation practices and growing of rice with short vegetative periods. Breeding of rice varieties for resistance toRYMVhas to be initiated.
Article
Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.
Article
When RNA from southern bean mosaic virus is fractionated on a sucrose gradient, the resulting absorbance profile shows a major peak corresponding to 1.4 x 10(6) MW together with a considerable amount of slower sedimenting material. The RNA from these gradient fractions was translated in wheat embryo extracts and reticulocyte lysates. Only RNA in the molecular weight range 0.3 - 0.4 x 10(6) was found to induce synthesis of coat protein (designated P3). RNA of molecular weight 1.4 x 10(6) induced synthesis of three proteins, P1, P2, and P4.
Article
We investigated the nutrient requirements of rice in liquid culture and developed a revised medium of mineral salts, sucrose, thiamine and 2,4-dichlorophenoxyacetic acid. The following major nutrients at the indicated concentrations were beneficial: NO 3 -N (40 mM), NH 4 -N (5.0 mM), P (2.0 mM) and K (40 mM). Cobalt, iodine, pyridoxine, nicotinic acid and m -inositol were not essential and were excluded from the revised R-2 medium. Growth was better with ammonium and nitrate together than with nitrate as the sole nitrogen source. Cell growth in the R-2 medium was superior to that obtained in B5, Heller, Murashige-Skoog and White media.
Article
Two genomic clones (lambda Ubi-1 and lambda Ubi-2) encoding the highly conserved 76 amino acid protein ubiquitin have been isolated from maize. Sequence analysis shows that both genes contain seven contiguous direct repeats of the protein coding region in a polyprotein conformation. The deduced amino acid sequence of all 14 repeats is identical and is the same as for other plant ubiquitins. The use of transcript-specific oligonucleotide probes shows that Ubi-1 and Ubi-2 are expressed constitutively at 25 degrees C but are inducible to higher levels at elevated temperatures in maize seedlings. Both genes contain an intron in the 5' untranslated region which is inefficiently processed following a brief, severe heat shock. The transcription start site of Ubi-1 has been determined and a transcriptional fusion of 0.9 kb of the 5' flanking region and the entire 5' untranslated sequence of Ubi-1 with the coding sequence of the gene encoding the reporter molecule chloramphenicol acetyl transferase (CAT) has been constructed (pUBI-CAT). CAT assays of extracts of protoplasts electroporated with this construct show that the ubiquitin gene fragment confers a high level of CAT expression in maize and other monocot protoplasts but not in protoplasts of the dicot tobacco. Expression from the Ubi-1 promoter of pUBI-CAT yields more than a 10-fold higher level of CAT activity in maize protoplasts than expression from the widely used cauliflower mosaic virus 35S promoter of a 35S-CAT construct. Conversely, in tobacco protoplasts CAT activity from transcription of pUBI-CAT is less than one tenth of the level from p35S-CAT.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.
Article
The differential rate of systemic symptom induction in zucchini squash by the Fny- and Sny-strains of cucumber mosaic virus (CMV) previously was mapped to RNA 1, which encodes a protein (1a) involved in virus replication. Examination of the kinetics of accumulation of the RNAs and the four encoded proteins in the inoculated cotyledons showed that the Fny-CMV-associated products generally appeared earlier than the Sny-CMV-associated products. In the systematically infected leaves, this difference was magnified, with a 2-day delay in the appearance of the Sny-CMV RNAs and encoded proteins. However, both Fny-CMV and Sny-CMV RNAs showed similar kinetics of RNA, 2a, 3a, and coat protein accumulation in protoplasts prepared from zucchini squash cotyledons. These data indicate that the differential rate of systemic symptom development was due to a difference in the rate of movement rather than the rate of replication. This was confirmed by a leaf-detachment assay, which showed a difference in the rate of systemic movement by Fny-CMV vs Sny-CMV, and by leaf-press blot hybridization of the inoculated cotyledons at different days postinoculation, which showed a difference in the rate of cell-to-cell movement by the two strains of CMV. Taken together, these data show that the rates of cell-to-cell and long-distance movement can be regulated by sequences in CMV RNA 1, previously thought to be involved only in virus replication.
Article
The coat protein of the cowpea strain of southern bean mosaic sobemovirus (SBMV-C) is translated from a subgenomic RNA (sgRNA) that is synthesized in the virus-infected cell. Like the SBMV-C genomic RNA, the sgRNA has a viral protein (VPg) covalently bound to its 5' end. The mechanism(s) by which ribosomes initiate translation on the SBMV-C RNAs is not known. To begin to characterize the translation of the sgRNA it was first necessary to precisely map its 5' end. Primer extension was used to identify SBMV-C nucleotide (nt) 3241 as the transcription start site. As a control, the 5' end of the genomic RNA was also mapped. Surprisingly, the 5' terminal nt of this RNA was identified as SBMV-C nt 2. The primary structure of the 5' ends of these two RNAs is therefore expected to be VPg-ACAAAA. Precise mapping of the 5' end of the sgRNA of the bean strain of SBMV (SBMV-B) demonstrated that it has these same elements. Translation of coat protein from the SBMV-C sgRNA and p21 from the SBMV-C genomic RNA was compared using a cell-free system. The results of these experiments were consistent with translation of these proteins by a 5' end-dependent scanning mechanism rather than by internal ribosome binding.
Article
The function of the 30-kilodalton movement protein (MP) of tobacco mosaic virus is to facilitate cell-to-cell movement of viral progeny in an infected plant. A novel method for delivering non-plasmalemma-permeable fluorescent probes to the cytosol of spongy mesophyll cells of tobacco leaves was used to study plasmodesmatal size exclusion limits in transgenic plants that express the MP gene. Movement of fluorescein isothiocyanate-labeled dextran (F-dextran) with an average molecular mass of 9400 daltons and an approximate Stokes radius of 2.4 nanometers was detected between cells of the transgenic plants, whereas the size exclusion limit for the control plants was 700 to 800 daltons. No evidence of F-dextran metabolism in the leaves of the transgenic plants was found. Thus, the tobacco mosaic virus movement protein has a direct effect on a plasmodesmatal function.
Virus-host interactions in southern bean mosaic virus gene expression and assembly
  • D.L. Hacker
  • K. Sivakumaran
  • D.L. Hacker
  • K. Sivakumaran
Schematic representations of mutants FL5⌬88-547, FL5mATG, FL5⌬83aa Characterization and ecological aspects of rice yel-low mottle virus in Kenya Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein
  • ±553 Bakker
  • W P1 Protein Brugidou
  • C Holt
  • C Ngon
  • M Yassi
  • S Zhang
  • R Beachy
  • C Fauquet
All mutations were confirmed by enzyme digestion and DNA sequencing. Schematic representations of mutants FL5⌬88-547, FL5mATG, FL5⌬83aa, p-ORF1(1±553) and REFERENCES Bakker, W. (1974). Characterization and ecological aspects of rice yel-low mottle virus in Kenya. Agric. Res. Rep. 829. EXPRESSION OF RYMV P1 PROTEIN Brugidou, C., Holt, C., Ngon A Yassi, M., Zhang, S., Beachy, R., and Fauquet, C. (1995). Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein. Virology 206, 108±115.
The sobemovirus group. In The Plant Viruses
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Hull, R. (1988). The sobemovirus group. In The Plant Viruses,'' Vol. 3. (Renate Koenig, Ed.), pp. 113±146. Plenum, New York.
Infection of protoplasts from soybean cell culture with southern bean mosaic and cowpea mosaic viruses Cleavage of structural proteins during the as-sembly of the head of bacteriophage T4
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Jarvis, N. P., and Murakishi, H. H. (1980). Infection of protoplasts from soybean cell culture with southern bean mosaic and cowpea mosaic viruses. J. Gen. Virol. 48, 365±376. Laemmli, U. K. (1970). Cleavage of structural proteins during the as-sembly of the head of bacteriophage T4. Nature 227, 680±685.
Sobemoviruses Pathogenesis and Host Specificity in Plant Diseases: Histological, Biochemical Genetic and Molecular Bases
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Seghal, O. P. (1995). Sobemoviruses. In``In`` Pathogenesis and Host Specificity in Plant Diseases: Histological, Biochemical Genetic and Molecular Bases'' (R. P. Singh, U. S. Singh, and K. Kohomota, Eds.), pp. 115±128. Pergamon Press, U.K.
Virus-host interactions in southern bean mosaic virus gene expression and assembly Bi ology of Plant±Microbe Interactions
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Virus-host interactions in southern bean mosaic virus gene expression and assembly
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