Rice yellow mottle virus is a suitable amplicon vector for an efficient production of an anti-leishmianiasis vaccine in Nicotiana benthamiana leaves.

BACKGROUND: Since the 2000's, plants have been used as bioreactors for the transient production of molecules of interest such as vaccines. To improve protein yield, "amplicon" vectors based on plant viruses are used. These viral constructs, engineered to carry the gene of interest replicate strongly once introduced into the plant cell, allowing significant accumulation of the protein. Here, we evaluated the suitability of the monocot-infecting RNA virus Rice yellow mottle virus (RYMV) as an amplicon vector. The promastigote surface antigen (PSA) of the protozoan Leishmania was considered as a protein of interest due to its vaccine properties against canine leishmaniasis. RESULTS: Since P1 (ORF1) and CP (ORF3) proteins are not strictly necessary for viral replication, ORF1 was deleted and the PSA gene was substituted to ORF3 in the RYMV-based vector. We evaluated its expression in the best described plant bioreactor system, Nicotiana benthamiana which, unlike rice, allows transient transformation by Agrobacterium. Despite not being its natural host, we demonstrated a low level of RYMV-based vector replication in N. benthamiana leaves. Under optimized ratio, we showed that the P19 silencing suppressor in combination with the missing viral CP ORF significantly enhanced RYMV amplicon replication in N. benthamiana. Under these optimized CP/P19 conditions, we showed that the RYMV amplicon replicated autonomously in the infiltrated N. benthamiana cells, but was unable to move out of the infiltrated zones. Finally, we showed that when the RYMV amplicon was expressed under the optimized conditions we set up, it allowed enhanced PSA protein accumulation in N. benthamiana compared to the PSA coding sequence driven by the 35S promoter without amplicon background. CONCLUSION: This work demonstrates that a non-dicot-infecting virus can be used as an amplicon vector for the efficient production of proteins of interest such as PSA in N. benthamiana leaves.

First Report of Rice stripe necrosis virus Infecting Rice in Burkina Faso.

Rice stripe necrosis virus (RSNV) was first described in 1977 as a new virus infecting rice in Cote d'Ivoire (3) and was subsequently observed in Liberia, Nigeria, and Sierra Leone (2). RSNV is a soil-borne virus transmitted by the fungus Polymyxa graminis (1) and belongs to the genus Benyvirus (4). During a survey carried out in April of 2013, severe symptoms characterized by seedling death, severe plant malformation, and foliar striping were observed on rice plants in an experimental field of INERA at Banfora located in western Burkina Faso. Disease incidence in the field was estimated to be 80 ± 5%. The symptoms of disease were successfully transmitted to the susceptible rice (Oryza sativa) cultivar IR64 by soil transmission experiments (1). RSNV was detected by ELISA using a polyclonal antiserum (1), kindly provided by Dr. Denis Fargette, IRD, Montpellier, France. Total nucleic acid was extracted with TRIzol reagent (Invitrogen) from IR64 and field infected samples. The presence of the virus was confirmed by RT-PCR using primers 5'-CATCTTGTCGAGATGAG-3' and 5'-GCGTTGTCTTTATCAGTG-3' for specific sequences flanking the RNA2 CP gene. The RT-PCR product was directly sequenced and the sequence was deposited in GenBank (Accession No. LK023710). Sequence analysis showed that the CP gene of the RSNV isolate from Burkina Faso shared the highest nucleotide sequence identity (97.6%) with the known RSNV CP gene sequence from the Colombian isolate (EU099845) available in GenBank, confirming the presence of RSNV in the rice crops in Burkina Faso. To our knowledge, this is the first confirmed report of RSNV in Burkina Faso. Further studies are needed to determine its incidence and spread in the country. Detection of RSNV in Burkina Faso signals the urgent need for adoption of appropriate measures to restrict the spread and impact of this virus within Africa. References: (1) C. Fauquet and J. C. Thouvenel. Proc. Acad. Sci. Ser. D 296:575, 1983. (2) C. Fauquet et al. Develop. Appl. Biol. 2:71, 1988. (3) D. Louvel and J.-M. Bidaux. Agronomie Tropicale 32:257, 1977. (4) I. Lozano and F. Morales. Eur. J. Plant Pathol. 124:673, 2009.

Expression of rice yellow mottle virus coat protein enhances virus infection in transgenic plants.

The disease caused by rice yellow mottle virus (RYMV) is a major, economically important constraint to rice production in Africa. RYMV is mechanically transmitted by a variety of agents, including insect vectors. The production of resistant rice varieties would be an important advance in the control of the disease and increase rice production in Africa. We produced transgenic plants of the Oryza sativa japonica variety, TP309, to express a RYMV coat protein gene (CP) and mutants of the CP under the control of a ubiquitin promoter. Transgenic plants expressing genes that encode wild-type CP (wt.CP), deleted CP (DeltaNLS.CP), mRNA of the CP, or antisense CP sequences of the CP gene were characterised. Eighty per cent (80%) of independent transgenic lines analysed contained CP gene sequences. Transgenic plants were challenged with RYMV and produced two types of reactions. Most of the plants expressing antisense sequences of the CP and untranslatable CP mRNA exhibited a delay in virus accumulation of up to a week, and the level of virus accumulation was reduced compared with non-transgenic TP309 plants. Transgenic plants expressing RYMV wild-type CP (wt.CP) and deleted CP (DeltaNLS.CP) accumulated the highest levels of virus particles. These results suggest that antisense CP and untranslatable CP mRNA induced moderate resistance, whereas transgenic CP enhanced virus infection.

Analysis of the transcriptional response to Rice Yellow Mottle Virus infection in Oryza sativa indica and japonica cultivars.

Several cDNA libraries were constructed using mRNA isolated from roots, panicles, cell suspensions and leaves of non-stressed Oryza sativa indica (IR64) and japonica (Azucena) plants, from wounded leaves, and from leaves of both cultivars inoculated with Rice Yellow Mottle Virus (RYMV). A total of 5549 cleaned expressed sequence tags (ESTs) were generated from these libraries. They were classified into functional categories on the basis of homology, and analyzed for redundancy within each library. The expression profiles represented by each library revealed great differences between indica and japonica backgrounds. EST frequencies during the early stages of RYMV infection indicated that changes in the expression of genes involved in energy metabolism and photosynthesis are differentially accentuated in susceptible and partially resistant cultivars. Mapping of these ESTs revealed that several co-localize with previously described resistance gene analogs and QTLs (quantitative trait loci).

Comparison of molecular and immunological typing of isolates of Rice yellow mottle virus.

Isolates of Rice yellow mottle virus (RYMV) were typed at the molecular level through the sequences of the open reading frame (ORF) 4 (coding for the coat protein) and ORF1 (coding for the movement protein), and serologically by means of polyclonal and monoclonal antibodies. The overall patterns of diversity shown by molecular and serological analyses were similar: East-African isolates differed from West-African ones, and the West-African isolates from forest differed from the savannah ones. Each major strain had a different serological profile. However, molecular typing was more discriminating than immunological typing since several sequence variants belonged to the same serotype. In rare instances, there were explainable discrepancies between molecular and serological typing. Two amino acids at positions 115 (alanine vs threonine) and 191 (valine vs threonine) consistently discriminated between the major serotypes. These positions were located in antigenic sites as revealed by Spot-scan method and were recognised by discriminating monoclonal antibodies. One shared epitope, lying within a conserved region, may be responsible for the cross-reactivity between RYMV isolates. A rationale for the correlation between molecular and immunological typing of RYMV and other sobemoviruses is proposed.

3D domain swapping modulates the stability of members of an icosahedral virus group.

BACKGROUND: Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS: The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS: The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Structure of native and expanded sobemoviruses by electron cryo-microscopy and image reconstruction.

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 A 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 Ca(2+) 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.

Expression of the rice yellow mottle virus P1 protein in vitro and in vivo and its involvement in virus spread.

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 in in vitro translation 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.

Movement of rice yellow mottle virus between xylem cells through pit membranes.

The translocation of rice yellow mottle virus (RYMV) within tissues of inoculated and systemically infected Oryza sativa L. leaves was characterized by Western immunoblotting, Northern blotting, and electron microscopy of thin sections. In inoculated leaves, RYMV RNA and coat protein first were detected at 3 and 5 days postinoculation, respectively. By 6 days postinoculation, RYMV had spread systemically to leaves, and virus particles were observed in most cell types, including epidermal, mesophyll, bundle sheath, and vascular parenchyma cells. Most of the virions accumulated in large crystalline patches in xylem parenchyma cells and sieve elements. Colocalization of a cell wall marker for cellulosic beta-(1-4)-D-glucans and anti-RYMV antibodies over vessel pit membranes suggests a pathway for virus migration between vessels. We propose that the partial digestion of pit membranes resulting from programmed cell death may permit virus migration through them, concomitant with autolysis. In addition, displacement of the Ca2+ from pit membranes to virus particles may contribute to the disruption of the pit membranes and facilitate systemic virus transport.

Mass spectrometry and viral analysis.

BACKGROUND: Electrospray ionization (ESI) mass spectrometry is a powerful new approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry; it is not clear, however, whether the native conformation of the biomolecules is maintained throughout the ionization and analysis process. We set out to address this question using live viruses. RESULTS: Viral ions have been generated in the gas phase using electrospray ionization mass spectrometry. These ions have been collected, following ion filtering through the mass analyzer, and then analyzed by transmission electron microscopy. Transmission electron microscopy revealed that rice yellow mottle virus and tobacco mosaic virus retained their respective spherical and rod-like ultrastructure. The viability of the isolated tobacco mosaic virus was confirmed by inoculation and infection of tobacco plants. CONCLUSIONS: These results demonstrate the utility of electrospray for supramolecular complexes with molecular weights of over 40 million Da and offer conclusive evidence that native biomolecular structures can be conserved through the electrospray process.

The complete nucleotide sequence of yam mosaic virus (Ivory Coast isolate) genomic RNA.

The complete nucleotidic sequence of the yam mosaic virus (YMV) RNA was determined following the cloning of partial segments of the genome by reverse transcription and polymerase chain reactions (RT-PCR) using degenerate and/or specific oligonucleotide primers. YMV genomic RNA is 9,608 nucleotides in length and contains one open reading frame (ORF) encoding a polyprotein of 3,103 amino acids (aa) with a calculated Mr of 350,915. The 5' leader sequence of YMV RNA preceding the ORF is 134 nucleotides (nt) long while the 3' untranslated region (UTR) is 165 nt excluding the poly(A) tail. A computer algorithm predicted that the 3'UTR forms four stem loop structures which form a cloverleaf-like secondary structure. These structures apparently share some homologies with those observed in the 3'UTR of the potato virus Y-NL1 strain. Seven potential recognition sites for the NIa protease were found: one putative cleavage site for the P1 proteinase and one for the HC proteinase. The organization of the YMV genome is therefore similar to the other members of the genus Potyvirus based upon conserved sequence motifs common amongst members of this group. Despite its similarity with the other potyviruses in these conserved regions, YMV appears to be a distinct potyvirus species based upon a comparison of its sequence with those of other potyviruses.

Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein.

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.

Nucleotide sequence and genome characterization of rice yellow mottle virus RNA.

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.

Growth-related gene expression in Nicotiana tabacum mesophyll protoplasts.

Eight cDNAs whose genes are more strongly expressed in suspension cells in growth phase than in stationary phase and at a low level in mature leaves have been isolated. The corresponding mRNAs are abundantly accumulated in young plant organs and in germinating seeds but are almost undetectable in mature plant tissues and dry seeds. Six of these cDNAs were characterized by comparison of nucleotide and protein sequences to the EMBL and SWISSPROT databanks. These eight growth-related genes are expressed in protoplasts isolated from Nicotiana tabacum mesophyll cells shortly after preparation (4 h). Two of them are expressed in freshly isolated protoplasts (early genes), while the other six are detected after 4 h of culture (late genes). Seven are more abundantly expressed in protoplasts than in growing plant organs while one growth-related gene is weakly expressed in protoplasts, as is the histone H4 gene. They seem to be induced in protoplasts by a synergistic effect of wounding and maceration. Sustained expression of the early genes is dependent on the presence of sucrose in the culture medium.

The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed.

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h2) was isolated using thioredoxin h1 cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 bp respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h1 (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h1 and h2, do not have any signal peptides and are most probably cytoplasmic. Using the 3' regions of the mRNAs, two probes specific for thioredoxins h1 and h2 have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h1 and a 4.5 kb fragment encodes h2. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h2 is present in N. sylvestris and N. tomentosiformis but that thioredoxin h1 is absent from both putative ancestors. Thus, the thioredoxin h1 gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for an Amiloride-Inhibited Mg/2H Antiporter in Lutoid (Vacuolar) Vesicles from Latex of Hevea brasiliensis.

Lutoids represent a lysosomal microvacuolar compartment of rubber-tree (Hevea brasiliensis) latex. We observed acidification of isolated vesicles after imposing an outward Mg(2+) diffusion gradient and dissipation of a preformed pH gradient in the presence of exogenous Mg(2+). These results suggest the presence of a Mg(2+)/H(+) antiporter. The maximum Mg(2+)/H(+) exchange rate was observed at pH 8.5. The K(m) values for Mg(2+) (2.6 mm) were identical for both influx and efflux experiments. When membrane potential was clamped at zero with K(+) and valinomycin, the response of the membrane potential probe oxonol VI showed that the Mg(2+)/H(+) exchange was electroneutral. Mg(2+)/H(+) exchange was inhibited by amiloride and imipramine. Both the inhibiting concentration range and the K(m) for Mg(2+) are similar to those reported for the Mg(2+)/2Na(+) antiporter in animals cell. These data are consistent with the existence of a Mg(2+)/2H(+) antiporter in a plant tonoplast.