Rice Hoja Blanca Virus Research Articles

Genetic bases of resistance to the rice hoja blanca disease deciphered by a quantitative trait locus approach.

Rice hoja blanca (RHB) is one of the most serious diseases in rice-growing areas in tropical Americas. Its causal agent is RHB virus (RHBV), transmitted by the planthopper Tagosodes orizicolus Müir. Genetic resistance is the most effective and environment-friendly way of controlling the disease. So far, only 1 major quantitative trait locus (QTL) of Oryza sativa ssp. japonica origin, qHBV4.1, that alters the incidence of the virus symptoms in 2 Colombian cultivars has been reported. This resistance has already started to be broken, stressing the urgent need for diversifying the resistance sources. In the present study, we performed a search for new QTLs of O. sativa indica origin associated with RHB resistance. We used 4 F2:3-segregating populations derived from indica-resistant varieties crossed with a highly susceptible japonica pivot parent. Besides the standard method for measuring disease incidence, we developed a new method based on computer-assisted image processing to determine the affected leaf area (ALA) as a measure of symptom severity. Based on the disease severity and incidence scores in the F3 families under greenhouse conditions and SNP genotyping of the F2 individuals, we identified 4 new indica QTLs for RHB resistance on rice chromosomes 4, 6, and 11, namely, qHBV4.2WAS208, qHBV6.1PTB25, qHBV11.1, and qHBV11.2, respectively. We also confirmed the wide-range action of qHBV4.1. Among the 5 QTLs, qHBV4.1 and qHBV11.1 had the largest effects on incidence and severity, respectively. These results provide a more complete understanding of the genetic bases of RHBV resistance in the cultivated rice gene pool and can be used to develop marker-aided breeding strategies to improve RHB resistance. The power of joint- and meta-analyses allowed precise mapping and candidate gene identification, providing the basis for positional cloning of the 2 major QTLs qHBV4.1 and qHBV11.1.

Rice hoja blanca virus (white leaf disease of rice)

Rice hoja blanca virus (white leaf disease of rice)

Molecular characterisation of a new tenuivirus from Festuca sp.

A novel virus with a quadruple genome of negative-sense, single-stranded RNA was identified by high-throughput sequencing (HTS) in a grass sample from Saxony-Anhalt, Germany, and tentatively called Festuca stripe-associated virus (FSaV). The genome of FSaV consists of four segments and a total of 16,535 nucleotides (nt) which encode seven open reading frames (ORF). FSaV shares highest nt identity (between 72.84% to 80.74%) to Iranian wheat stripe virus (IWSV) and rice hoja blanca virus (RHBV). Additionally, pairwise comparisons between the amino acid sequences of the ORFs on the genome of FSaV and the corresponding ones on the genomes of the members of the Tenuvirus genus showed that FSaV shared 83.17% and 90.85% (amino acid) aa identity to IWSV. Moreover, the non-coding intergenic regions (ncIR) shared only between 49.5% to 60.87% nt identity to the corresponding regions on the IWSV genome. Based on the ICTV species demarcation, the results suggest that FSaV may represent a new species of the genus Tenuivirus. Plastid sequence analysis of the HTS data showed that the original host is a member of the genus Festuca most likely the species Festuca pratensis.

Assessing the Potential Infection of Tagosodes orizicolus (Hemiptera: Delphacidae) by Rice Hoja Blanca Virus in Texas.

Tagosodes orizicolus (Muir) is the most important pest of rice in Latin America. Besides causing direct damage called hopperburn from feeding on and ovipositing in rice leaves, this insect pest also transmits rice hoja blanca virus (RHBV, Family Phenuiviridae, Genus Tenuivirus) in a persistent-propagative manner. This pathosystem can cause up to 100% yield loss in Latin American rice fields. T. orizicolus and RHBV symptoms were detected in Louisiana, Mississippi, and Florida rice fields in the 1950s, 1960s, and 1980s. However, neither has been detected in the United States since. Two outbreaks of T. orizicolus on ratoon rice occurred in the fall of 2015 and 2018 in counties southwest and south of Houston, TX. Insects were collected from ratoon rice fields by sweep net methods. Insects from the 2015 and 2018 outbreaks were tested individually and in pools of 10, respectively, for RHBV infection and the cytochrome oxidase 1 (CO1) gene from Delphacidae. No insects were positive for RHBV, however, all samples yielded amplicons for the CO1 gene. Furthermore, the CO1 gene from five 2015 individuals was sequenced and found to have a 100% identity to the Fer26_Argentina and 99.81% identity to the DEL074 Venezuela isolates of T. orizicolus. Five new sequences from 2015 individuals have now been deposited in GenBank. It is imperative to stay up to date on the potential invasion and establishment of this exotic pest of rice in Texas and other rice-growing regions of the United States through continued monitoring and research.

Conformationally Restricted Peptides from Rice Proteins Elicit Antibodies That Recognize the Corresponding Native Protein in ELISA Assays.

The rice hoja blanca virus (RHBV), transmitted by the planthopper insect Tagosodes orizicolus, is a disease that attacks rice and generates significant production losses in Colombia. Fedearroz 2000 and Colombia I commercial rice varieties, which have different resistance levels to the disease, were selected in this study. To identify proteins associated to the insect and virus signaling, a comparative proteomics study was performed. By comparing proteomic profiles, between virus-infected and control group plants in two-dimensional electrophoresis, proteins exhibiting significant changes in abundance were found. In another test, peptide dendrimers containing sequences conformationally restricted to α-helix from four of those rice proteins were synthesized. In the experiment, sera from mice inoculated with peptide dendrimers could recognize the corresponding native protein in ELISA assays. Reported comparative proteomic results provide new insights into the molecular mechanisms of plant response to the RHBV and comprehensive tools for the analysis of new crop varieties. Besides, results from conformational peptide dendrimer approach are promising and show that it is feasible to detect proteins as markers, and may have biological applications by decreasing the susceptibility to proteolytic degradation.

Identification of new sources of resistance to RHBV- rice hoja blanca virus

Con el objetivo de encontrar nuevas fuentes de resistencia a la enfermedad de la hoja blanca del arroz, transmitida por el insecto Tagosodes orizicolus, se evaluaron 660 genotipos en condiciones de invernadero y campo. Se identificaron siete genotipos con resistencia a la enfermedad y se realizaron estudios del genoma para evidenciar que eran genéticamente diferentes a Fedearroz 2000, la variedad de mejor comportamiento ante el virus, en el momento. Estas nuevas fuentes de resistencia constituyen un recurso que puede utilizarse para extender un manejo sostenible de la enfermedad, en todas las regiones productoras de arroz en América tropical. Este es el primer reporte de fuentes de resistencia, tipo indica, diferentes a Fedearroz 2000.

Occurrence and Molecular Detection of Rice hoja blanca virus (Genus Tenuivirus) in Peru

Occurrence and Molecular Detection of <i>Rice hoja blanca virus</i> (Genus <i>Tenuivirus</i>) in Peru

Expression of the &lt;i&gt;rice hoja blanca virus&lt;/i&gt; (RHBV) non-structural protein 3 (NS3) in Escherichia coli and its &lt;i&gt;in situ&lt;/i&gt; 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.

Micromorfología de la epidermis foliar de cultivares de arroz Venezolano (Poaceae) asociado con el daño mecánico de sogata Tagosodes orizicolus (Homoptera: Delphacidae)

Rice cultivars are affected directly and indirectly by the insect sogata. The mechanical damage or direct loss, is produced after feeding and ovoposition on the young leaves tissues, while the indirect damage is produced after the transmission of the Rice hoja blanca virus. We studied the morpho-anatomic structures associated with the resistance of the mechanical damage produced by the insect, in six rice cultivars, including controls for resistance and susceptibility (Makalioka and Bluebonnet 50), during August 2011, in Fundacion Danac, Venezuela. Samples were taken from leaf 3, where cuticle thickness, presence of macrohair, microhair and silica bodies in the second third of the leaf was evaluated. A significant difference to thickness of the cuticle, the presence of microhair in the leaves, and presence of silica bodies was observed among cultivars, determining a significant correlation among the number of macrohair and microhair in the adaxial leaf blade with the presence of silica bodies, and thickness of the cuticle and number of posed insects. Thickness of the cuticle and presence of silica bodies in the intercostals space of microhair and macrohair showed to be the variables most related to mechanical damage and resistance mechanism.

Major QTLs Control Resistance to Rice Hoja Blanca Virus and Its VectorTagosodes orizicolus

Rice hoja blanca (white leaf) disease can cause severe yield losses in rice in the Americas. The disease is caused by the rice hoja blanca virus (RHBV), which is transmitted by the planthopper vector Tagosodes orizicolus. Because classical breeding schemes for this disease rely on expensive, time-consuming screenings, there is a need for alternatives such as marker-aided selection. The varieties Fedearroz 2000 and Fedearroz 50, which are resistant to RHBV and to the feeding damage caused by T. orizicolus, were crossed with the susceptible line WC366 to produce segregating F2:3 populations. The F3 families were scored for their resistance level to RHBV and T. orizicolus. The F2:3 lines of both crosses were genotyped using microsatellite markers. One major QTL on the short arm of chromosome 4 was identified for resistance to RHBV in the two populations. Two major QTL on chromosomes 5 and 7 were identified for resistance to T. orizicolus in the Fd2000 × WC366 and Fd50 × WC366 crosses, respectively. This comparative study using two distinct rice populations allowed for a better understanding of how the resistance to RHBV and its vector are controlled genetically. Simple marker-aided breeding schemes based on QTL information can be designed to improve rice germplasm to reduce losses caused by this important disease.

Mechanisms of &lt;i&gt;Oryza sativa&lt;/i&gt; (Poaceae) resistance to &lt;i&gt;Tagosodes orizicolus&lt;/i&gt; (Homoptera: Delphacidae) under greenhouse condition in Venezuela

Tagosodes orizicolus is one of the main plagues of rice in tropical America causing two types of damages, the direct one, feeding and oviposition effect, and an indirect one, by the transmission of the "Rice hoja blanca virus". During 2006-2007 we carried out research under greenhouse conditions at Fundaci6n Danac, Venezuela, in order to determine the mechanisms of antixenosis, antibiosis and tolerance to T. orizicolus, which could be acting in commercial varieties and advanced lines of the rice genetic breeding programs of INIA and Fundaci6n Danac. The method of free feeding was used for the antixenosis evaluation, whereas the method of forced feeding was used for antibiosis evaluation (effect on survival and oviposition). Additionally, we used the indirect method based on biomass depression to estimate the tolerance. Some of the evaluated traits included: grade of damage, number of insects settling on rice plants, percentage of sogata mortality at the mature state, number of eggs in the leaf midrib and an index of tolerance. The results showed that rice genotypes possess different combinations of resistance mechanisms, as well as different grades of reactions. The susceptible control 'Bluebonnet 50' was consistently susceptible across experiments and the resistant control 'Makalioka' had high antixenosis and high antibiosis based on survival and oviposition. The rest of the genotypes presented lower or higher degrees of antixenosis and antibiosis for survival and oviposition. The genotype 'FD0241-M-17-6-1-1-1-1' was identified with possible tolerance to the direct damage of sogata.

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.

Binding of small interfering RNA molecules is crucial for RNA interference suppressor activity of rice hoja blanca virus NS3 in plants

The NS3 protein of rice hoja blanca virus represents a viral suppressor of RNA interference (RNAi) that sequesters small interfering (si)RNAs in vitro. To determine whether this siRNA binding property is the critical determinant for the suppressor activity of NS3, NS3 was altered by alanine point mutations and the resulting mutant proteins were tested for both siRNA binding ability and RNAi suppressor activity in plants. Alanine substitutions of lysine residues at positions 173-175 resulted in mutant proteins that lost both their affinity for siRNAs and their RNAi suppressor activity in planta. This indicates that siRNA binding of NS3 is indeed essential for the suppressor function of NS3 and that residues at positions 173-175 are involved in the siRNA binding and suppressor activities.

The NS3 protein of rice hoja blanca virus complements the RNAi suppressor function of HIV‐1 Tat

The question of whether RNA interference (RNAi) acts as an antiviral mechanism in mammalian cells remains controversial. The antiviral interferon (IFN) response cannot easily be distinguished from a possible antiviral RNAi pathway owing to the involvement of double-stranded RNA (dsRNA) as a common inducer molecule. The non-structural protein 3 (NS3) protein of rice hoja blanca virus (RHBV) is an RNA silencing suppressor (RSS) that exclusively binds to small dsRNA molecules. Here, we show that this plant viral RSS lacks IFN antagonistic activity, yet it is able to substitute the RSS function of the Tat protein of human immunodeficiency virus type 1. An NS3 mutant that is deficient in RNA binding and its associated RSS activity is inactive in this complementation assay. This cross-kingdom suppression of RNAi in mammalian cells by a plant viral RSS indicates the significance of the antiviral RNAi response in mammalian cells and the usefulness of well-defined RSS proteins.

The NS3 protein of rice hoja blanca virus suppresses RNA silencing in mammalian cells

The NS3 protein of the tenuivirus rice hoja blanca virus (RHBV) has previously been shown to represent the viral RNA interference (RNAi) suppressor and is active in both plant and insect cells by binding short interfering RNAs (siRNAs) in vitro. Using a firefly luciferase-based silencing assay it is described here that NS3 is also active in mammalian cells. This activity is independent of the inducer molecule used. Using either synthetic siRNAs or a short hairpin RNA construct, NS3 was able to significantly suppress the RNAi-mediated silencing of luciferase expression in both monkey (Vero) and human (HEK293) cells. These results support the proposed mode of action of NS3 to act by sequestering siRNAs, the key molecules of the RNAi pathway conserved in all eukaryotes. The possible applications of this protein in modulating RNAi and investigating the proposed antiviral RNAi response in mammalian cell systems are discussed.

The NS3 protein of Rice hoja blanca tenuivirus suppresses RNA silencing in plant and insect hosts by efficiently binding both siRNAs and miRNAs

RNA silencing plays a key role in antiviral defense as well as in developmental processes in plants and insects. Negative strand RNA viruses such as the plant virus Rice hoja blanca tenuivirus (RHBV) replicate in plants and in their insect transmission vector. Like most plant-infecting viruses, RHBV encodes an RNA silencing suppressor, the NS3 protein, and here it is demonstrated that this protein is capable of suppressing RNA silencing in both plants and insect cells. Biochemical analyses showed that NS3 efficiently binds siRNA as well as miRNA molecules. Binding of NS3 is greatly influenced by the size of small RNA molecules, as 21 nucleotide (nt) siRNA molecules are bound > 100 times more efficiently than 26 nt species. Competition assays suggest that the activity of NS3 is based on binding to siRNAs prior to strand separation during the assembly of the RNA-induced silencing complex. In addition, NS3 has a high affinity for miRNA/miRNA* duplexes, indicating that its activity might also interfere with miRNA-regulated gene expression in both insects and plants.

Molecular characterization of Iranian wheat stripe virus shows its taxonomic position as a distinct species in the genus Tenuivirus

The full lengths of three genome segments of Iranian wheat stripe virus (IWSV) were amplified by reverse transcription (RT) followed by polymerase chain reaction (PCR) using a primer complementary to tenuivirus conserved terminal sequences. The segments were sequenced and found to comprise 3469, 2337, and 1831 nt, respectively. The gene organization of these segments is similar to that of other known tenuiviruses, each displaying an ambisense coding strategy. IWSV segments, however, are different from those of other viruses with respect to the number of nucleotides and deduced amino acid sequence for each ORF. Depending on the segment, the first 16-22 nt at the 5' end and the first 16 nt at the 3' end are highly conserved among IWSV and rice hoja blanca virus (RHBV), rice stripe virus (RSV) and maize stripe virus (MStV). In addition, the first 15-18 nt at the 5' end are complementary to the first 16-18 nt at the 3' end. Phylogenetic analyses showed close similarity and a common ancestor for IWSV, RHBV, and Echinochloa hoja blanca virus (EHBV). These findings confirm the position of IWSV as a distinct species in the genus Tenuivirus.

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.

Association of a Virus with Wheat Displaying Yellow Head Disease Symptoms in the Great Plains.

Wheat with yellow head disease (YHD) (yellow heads and mosaic leaf symptoms) has been observed in Kansas since 1997. A pathogen was transmitted from the infected wheat to maize by vascular puncture inoculation and to Nicotiana benthamiana by rub inoculation. The original infected wheat and infected maize and N. benthamiana test plants all produced a unique 32- to 34-kDa protein when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Time-of-flight mass spectrometry analysis of the unique 32- to 34-kDa protein showed that the amino acid sequence was most closely related to the nucleoprotein of Rice hoja blanca virus, indicating that the virus causing YHD symptoms in wheat is a tenuivirus. Antiserum made to this protein failed to react with extracts made from healthy wheat or wheat infected with Wheat streak mosaic virus or the High Plains virus. The antiserum did react to extracts made from symptomatic wheat, maize, and N. benthamiana, shown by SDS-PAGE to contain the unique protein, and to extracts of wheat with YHD symptoms from Kansas, North Dakota, South Dakota, and Oklahoma. The name Wheat yellow head virus is proposed for this virus.

Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions.

Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the BUNYAVIRIDAE: Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS(S) gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS(S)-expressing leaf sectors suggests that the tospoviral NS(S) protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.