Rice grassy stunt virus p5 interacts with two protein components of the plant-specific CBL-CIPK Ca+2 signaling network of rice.

Rice grassy stunt virus (RGSV) is a tenuivirus posing a threat to rice production in many South, Southeast, and East Asian countries. To date, no host factor interacting with RGSV has been reported. In this study, we screened a rice cDNA library with the GAL4-based yeast two-hybrid system using RGSV p5 as the bait. One of the candidate host factors interacting with RGSV p5 was found to be CBL-interacting protein kinase 25 (OsCIPK25), a member of the plant-specific CBL-CIPK Ca2+ signaling network. The interaction between RGSV p5 and OsCIPK25, as well as OsCIPK5, which is closely related to OsCIPK25, was confirmed by their cellular co-localization and by a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Given the importance of CIPKs in the regulation of ion homeostasis and the resemblance of RGSV symptoms to potassium deficiency in rice, we evaluated potassium content of RGSV-infected rice and found it to be much lower than that in the healthy rice.

Screening anti-southern rice black-streaked dwarf virus drugs based on S7-1 gene expression in rice suspension cells.

Southern rice black-streaked dwarf virus (SRBSDV) is a rice pathogen that had an outbreak in southern China in 2010 and caused significant crop losses. Therefore, screening for effective antiviral drugs against SRBSDV is very important. This study used rice suspension cells infected with SRBSDV by polyethylene glycol-mediated uptake for screening antiviral drugs. SRBSDV P7-1, which is coded by the S7-1 gene, has an intrinsic ability to self-interact to form tubules that play an important role in viral infection. Therefore, relative expression level of the SRBSDV S7-1 gene in infected rice suspension cells was assayed by real-time quantitative polymerase chain reaction to evaluate the antiviral activities of various drugs. Dufulin displayed the highest inhibitory activity against SRBSDV S7-1 expression. In addition, changes in peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) activities were determined in inoculated and noninoculated cells. The results showed that both POD and PPO activities increased upon dufulin treatment. Furthermore, the validity of this approach was confirmed in an in vivo experiment in which dufulin was found to effectively inhibit SRBSDV.

Dot immunobinding assay method with chlorophyll removal for the detection of southern rice black-streaked dwarf virus.

Southern rice black-streaked dwarf virus (SRBSDV), a new virus from Fiji, has seriously damaged rice crops in southern China and northern Vietnam in recent years. This virus is difficult to diagnose in the early stages of infection, and is very destructive at the late stage. In the present study, a dot immunobinding assay (DIBA) that has a high sensitivity for diagnosing SRBSDV was developed. Two kinds of treatment for the DIBA were evaluated to determine the most effective one for removing chlorophyll interferences via rice extraction. The first included several reagents to remove chlorophyll, namely, the alkaline reagents like magnesium oxide and alumina oxide, the adsorbent reagents like activated carbon and bentonite, as well as the extraction agent acetone. The second and third treatments, which were used to remove chlorophyll in blot membrane-nitrocellulose and polyvinylidene fluoride (PVDF), included several organic solvents containing methanol, ethanol, acetone, ethyl acetate, and diethyl ether. The results showed that activated carbon and methanol yielded the best contrasting purple color for the infected samples by decreasing the chlorophyll content.

A negative element in the downstream region of the Rice tungro bacilliform virus promoter is orientation- and position-independent and is active with heterologous promoters.

The promoter of an Indian isolate of the pararetrovirus Rice tungro bacilliform virus (RTBV-WB) contains a negative element downstream of the transcription start site (TSS), between nucleotide residues +58 and +195 (Mathur and Dasgupta, 2007). To further characterize the element, we show, by using transient gus reporter gene assays in the cells of onion peel, rice calli and Arabidopsis leaves, that it down-regulates heterologous promoters CaMV35S and Maize ubiquitin. Quantitative measurements of transient GUS activity indicated more than 90% inhibition of reporter gene expression by the negative element. We also show, by reversing the orientation of the element downstream and by placing it in a position upstream to a constitutively expressing RTBV promoter, that the negative element is orientation- and position-independent, pointing towards its activity at the transcriptional and not post-transcriptional level.

Host range of rice yellow mottle virus in Sudano-Sahelian Savannahs.

In the present study, we investigated on the experimental host range of RYMV among plant species most of which are frequently encountered in rice-growing environments of west and central African savannahs. Only seven out of 66 plant species inoculated were infected by RYMV. All susceptible plant species belonged to the Poaceae family and three of them (Chloris prieuri, Eragrostis cilianensis and Shoenefeldia gracilis) were reported for the first time. Symptoms were conspicuous and persistent in most species but disappeared totally in older plants of some host species such as S. gracilis and Eragrostis tenella. Therefore, surveys for identification ofRYMV wild hosts should be conducted before the flowering stage. Virus-host Interactions were studied between 15 RYMV isolates of different strains and 10 wild host species. Differential reactions were obtained in the crow-foot grass Dactyloctenium aegyptium which was susceptible to five of the fifteen isolates. All other plants were susceptible to the whole set of virus isolates. Altogether, this study underlined the narrowness of RYMV host range and pointed out the complexity of interactions between the virus and its hosts, especially the rationale behind overcoming host barriers.

Mechanism of ribosome shunting in Rice tungro bacilliform pararetrovirus.

In plant pararetroviruses, pregenomic RNA serves both as a template for replication through reverse transcription and a polysictronic mRNA. This RNA has a complex leader sequence preceding the first large ORF. The leader contains multiple short ORFs and strong secondary structure, both inhibiting ribosome scanning. Translation on this RNA is initiated by shunting, in which scanning ribosomes bypass a large portion of the leader with the inhibitory secondary structure and short ORFs. In Cauliflower mosaic virus (CaMV), the ribosome shunting mechanism involves translation of the 5'-proximal short ORF terminating in front of the secondary structure that appears to force ribosomes to take off and resume scanning at a landing site downstream of the structure. Using two plant protoplast systems and shunt-competent wheat-germ extracts, we demonstrate that in Rice tungro bacilliform virus (RTBV) shunting also depends on the first short ORF followed by strong secondary structure. Swapping of the conserved shunt elements between CaMV and RTBV revealed the importance of nucleotide composition of the landing sequence for efficient shunting. The results suggest that the mechanism of ribosome shunting is evolutionary conserved in plant pararetroviruses.

Detection of rice grassy stunt tenuivirus nonstructural proteins p2, p5 and p6 from infected rice plants and from viruliferous brown planthoppers.

The genome of Rice grassy stunt virus (RGSV) consists of 6 ambisense RNA segments, among which RNAs 1, 2, 5 and 6 are equivalent to RNAs 1, 2, 3 and 4, respectively, of Rice stripe virus, the type species of the genus Tenuivirus. The RGSV 36-kDa nucleocapsid protein (N) is encoded on the complementary strand of RNA 5. Here, we studied accumulation of three nonstructural proteins, a 23-kDa p2 protein encoded on vRNA 2 (virus genomic strand), a 22-kDa p5 protein encoded on vRNA 5, and a 21-kDa p6 protein encoded on vRNA 6, from RGSV-infected rice leaf tissues and from viruliferous vector insects (brown planthopper, Nilaparvata lugens) by Western blot analyses. p2, p5 and p6 were detected from RGSV-infected rice leaf homogenates; p2 was detected mostly in the cytoplasmic soluble fraction but also a small amount was detected in the cell-wall, organelle-enriched and crude membrane fractions; p5 and p6 were detected from the cytoplasmic soluble fraction in large amounts. Among individual nymphs of N. lugens raised on RGSV-infected rice leaves, only 20% of insects were positive with the N protein. A large amount of p5 was detected from all the N-positive insects. Small amounts of p2 and p6 were detected only from a subset of the N- and p5-positive insects. p5 may have an essential role in virus infection in both plant and insect hosts, whereas p2 may function in plants such as a cell-to-cell movement protein.

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.

Detection of rice tungro bacilliform virus in field and glasshouse samples from India using the polymerase chain reaction.

Rice tungro bacilliform virus (RTBV) together with rice tungro spherical virus (RTSV) is the causal agent for the rice tungro disease. A rapid technique was developed to detect RTBV DNA in the crude extract of freshly collected leaf samples by polymerase chain reaction (PCR). This technique can detect the viral DNA in 1000-fold diluted leaf extract. Detection has been possible in samples stored upto 5 days after the collection. This technique may have wide application for the field diagnosis of RTBV infection.

Structure of sesbania mosaic virus at 3 A resolution.

BACKGROUND: Sobemoviruses are a group of RNA plant viruses that have a narrow host range. They are characterized in vitro by their stability, high thermal inactivation point and longevity. The three-dimensional structure of only one virus belonging to this group, southern bean mosaic virus (SBMV), is known. Structural studies on sesbania mosaic virus (SMV), which is closely related to SBMV, will provide details of the molecular interactions that are likely to be important in the stability and assembly of sobemoviruses. RESULTS: We have determined the three-dimensional structure of SMV at 3 A resolution. The polypeptide fold and quaternary organization are very similar to those of SBMV. The capsid consists of sixty icosahedral asymmetric units, each comprising three copies of a chemically identical coat protein subunit, which are designated as A, B and C and are in structurally different environments. Four cation-binding sites have been located in the icosahedral asymmetric unit. Of these, the site at the quasi-threefold axis is not found in SBMV. Structural differences are observed in loops and regions close to this cation-binding site. Preliminary studies on ethylene diamine tetra acetic acid (EDTA) treated crystals suggest asymmetry in removal of the quasi-equivalent cations at the AB, BC, and AC subunit interfaces. CONCLUSIONS: Despite the overall similarity between SMV and SBMV in the nature of the polypeptide fold, these viruses show a number of differences in intermolecular interactions. The polar interactions at the quasi-threefold axis are substantially less in SMV and positively charged residues on the RNA-facing side of the protein and in the N-terminal arm are not particularly well conserved. This suggests that protein-RNA interactions are likely to be different between the two viruses.

Detection of rice tungro bacilliform virus gene products in vivo.

To study the products of the open reading frames (ORFs) of rice tungro bacilliform virus in rice plants the sequences containing ORFs I (encoding a 24-kDa protein, P24) and IV (P46) and the protease and polymerase (reverse transcriptase+RNaseH) domains of ORF III were cloned into a pGEX expression vector. The proteins, which were C-terminal fusions to glutathione S-transferase, were expressed in Escherichia coli and antisera were raised against them which, together with an antiserum against virus particles, was used to probe blots of proteins from infected and uninoculated plants and from virus preparations. The P24 antiserum detected virus-specific proteins of 74, 60, and 52 kDa, which are much bigger than expected. These proteins were found in virus preparations and immunogold labeling suggested that they might be internal in the particles. Virus-specific proteins of 33, 37, 62, and > 150 kDa were revealed by antiserum to virus particles. The antiserum to the protease revealed proteins of 13.5, 37, and 68 kDa both in extracts from infected plants and in purified virus preparations. This antiserum decorated intact virus particles as did the particle antiserum. The polymerase domain antiserum reacted with products of 56, 65, and 68 kDa in extracts from infected plants but not in virus particles. The antiserum to the ORF IV product did not detect any bands in either infected plant extracts or virus preparations. The significance of these products is discussed.