Rice Hoja Blanca Virus Research Articles

Expression and inheritance of hypersensitive resistance to rice hoja blanca virus mediated by the viral nucleocapsid protein gene in transgenic rice.

Rice hoja blanca virus (RHBV) is a major virus disease of economic importance affecting rice in northern South America, Central America and the Caribbean. This is the first report of transgenic resistance to RHBV and the transformation of an indica rice variety from Latin America. Rice transformed with the RHBV nucleocapsid protein ( N) gene had a significant reduction in disease development. Several reactions were observed that ranged from susceptible to completely resistant plants (immunity). The resistant reactions were characterized by the production of local lesions like a hypersensitive reaction or a recovery phenotype with the emergence of symptom-less new leaves. These transgenic RHBV-resistant rice lines expressed the N gene RNA at low levels that were below the detection limit by Northern blots and only resolved by RT-PCR. The nucleocapsid protein could not be detected in any of the transgenic plants either by Western or ELISA tests. These results suggest that the resistance encoded by the N gene in these plants appears to be mediated by RNA. When challenged with RHBV, the resistant transgenic lines showed a significant increased performance for important agronomic traits including the number of tillers, the number of grains per plant and the yield as compared to the susceptible control. Furthermore, upon inoculation some of the most-resistant transgenic lines showed agronomic traits similar to the uninoculated non-transgenic Cica 8 control. Using both agronomic traits and disease severity as criteria, several of the most-resistant lines were followed through the R(4) generation and demonstrated that the N gene and RHBV resistance was inherited in a stable manner. These transgenic rice lines could become a new genetic resource in developing RHBV-resistant cultivars.

Phylogenetic placement of a novel tenuivirus from the grass Urochloa plantaginea.

Evidence is presented that a tenuivirus recovered from the grass Urochloa plantaginea is probably a novel tenuivirus species, to be called Urochloa hoja blanca virus (UHBV). It is related to both Echinochloa hoja blanca virus (EHBV) and Rice hoja blanca virus (RHBV), and these three form a group distinct from Maize stripe virus (MStV) and Rice stripe virus (RStV). Phylogenetic analysis of the sequence data for RNA-3 and RNA-4 of these viruses supports the hypothesis that EHBV and UHBV may have evolved from an ancestral form of RHBV, precipitated by the introduction of Echinochloa colona and Urochloa plantaginea to America.

Infection of barley protoplasts with rice hoja blanca tenuivirus. Brief report.

A barley protoplast system has been established that supports replication of Rice hoja blanca tenuivirus (RHBV). Following polyethylene glycol-mediated RHBV inoculation of barley protoplasts, newly synthesized viral RNAs and proteins could be detected. Time course analyses revealed de novo synthesis of genome length viral RNA4, as well as subgenomic-sized RNA4 molecules of both polarities. Two proteins, N and NS4, encoded by viral complementary RNA3 and viral RNA4 respectively, were detected by Western immunoblot analysis. The barley protoplast system thus constitutes a promising tool for in vivo studies of the sequential steps involved in the multiplication cycle of RHBV.

Rice Virus Diseases: Epidemiology and Management Strategies

ABSTRACT There are now over 30 viruses reported to infect rice through experimental tests and in nature. However, only 25 are of any direct economic impact to rice production. And most of them are transmitted by either plant or leaf hoppers. The rice grassy stunt tenuivirus (RGSV), rice hoja blanca tenuivirus (RHBV), rice ragged stunt phytoreovirus (RRSV), rice wilted stunt virus (RWSV) (a relative of Rice grassy stunt tenuivirus (RGSV)), rice stripe tenuivirus (RSV), rice black streaked dwarf fijivirus (RBSDV) are plant hopper borne, while rice bunchy stunt pytoreovirus (RBSV), rice dwarf phytoreovirus (RDV), rice gall dwarf phytoreovirus (RGDV), rice transitory yellowing rhabdovirus (RTYV), rice yellow dwarf mycoplasma-like organisms (RYDMLO's), rice waika machlovirus (RWV), rice orange leaf virus (ROLV), maize streak geminivirus (MSV) strain A, and rice tungro machlovirus/badnavirus (RTV) are transmitted by leafhoppers. The African specific rice yellow mottle sobemovirus (RYMV) is transmitted by the Chrysomelid beetles, whereas rice chlorotic streak virus (RCSV) is transmitted by the rice mealybug. The rice guillame luteovirus (RGV) is aphidborne while rice necrosis mosaic luteovirus (RNMV) and rice necrosis furovirus (RNV) are transmitted by the fungus Polymyxa graminis. Two viruses, rice wrinkled stunt disease (RWSD) and witches' broom disease (WBD) are seedborne. The rice crinkle disease (RCD) is transmitted through soil while rice mosaic virus (RMV) is so far associated with contact transmission alone. While the majority of these viruses occur in countries of Asia and the Americas, only 5 (rice stripe necrosis furovirus, rice crinkle disease, maize streak geminivirus strain A, African cereal streak virus, and rice yellow mottle sobemo-virus) are known to infect rice in nature in Africa and its neighbouring islands. The underlying causes of epidemics of these viruses and their pests are primarily based on the intensification of rice cultivation in these areas with the introduction of new high yielding varieties in conjunction with increased use of irrigation facility, fertilizer, pesticides, changing cultural practices, crop monoculture, and obvious mechanical contamination. It is worthwhile to note that in Asia and the Americas it is the japonica rice types that are mostly susceptible to the viruses whereas in Africa and the neighbouring islands it is the indica rice types that are highly susceptible to the rice yellow mottle sobemovirus. The management strategies for rice virus diseases involve the use of screening and breeding for varietal resistance, chemical treatment, plant quarantine, efficient cultural practices, integrated pest management, training for disease management, genetically engineered resistance and constant collaboration with other laboratories working to achieve the same goal of disease management.

Characterization of the in vitro activity of the RNA-dependent RNA polymerase associated with the ribonucleoproteins of rice hoja blanca tenuivirus.

An RNA-dependent RNA polymerase (RdRp) activity associated with the ribonucleoproteins of rice hoja blanca tenuivirus (RHBV) was detected and analyzed. Conditions for in vitro RNA synthesis and for coupled RNA synthesis-translation of RHBV were established. In both cases, synthesis of the viral and viral complementary genomic and subgenomic RNA3 and RNA4 were observed, demonstrating that both transcription and replication occurred. Though coupling of RNA synthesis to translation allowed efficient translation of the newly synthesized subgenomic RNAs, studies of the effect of various inhibitors of protein synthesis revealed that RNA synthesis was independent of translation. Primer extension experiments demonstrated that in the presence of capped exogenous RNAs, a stretch of 10 to 16 nonviral nucleotides was added to the 5' end of a population of newly synthesized viral complementary RNA4. It appears that in addition to RdRp activity, RHBV-associated protein(s) also possessed cap-snatching capacity.

Sequence of echinochloa hoja blanca tenuivirus RNA-5.

The sequence is presented of RNA-5 of Echinochloa hoja blanca tenuivirus, a second tenuivirus associated with rice cultivation in Latin America (after rice hoja blanca virus). The RNA is 1334 nucleotides long and contains in the complementary sense RNA a single long open reading frame. The deduced amino acid sequence of this open reading frame shows that it encodes a highly basic and hydrophilic 44 kD protein (pc5) with about 50% similarity to the pc5 protein of maize stripe virus (MStV). This and other features of the RNA are discussed.

Sequence of Echinochloa hoja blanca tenuivirus RNA-4.

The sequence is presented of RNA-4 of Echinochloa hoja blanca tenuivirus (EHBV), one of two tenuiviruses associated with rice cultivation in Latin America (together with rice hoja blanca virus; RHBV). Analysis of the sequence shows that the coding regions of EHBV RNA-4 are closely related to those of RHBV RNA-4. However, the intergenic region separating the two ambisense open reading frames, are highly distinct for the two viruses. The features of the RNA and the comparisons with the sequences of RNA-4 of RHBV, rice stripe virus (RStV) and maize stripe virus (MStV) are discussed.

Sequence of Echinochloa hoja blanca tenuivirus RNA-3.

Analysis of the sequence of the 2336 nucleotide RNA-3 of Echinochloa hoja blanca tenuivirus shows that it is closely related to RNA-3 of rice hoja blanca tenuivirus, the principal virus disease of rice in Latin America. This is especially true for the coding regions, where the viruses are almost 90% similar. However, the non-coding regions of RNA-3 of these viruses, principally the intergenic region separating the two ambisense open reading frames, are only about 50% similar, suggesting that these are distinct viruses. The results closely resemble those obtained for the analysis of RNA-4 of these viruses, both in the absolute and relative percentage similarities of the coding and non-coding regions. This implies a coordinated evolution of the different tenuivirus RNA segments. The features of the RNA and the comparisons with the sequences of RNA-3 of RHBV, rice stripe virus (RStV) and maize stripe virus (MStV) are discussed.

Rapid, small scale purification of rice hoja blanca and Echinochloa hoja blanca tenuivirus ribonucleoprotein

Highly purified tenuivirus ribonucleoprotein was obtained from small amounts of leaf tissue by sedimenting the ribonucleoprotein particles from debris-free plant extract into a 30% sucrose cushion, in 1.5-mL microfuge tubes. Using this protocol, significant size differences were discovered in the double-stranded forms of the viral genomic RNAs of rice hoja blanca tenuivirus and a tenuivirus isolated from Echinochloa colonum, a common weed of rice cultivation.

Sequence of rice hoja blanca tenuivirus RNA-2.

The sequence of rice hoja blanca tenuivirus RNA-2 is analysed and compared to its counter-part in rice stripe tenuivirus. The RNA encodes two proteins, in an ambisense arrangement. The 94 kD pc2, located in the complementary sense RNA, has several features typical of viral membrane (glyco)proteins, and also has regions of local homology to the glycoproteins of the Phleboviruses (Bunyaviridae). The 23 kD pv2 lies in the viral sense RNA and has two small conserved domains that are almost exclusively found in retro-viral membrane glycoproteins. Its genome location is analogous to the NSm protein of several of the Bunyaviridae species, which is thought to have a membrane-related function. The two open reading frames are separated by a large intergenic region which, in common with the other tenuivirus ambisense RNA segments, has a short region that is highly conserved between RStV and RHBV. The significance of these results with respect to the virus structure and gene expression is discussed.

Sequence of the PV2 gene of rice hoja blanca tenuivirus RNA-2.

Comparison of a partial sequence of rice hoja blanca tenuivirus RNA-2 with 40% similarity to rice stripe tenuivirus RNA-2 revealed regions of high local sequence homology at the 5' terminus, within the coding region (the pv2 gene), and in the intergenic region separating this gene from the other protein (pc2) encoded by this ambisense RNA. Analysis of the conserved regions of the pv2 protein identified two motifs found principally in viral membrane glycoproteins and six motifs found each in a wide variety of proteins. The possible significance of these results is discussed.

Capped nonviral sequences at the 5' end of the mRNAs of rice hoja blanca virus RNA4.

Subgenomic RNAs of both polarities corresponding to rice hoja blanca virus (RHBV) ambisense RNA4 were detected in RHBV-infected rice tissues. Total RNA extracted from RHBV-infected and noninfected rice tissues and RNA4 purified from RHBV ribonucleoprotein particles were used as templates for primer extension studies. The RNAs extracted from RHBV-infected tissues contain a population of RNA molecules with 10 to 17 nonviral nucleotides at their 5' end. The RNA-cDNA hybrids resulting from primer extension of such RNA molecules were specifically immunoselected with anti-cap antibodies, indicating that the subgenomic RNAs are capped and probably serve as mRNAs and that the additional nucleotides at their 5' end possibly derive from host mRNAs via a cap-snatching mechanism.

Characteristics of resistance in rice to rice hoja blanca virus (RHBV) and its vector, Tagosodes orizicolus (Muir)

SummaryThis paper characterises resistance to rice hoja blanca virus (RHBV) which is transmitted by the planthopper Tagosodes orizicolus (Muir). Resistance is expressed as decreased proportion of plants infected compared to susceptible lines, although severity of symptom expression is similar in both types. This resistance is not due to differences in vector feeding behaviour. Vectors reared eight generations on resistant plants showed no increased ability to transmit to resistant lines or decreased ability to transmit to susceptible ones. Longevity of vectors was similar when reared either on virus‐resistant or susceptible plants. Incubation period of the virus in resistant plants are significantly longer than in susceptible plants. Resistance increased with plant age in resistant and susceptible cultivars. Increased virus dosage, as determined by increased number of viru‐liferous vectors per inoculated plant, caused an increase in transmission to both resistant and susceptible cultivars. However, the ranking of resistant and susceptible remained the same across the experimental range of dosage and plant age. It is concluded that the resistance studied is to virus infection and there is little risk of “breakdown” occurring as a result of genetic or behavioural changes in the vector population. This will permit the use of economic thresholds to planthopper feeding damage with little risk of RHBV epidemic outbreak.

Complete nucleotide sequence and coding strategy of rice hoja blanca virus RNA4.

The complete sequence of rice hoja blanca virus (RHBV) RNA4 has been determined, based on the sequence of the corresponding cDNA clones. RNA4 consists of 1991 nucleotides with two open reading frames (ORFs). One putative ORF is located in the 5'-proximal region of the viral RNA4; it encodes a protein of predicted M(r) 20076 which corresponds to the major non-structural protein that accumulates in RHBV-infected rice plants, and which bears limited sequence identity with the helper component of tobacco vein mottling potyvirus. The other ORF is located in the 5'-proximal region of the viral complementary RNA4 and encodes a protein of predicted M(r) 32,469. Between the two ORFs is an intergenic region of 524 nucleotides, part of which can theoretically adopt a stable stem-loop structure; the 5' and 3' ends can potentially base-pair over 16 nucleotides, producing a pan-handle configuration. These characteristics are in favour of an ambisense coding strategy for RHBV RNA4.

In situ immunogold labeling analysis of the rice hoja blanca virus nucleoprotein and major noncapsid protein

Ribonucleoprotein particles (RNPs) of rice hoja blanca virus (RHBV) were purified and used for electron microscopic analysis and antibody production. Antibodies made to RNPs specifically decorated purified RNPs. The RNPs typically showed characteristic tenuivirus morphologies. They were approximately 8 nm in diameter, mostly circular in nature, and exhibited branching and a high degree of superhelicity. When the RNP antibodies were used for in situ immunogold labeling analysis of RHBV-infected tissues, no specific structures were identified, but gold particles were distributed throughout the cytosol of RHBV-infected but not healthy plants. However, amorphous semi-electron opaque inclusion bodies (ASO-IBs) were abundant in cells of RHBV-infected plants. While the ASO-IBs were not labeled with the anti-RNP antiserum, they were specifically labeled with antibodies to the RHBV major noncapsid protein (NCP) and with antibodies to the NCP of another tenuivirus, maize stripe virus.

Isolation and Partial Characterization of a Tenuivirus from Wheat in Iran

A tenuivirus, tentatively designated Iranian wheat stripe virus (IWSV), was transmitted by the delphacid Unkanodes tanasijevici and induced dwarfing, striping and yellowing symptoms in wheat. It could also infect barley, oat, rice, rye, sorghum and a number of other gramineous species. The virus was purified from wheat by chloroform clarification and differential and density-gradient centrifugation. It formed several layers in density-gradient columns. Purified virus preparations had a UV absorption spectrum typical of nucleoproteins and contained flexuous supercoils of 8.7 nm and fine filaments of 4.3 nm width. The antiserum produced against the virus reacted with infected plant sap in agar-gel diffusion and enzyme-linked immunosorbent assay (ELISA). The virus could be readily detected in individual viruliferous planthoppers by ELISA. IWSV was serologically related to rice hoja blanca virus but not to maize stripe virus. The relationship of IWSV with other tenuiviruses is discussed.

Rice hoja blanca virus genome characterization and expression in vitro.

No information exists on the organization and mechanisms of expression of the genome of rice hoja blanca virus (RHBV), a member of the tenuivirus group, but here we describe the first steps in its characterization. RHBV contains four ssRNA and three dsRNA species, the sizes of which were estimated by native and denaturing gel electrophoresis. Hybridization analyses using 32P-labelled riboprobes of viral and viral complementary polarities showed that unequal amounts of the two polarities of at least the smallest RNA are present in the virion, and indicated that the dsRNA species contain the same information as the ssRNA species of corresponding size. Total RHBV RNA directs the synthesis of two major proteins of 23K and 21K in vitro. RNA3 directs the synthesis of a 23K protein designated NS3, and RNA4 of a 21K protein designated NS4. The NS4 protein corresponds to the non-structural protein that accumulates in RHBV-infected rice tissue. The nuclecocapsid protein is not translated from either total RHBV RNA or any individual RHBV RNA in vitro.

A field screening method to evaluate rice breeding lines for resistance to the hoja blanca virus

SUMMARYA method is presented for rearing large colonies of viruliferous Sogatodes oryzicola, vector of the rice hoja blanca virus (RHBV). These colonies were used for field screening up to 10 000 rice breeding lines per season for resistance to RHBV. Uniform infection of check varieties in the field indicated that the method was adequate. Field release of vectors when plants were 14 days old resulted in satisfactory disease incidence, after 21 days, to distinguish lines segregating for resistance from lines uniformly resistant or susceptible. Various sources of resistance identified earlier continued to be resistant under the screening conditions. Progeny of lines identified as non‐segregating resistant continued as non‐segregating resistant. Resistant plants from lines segregating for resistance produced progeny lines that were segregating and non‐segregating. Ratios of resistant to susceptible plants in F1 progeny of three‐way crosses were consistent with earlier observations that RHBV resistance is a dominant character. The susceptibility of the commercial checks indicates that rice production in RHBV areas of tropical Latin America continues to be at risk from the virus. Virus‐resistant commercial cultivars resulting from this method should be available in 2 years.

Fine flexuous threadlike particles in cells of plants and insect hosts infected with rice hoja blanca virus

Numerous bundles of long flexuous threadlike particles were found both in cells of rice leaves infected with hoja blanca and in its infective insect vector. The threadlike particles are approximately 8 to 10 mμ in diameter and variable in length. They appeared in the epidermis, palisade cells, spongy parenchyma, phloem, and vessels of diseased leaves. In the plant cells, the threadlike particles appeared in the nuclei and cytoplasm, but not in the chloroplasts, or mitochondria. In the insects, the threads were found in the lumen and epithelial cells of the intestine, probably in the filter chamber. These threads may be the particles of the rice hoja blanca virus.