Targeting of rice grassy stunt virus pc6 protein to plasmodesmata requires the ER-to-Golgi secretory pathway and an actin-myosin VIII motility system.

The nonstructural protein pc6 encoded by rice grassy stunt virus (RGSV) plays a significant role in viral cell-to-cell movement, presumably by transport through plasmodesmata (PD). We confirmed the association of pc6 with PD, and also elucidated the mechanisms of protein targeting to PD. Several inhibitor treatments showed conclusively that pc6 is targeted to PD via the ER-to-Golgi secretory system and actin filaments. In addition, VIII-1 myosin was also found to be involved in pc6 PD targeting. Deletion mutants demonstrated that C-terminal amino acid residues 209-229 (transmembrane domain 2; TM2) are essential for pc6 to move through PD.

Model-based structural and functional characterization of the Rice stripe tenuivirus nucleocapsid protein interacting with viral genomic RNA.

Rice stripe tenuivirus (RSV) is a filamentous, negative-strand RNA virus causing severe diseases on rice in Asian countries. The viral particle is composed predominantly of a nucleocapsid protein (NP) and genomic RNA. However, the molecular details of how the RSV NP interacts with genomic RNA during particle assembly remain largely unknown. Here, we modeled the NP-RNA complex and show that polar amino acids within a predicted groove of NP are critical for RNA binding and protecting the RNA from RNase digestion. RSV NP formed pentamers, hexamers, heptamers, and octamers. By modeling the higher-order structures, we found that oligomer formation was driven by the N-terminal amino arm of the NP. Deletion of this arm abolished oligomerization; the N-terminally truncated NP was less able to interact with RNA and protect RNA than was the wild type. These findings afford valuable new insights into molecular mechanism of RSV NPs interacting with genomic RNA.

Artificial feeding Rice stripe virus enables efficient virus infection of Laodelphax striatellus.

Rice stripe virus (RSV), the causative agent of rice stripe disease, is transmitted by Laodelphax striatellus in a persistent-propagative manner. Efficient virus acquisition is primary for studies of virus transmission and virus-insect vector interactions. However, under greenhouse conditions, less than 30% of the L. striatellus population, on average, become viruliferous during feeding on RSV-infected plants. Here, we explored a method for efficient RSV acquisition by feeding the insects with a virus-containing artificial diet. Virus particles were partially purified from frozen infected rice leaves. A series of RSV concentrations in a 5% sucrose solution were tested in the feed of L. striatellus nymphs. The percentage of infected insects increased along with the increasing viral concentration, and the highest infection percentage 96% was achieved using a 1200ngµL(-1) crude RSV suspension after 48h feeding. RSV particles acquired in this manner were able to spread to L. striatellus salivary glands. This improved method of obtaining viruliferous insects should assist the study of RSV transmission mechanisms in L. striatellus.

Mapping of the regions involved in self-interaction of rice stripe virus P3 protein.

Rice stripe virus (RSV) protein P3 is a suppressor of RNA silencing in plants. P3 has been shown by biomolecular fluorescence complementation assay to self-interact in planta but the regions responsible for homotypic interaction have not been determined. Here we analyzed the domains for the self-interaction of P3 by using yeast two-hybrid, co-immunoprecipitation and fluorescence experiments. The results showed that P3 was also able to interact with itself in yeast and insect cells. The domain responsible for P3-P3 interaction was mapped to amino acids 15-30 at the N-terminal region of P3. Furthermore, subcellular localization suggested that the homo-oligomerization was the prerequisite for P3 to form larger protein aggregates in the nucleus of insect cell.

Rice stripe tenuivirus NSvc2 glycoproteins targeted to the golgi body by the N-terminal transmembrane domain and adjacent cytosolic 24 amino acids via the COP I- and COP II-dependent secretion pathway.

UNLABELLED: The NSvc2 glycoproteins encoded by Rice stripe tenuivirus (RSV) share many characteristics common to the glycoproteins found among Bunyaviridae. Within this viral family, glycoproteins targeting to the Golgi apparatus play a pivotal role in the maturation of the enveloped spherical particles. RSV particles, however, adopt a long filamentous morphology. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. Here, we demonstrate that the amino-terminal NSvc2 (NSvc2-N) targets to the Golgi apparatus in Nicotiana benthamiana cells, whereas the carboxyl-terminal NSvc2 (NSvc2-C) accumulates in the endoplasmic reticulum (ER). Upon coexpression, NSvc2-N redirects NSvc2-C from the ER to the Golgi bodies. The NSvc2 glycoproteins move together with the Golgi stacks along the ER/actin network. The targeting of the NSvc2 glycoproteins to the Golgi bodies was strictly dependent on functional anterograde traffic out of the ER to the Golgi bodies or on a retrograde transport route from the Golgi apparatus. The analysis of truncated and chimeric NSvc2 proteins demonstrates that the Golgi targeting signal comprises amino acids 269 to 315 of NSvc2-N, encompassing the transmembrane domain and 24 adjacent amino acids in the cytosolic tail. Our findings demonstrate for the first time that the glycoproteins from an unenveloped Tenuivirus could target Golgi bodies in plant cells. IMPORTANCE: NSvc2 glycoprotein encoded by unenveloped Rice stripe tenuivirus (RSV) share many characteristics in common with glycoprotein found among Bunyaviridae in which all members have membrane-enveloped sphere particle. Recently, RSV NSvc2 glycoproteins were shown to localize exclusively to the ER in Sf9 insect cells. In this study, we demonstrated that the RSV glycoproteins could target Golgi bodies in plant cells. The targeting of NSvc2 glycoproteins to the Golgi bodies was dependent on active COP II or COP I. The Golgi targeting signal was mapped to the 23-amino-acid transmembrane domain and the adjacent 24 amino acids of the cytosolic tail of the NSvc2-N. In light of the evidence from viruses in Bunyaviridae that targeting Golgi bodies is important for the viral particle assembly and vector transmission, we propose that targeting of RSV glycoproteins into Golgi bodies in plant cells represents a physiologically relevant mechanism in the maturation of RSV particle complex for insect vector transmission.

The Rice stripe virus pc4 functions in movement and foliar necrosis expression in Nicotiana benthamiana.

The Rice stripe virus (RSV) pc4 has been determined as the viral movement protein (MP). In this study, the pc4 gene was cloned into a movement-deficient Tobacco mosaic virus (TMV). The resulting hybrid TMV-pc4, in addition to spreading cell to cell in Nicotiana tabacum, moved systemically and induced foliar necrosis in Nicotiana benthamiana, indicating novel functions of the RSV MP. A systematic alanine-scanning mutagenesis study established the region K(122)-D(258) of the pc4 substantially associated with cell-to-cell movement, and mutants by replacement of KGR(122-124), D(135), ED(170-171), ER(201-202), EFE(218-220) or ELD(256-258) with alanine(s) no longer moved cell to cell. However, only one amino acid group KGR(122-124) was linked with long-distance movement. The region D(17)-K(33) was recognized as a crucial domain for leaf necrosis response, and mutagenesis of DD(17-18) or RK(32-33) greatly attenuated necrosis. The overall data suggested manifold roles of the pc4 during the RSV infection in its experimental host N. benthamiana.

Concentración mínima inhibitoria de higromicina B en callos embriogénicos de arroz -Oryza sativa L / Minimum inhibitory concentration of higromycin B in rice embryogenic calli -Oryza sativa L

Uno de los elementos imprescindibles en la ingeniería genética de plantas es un sistema de selección eficiente. El propósito de este trabajo fue evaluar la sensibilidad al marcador de selección higromicina B, de callos embriogénicos obtenidos a partir del escutelo de semilla de tres variedades colombianas de arroz (FEDEARROZ 2000, FEDEARROZ 50 y FEDEARROZ 369). Además, se validó la respuesta de estas variedades al protocolo de regeneración empleado. Se probaron cuatro concentraciones del antibiótico (25 mg/L, 50 mg/L, 75 mg/L y 100 mg/L) más un control sin higromicina B. Los resultados obtenidos mostraron que una concentración de 50 mg/L de antibiótico en el medio de regeneración es adecuada para la selección. Con esta concentración se impide la formación de brotes, aunque los callos no mueren completamente. Por otra parte, se estableció que el protocolo de regeneración utilizado es de baja eficiencia y, por consiguiente, es necesario optimizarlo para poder usarlo en procesos de ingeniería genética de cultivares colombianos de arroz.

An efficient selection system is one of the most important elements of plant genetic engineering. The purpose of this study was to evaluate the sensitivity of scutellum-derived embriogenic calli obtained from three colombian rice varieties (FEDEARROZ 2000, FEDEARROZ 50 and FEDEARROZ 369), to the selection marker hygromycin B. Aditionally, the response of these varieties to the regeneration protocol was measured. Four antibiotic concentrations were tested (25 mg/L, 50 mg/L, 75 mg/L and 100 mg/L) plus one control without hygromycin B. The results show that 50 mg/L of antibiotic in the regeneration medium is adequate for selection. This concentration prevents the formation of shoots, though the calli do not die. It was also established that the regeneration protocol is a low-efficiency system and it needs to be improved, in order to use it for colombian rice genetic engineering.

Structural implications into dsRNA binding and RNA silencing suppression by NS3 protein of Rice Hoja Blanca Tenuivirus.

Rice Hoja Blanca Tenuivirus (RHBV), a negative strand RNA virus, has been identified to infect rice and is widely transmitted by the insect vector. NS3 protein encoded by RHBV RNA3 was reported to be a potent RNAi suppressor to counterdefense RNA silencing in plants, insect cells, and mammalian cells. Here, we report the crystal structure of the N-terminal domain of RHBV NS3 (residues 21-114) at 2.0 Å. RHBV NS3 N-terminal domain forms a dimer by two pairs of α-helices in an anti-parallel mode, with one surface harboring a shallow groove at the dimension of 20 Å × 30 Å for putative dsRNA binding. In vitro RNA binding assay and RNA silencing suppression assay have demonstrated that the structural conserved residues located along this shallow groove, such as Arg50, His51, Lys77, and His85, participate in dsRNA binding and RNA silencing suppression. Our results provide the initial structural implications in understanding the RNAi suppression mechanism by RHBV NS3.

Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing.

RNA interference is a natural defense against viruses in plants. To date, the only viral siRNAs characterized have been those for positive-sense RNA viruses with one or two genome components. Here, we characterized siRNAs derived from rice stripe virus (RSV), a member of the genus Tenuivirus with four genomic RNAs and an ambisense coding strategy. Deep sequencing of small RNAs from infected rice leaves showed that siRNAs were derived almost equally from virion and complementary RNA strands and were mostly 20-22 nucleotides long. Most viral siRNAs were produced within the coding sequences and 5' termini of the RSV genome. RSV siRNAs had a higher G and lower C content than the viral genome but a strong A/U bias at the first nucleotide and a U bias at the final one, suggesting preferential targeting of such sequences by rice Dicer-like proteins.

Nucleic acid binding property of the gene products of rice stripe virus.

GST fusion proteins of the six gene products from RNAs 2,3 and 4 of the tenuivirus, Rice stripe virus (RSV), were used to study the nucleic acid binding activities in vitro. Three of the proteins, p3, pc3 and pc4, bound both single- and double-stranded cDNA of RSV RNA4 and also RNA3 transcribed from its cDNA clone, while p2, pc2-N (the N-terminal part of pc2) nor p4 bound the cDNA or RNA transcript. The binding activity of p3 is located in the carboxyl-terminus amino acid 154-194, which contains basic amino acid rich beta-sheets. The acidic amino acid-rich amino-terminus (amino acids 1-100) of p3 did not have nucleic acid binding activity. The related analogous gene product of the tenuivirus, Rice hoja blanca virus, is a suppressor of gene silencing and the possibility of the nucleic acid binding ability of RSV p3 being associated with this property is discussed. The C-terminal part of the RSV nucleocapsid protein, which also contains a basic region, binds nucleic acids, which is consistent with its function. The central and C-terminal regions of pc4 bind nucleic acid. It has been suggested that this protein is a cell-to-cell movement protein and nucleic acid binding would be in accord with this function.

Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.

Expression of the rice hoja blanca virus (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its in situ localization in RHBV-infected rice tissues.

The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Rice grassy stunt tenuivirus nonstructural protein p5 interacts with itself to form oligomeric complexes in vitro and in vivo.

We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).

[Sequence aanlysis of intergenic region of rice stripe virus RNA4: evidence for mixed infection and genomic variation].

The intergenic region (IR) of the RNA4 of 22 isolates of Rice stripe virus (RSV) in China was cloned and sequenced. The IR sequences were compared with one another and with that from Japan. Sequence comparisons showed that these isolates could be divided into three different types, with the IR length of 634 bp, 654 bp and 732 bp, respectively. It is interesting to note three different types all occurred in Yunnan RSV natural population, whereas other province only existed 654 bp type length isolates. Mixed infections with different types of IR length coexisting in some isolates in Yunnan was observed. IR sequences were not more conserved (83% - 100%) among the populations of RSV from China than with those of RSV isolates from Japan (83% - 94%). There were two important structure characteristics in IRs sequences. Firstly, there was a-19 nt insertion in 654 bp type isolates and a-103 nt in 732 bp type isolates in comparison to 634 bp type isolates. This inserted sequences were rather highly conserved. Blast analysis indicates the 16 nt (AGAAACATGAGAGTA) in 19 nt insertation was very similar in sequence to wheat cDNA library; and the 20 nt (AGAATTGCCTTGGTGTTAT) in 103 nt insertion was identical to a stretch sequences of barley cDNA library. Recombination hot-spot sequences existed in RNA4 IR. Secondly, IRs sequence was rich in U and A residues where two distant hairpin structures could be formed with computer-assisted folding analysis. One was highly conserved and stable, but the other was rather unstable because of bases variation. It is believed that this stabilised hairpin structure, rather than a sequence motif, might serve as a transcription terminator during the synthesis of mRNAs from the ambisense segments. Negative selection constraints imposed by secondary structure might have maintained the conserved sequences. In this paper, the relationship between the lowest free energy of the unstable hairpin structures and the different pathogenesis among some isolates was also discussed in this paper.

Selection and expression of peptides which can change the conformation of p20 protein of rice stripe virus.

Phages with high affinity to the P20 protein of rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of phage display screening. Nine different peptides from the enriched library were selected by enzyme-linked immunosorbent assay (ELISA). The P20 protein from raw extracts of rice leaves infected with RSV could be detected by those 9 peptides displayed on the phage, which suggested that a peptide could be an effective tool for diagnosis of RSV in rice and planthopper. Circular dichroism (CD) spectra of P20 fusion proteins with the binding phages and non-binding phages showed that the conformation of P20 protein was changed after binding to each of the 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of the P20 protein. Thereafter, those peptides might be used to develop plant resistance and disrupt virus transmission. Three of the 12-mer peptide genes were fused with the glutathione-S-transferase (GST) gene in the vector pGEX 3X. The fusion proteins were obtained from an Escherichia coli expression system and purified. The fusion proteins might have a potential to develop a plant peptide-based resistance to its pathogens and virus diagnosis. It also provided a tool (i) to confirm the inhibition of the function of P20 protein by the fusion peptides in vivo, and (ii) to detect the function of P20 protein and the interaction between the virus and its vector.