Plant Reoviruses Research Articles

Nonstructural protein Pns12 of rice dwarf virus is a principal regulator for viral replication and infection in its insect vector

Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. The molecular mechanisms underling the formation of the viroplasm during infection of rice dwarf virus (RDV), a plant reovirus, in its leafhopper vector cells remain poorly understood. Viral nonstructural protein Pns12 forms viroplasm-like inclusions in the absence of viral infection, suggesting that the viroplasm matrix is basically composed of Pns12. Here, we demonstrated that core capsid protein P3 and nonstructural protein Pns11 were recruited in the viroplasm by direct interaction with Pns12, whereas nonstructural protein Pns6 was recruited through interaction with Pns11. The introduction of dsRNA from Pns12 gene into cultured insect vector cells or intact insect strongly inhibited such viroplasm formation, preventing efficient viral spread in the leafhopper in vitro and in vivo. Thus, nonstructural protein Pns12 of RDV is a principal regulator for viral replication and infection in its insect vector.

Interaction between non-structural protein Pns10 of rice dwarf virus and cytoplasmic actin of leafhoppers is correlated with insect vector specificity.

Many insect-transmissible pathogens are transmitted by specific insect species and not by others, even if the insect species are closely related. The molecular mechanisms underlying such strict pathogen-insect specificity are poorly understood. Rice dwarf virus (RDV), a plant reovirus, is transmitted mainly by the leafhopper species Nephotettix cincticeps but is transmitted ineffectively by the leafhopper Recilia dorsalis. Here, we demonstrated that virus-containing tubules composed of viral non-structural protein Pns10 of RDV associated with the intestinal microvilli of N. cincticeps but not with those of R. dorsalis. Furthermore, Pns10 of RDV specifically interacted with cytoplasmic actin, the main component of microvilli of N. cincticeps, but not with that of R. dorsalis, suggesting that the interaction of Pns10 with insect cytoplasmic actin is consistent with the transmissibility of RDV by leafhoppers. All these results suggested that the interaction of Pns10 of RDV with insect cytoplasmic actin may determine pathogen-vector specificity.

Assembly of viroplasms by viral nonstructural protein Pns9 is essential for persistent infection of rice gall dwarf virus in its insect vector

Rice gall dwarf virus (RGDV), a plant reovirus, is transmitted by leafhopper vector Recilia dorsalis in a persistent-propagative manner. In a sequential study of RGDV infection of its insect vector, the virus initially infected the filter chamber epithelium, then directly crossed the basal lamina into the visceral muscles, from where it spread throughout the entire midgut and hindgut. Finally, RGDV spread into the salivary glands. During RGDV infection of the continuous cultured cells of R. dorsalis, viroplasm that was mainly comprised of viral nonstructural protein Pns9 was formed and acted as the site of viral replication and assembly of progeny virions. Knockdown of Pns9 expression in cultured insect vector cells using synthesized dsRNAs from the Pns9 gene strongly inhibited viroplasm formation and viral infection. The microinjection of dsRNAs from the Pns9 gene strongly abolished viroplasm formation in the initially infected filter chamber epithelium and prevented viral spread into leafhopper visceral muscles. These results indicated that the assembly of viroplasms was essential for the persistent infection and spread of RGDV in its insect vector.

Virus-induced tubule: a vehicle for rapid spread of virions through basal lamina from midgut epithelium in the insect vector.

The plant reoviruses, plant rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent propagative manner. These viruses induce the formation of viral inclusions to facilitate viral propagation in insect vectors. The intestines of insect vectors are formed by epithelial cells that lie on the noncellular basal lamina surrounded by visceral muscle tissue. Here, we demonstrate that a recently identified plant reovirus, southern rice black-streaked dwarf virus (SRBSDV), exploits virus-containing tubules composed of virus-encoded nonstructural protein P7-1 to directly cross the basal lamina from the initially infected epithelium toward visceral muscle tissues in the intestine of its vector, the white-backed planthopper (Sogatella furcifera). Furthermore, such tubules spread along visceral muscle tissues through a direct interaction of P7-1 and actin. The destruction of tubule assembly by RNA interference with synthesized double-stranded RNA targeting the P7-1 gene inhibited viral spread in the insect vector in vitro and in vivo. All these results show for the first time that a virus employs virus-induced tubule as a vehicle for viral spread from the initially infected midgut epithelium through the basal lamina, facilitating the rapid dissemination of virus from the intestine of the insect vector. Numerous plant viruses are transmitted in a persistent manner by sap-sucking insects, including thrips, aphids, planthoppers, and leafhoppers. These viruses, ingested by the insects, establish their primary infection in the intestinal epithelium of the insect vector. Subsequently, the invading virus manages to transverse the basal lamina, a noncellular layer lining the intestine, a barrier that may theoretically hinder viral spread. The mechanism by which plant viruses cross the basal lamina is unknown. Here, we report that a plant virus has evolved to exploit virus-induced tubules to pass through the basal lamina from the initially infected midgut epithelium of the insect vector, thus revealing the previously undescribed pathway adapted by the virus for rapid dissemination of virions from the intestine of the insect vector.

New model for the genesis and maturation of viroplasms induced by fijiviruses in insect vector cells.

Plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structures called viroplasms. Here, we established continuous cell cultures of the white-backed planthopper (Sogatella furcifera Horváth) to investigate the mechanisms for the genesis and maturation of the viroplasm induced by Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus in the family Reoviridae, during infection of its insect vector. Electron and confocal microscopy revealed that the viroplasm consisted of a granular region, where viral RNAs and nonstructural proteins P6 and P9-1 accumulated, and a filamentous region, where viral RNAs, progeny cores, viral particles, as well as nonstructural proteins P5 and P6 accumulated. Our results suggested that the filamentous viroplasm matrix was the site for the assembly of progeny virions. Because viral RNAs were produced by assembled core particles within the filamentous viroplasm matrix, we propose that these viral RNAs might be transported to the granular viroplasm matrix. P5 formed filamentous inclusions and P9-1 formed granular inclusions in the absence of viral infection, suggesting that the filamentous and granular viroplasm matrices were formed primarily by P5 and P9-1, respectively. P6 was apparently recruited in the whole viroplasm matrix by direct interaction with P9-1 and P5. Thus, the present results suggested that P5, P6, and P9-1 are collectively required for the genesis and maturation of the filamentous and granular viroplasm matrix induced by SRBSDV infection. Based on these results, we propose a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses in insect vector cells.

Transmission Biology of Raspberry latent virus, the First Aphid-Borne Reovirus

Raspberry latent virus (RpLV) is a newly characterized reovirus found in commercial raspberry fields in the Pacific Northwest (PNW). Thus far, all members of the plant reoviruses are transmitted in a replicative, persistent manner by several species of leafhoppers or planthoppers. After several failed attempts to transmit RpLV using leafhoppers, the large raspberry aphid, commonly found in the PNW, was tested as a vector of the virus. The virus was transmitted to new, healthy raspberry plants when inoculated with groups of at least 50 viruliferous aphids, suggesting that aphids are vectors of RpLV, albeit inefficient ones. Using absolute and relative quantification methods, it was shown that the virus titer in aphids continued to increase after the acquisition period even when aphids were serially transferred onto fresh, healthy plants on a daily basis. Transmission experiments determined that RpLV has a 6-day latent period in the aphid before it becomes transmissible; however, it was not transmitted transovarially to the next generation. To our knowledge, this is the first report of a plant reovirus transmitted by an aphid. Phylogenetic analyses showed that RpLV is related most closely to but distinct from Rice ragged stunt virus (RRSV), the type member of the genus Oryzavirus. Moreover, the conserved nucleotide termini of the genomic segments of RpLV did not match those of RRSV or other plant reoviruses, allowing us to suggest that RpLV is probably the type member of a new genus in the Reoviridae comprising aphid-transmitted reoviruses.

Immunity to Rice black streaked dwarf virus, a plant reovirus, can be achieved in rice plants by RNA silencing against the gene for the viroplasm component protein

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants.

Functional and biochemical properties of Mal de Río Cuarto virus ( Fijivirus, Reoviridae) P9-1 viroplasm protein show further similarities to animal reovirus counterparts

Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.

Release of Rice dwarf virus from insect vector cells involves secretory exosomes derived from multivesicular bodies

Plant reoviruses in insect vector cells are sequestered in spherical multivesicular compartments. We demonstrated previously that the plant-infecting reovirus Rice dwarf virus (RDV) exploits multivesicular compartments for the transport and release of viral particles from infected insect vector cells. These multivesicular compartments contain small vesicles and, morphologically, they resemble previously reported endosomal multivesicular bodies (MVBs) exploited by enveloped RNA viruses during budding from the plasma membrane of infected cells. Electron microscopy revealed that, at a late stage of infection, RDV virions are released, together with small vesicles similar to secreted vesicles (exosomes), from infected cells. The incorporation of lysosomes into the multivesicular compartments raised the possibility that functions of host MVBs are required for the efficient release of RDV virions from infected insect vector cells. An actin-myosin transport system has been shown to mediate the transport of these multivesicular compartments. In this addendum, we provide evidence for the proposed model of release of RDV virions from infected insect vector cells that exploits secretory exosomes derived from MVBs.

A black-streaked dwarf disease on rice in China is caused by a novel fijivirus

An isolate of a plant reovirus causing severe stunting and dark leaf symptoms on rice from Guangdong, China, was similar in virion morphology and serologically related to rice black-streaked dwarf virus (RBSDV). The electrophoretic profiles of genome segments of the two viruses in agarose or polyacrylamide gel were indistinguishable. The four genome segments of the new isolate corresponding to RBSDV S7-S10 were amplified by ligation RT-PCR and sequenced. The size and organization of each genome segment was very similar to its counterparts in RBSDV, maize rough dwarf virus (MRDV), and mal de Rio Cuarto virus (MRCV). Sequence identity was greatest to RBSDV and MRDV (ranging from about 60-85% depending on the protein), but identities were always much lower than those between RBSDV and MRDV. These comparisons and phylogenetic analyses suggested that the virus represents a new species in genus Fijivirus group 2, tentatively named Rice black-streaked dwarf virus-2.

PHYTOREOVIRUS-LIKE SEQUENCES ISOLATED FROM SALIVARY GLANDS OF THE GLASSY-WINGED SHARPSHOOTER HOMALODISCA VITRIPENNIS (HEMIPTERA: CICADELLIDAE)

The salivary glands of the Glassy-winged sharpshooter (GWSS), Homalodisca vitripennis Germar 1821, (syn. H. coagulata, Hemiptera: Cicadellidae) were collected and used to produce a cDNA library. Examination by BLASTX analyses identified 2 viral sequences, one a 610-base pair fragment and a second 839-base pair fragment, both of which had significant homology to viruses within the genus Phytoreovirus. Resequencing of the fragments confirmed sequence validities. These sequences were used for in silico protein translation and BLASTP analysis confirming the established homology. While the GWSS is the primary vector of Pierce’s disease of grapes, this is the first report that GWSS may be a vector of a phytoreoviruses. Phylogenetic and homology comparisons with BLASTX, BLASTP, and PAUP analyses indicated that the viral sequences isolated from GWSS were closely related to the viruses in the Family Reoviridae, Genus Phytoreovirus, specifically Rice Dwarf Phytoreovirus (RDV). RDV is the only plant reovirus that is not limited to the phloem. Phytoreoviruses are transmitted in a propagative manner by cicadellid leafhoppers (Hemiptera: Cicadellidae), which acquire and transmit them during feeding. Phytoreoviruses have been reported from Agallian, Agalliopsis, Nephotettix, and Recilia, genera of leafhoppers, with evidence for transovarial transmission. The GWSS, although considered to feed primarily from the xylem, ingests from other plant tissues, such as the phloem and mesophyll during probing similar to other leafhoppers. The feeding behavior and wide host range of the GWSS provides an overlapping condition for these two organisms, leafhopper and virus. GWSS will feed from grasses as a transitory host, and on herbaceous and woody plants as primary hosts, which may favor the acquisition and transmission of Phytoreovirus by this leafhopper. Monitoring for an increase of Phytoreovirus spread in graminaceous crops that are in proximity to vineyards or tree crop orchards, where GWSS occurs, such as in southern California, will provide a better understanding of the potential role of the GWSS as a disease vector in the spread of phytoreoviruses and other plant pathogens. The sequences have been deposited in NCBI database under the accession numbers (EF058280) for GWSS-V1, WHSg013C11 and (EF058281) for GWSS-V2, WHSg024H02.

Molecular Characterization of Bombyx mori Cytoplasmic Polyhedrosis Virus Genome Segment 4

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra.

Sequence Analysis Shows that a Dwarfing Disease on Rice, Wheat and Maize in China is Caused by Rice Black-streaked Dwarf Virus

Isolates of plant reoviruses causing severe stunting and dark leaf symptoms on wheat in Hebei province, on maize in Hubei province and on rice in Zhejiang province, China have been characterized. Four of the ten genome segments corresponding to Rice black-streaked dwarf virus (RBSDV) S7, S8, S9, S10 were amplified by RT-PCR from the Hebei and Zhejiang isolates and sequenced. Sequences of S9 and S10 were also obtained from the Hubei isolate. Sequences of corresponding segments of the Chinese isolates were very similar to each other (94.0–99.0% identical nucleotides and 96.3–100% identical amino acids) and were closer to those of a previously reported Japanese RBSDV isolate (90.0–94.9% identical nucleotides and 91.1–98.6% identical amino acids) than to those of an Italian Maize rough dwarf virus isolate (85.1–88.1% identical nucleotides and 85.5–94.3% identical amino acids). The Chinese isolates should be classified as RBSDV.

Rice Ragged Stunt Oryzavirus: role of the viral spike protein in transmission by the insect vector

SUMMARYA 39 kDa protein, known as the viral spike protein or one of the protein components forming the viral spike, encoded by genomic segment 9 (S9) of Rice Ragged Stunt Oryzavirus (RRSV) was obtained by enzymatic cleavage of a fusion protein expressed by S9 cDNA in bacteria with proteinase factor Xa. The feeding of an insect vector — the rice brown planthopper (Nilaparvata lugens) on purified expressed 39 kDa protein before the inoculation of the insects on diseased rice plants could completely inhibit the vector transmission ability of the insect. The presence of a 32 kDa insect cell membrane protein which could bind to 39 kDa viral spike protein indicated that the inhibition might be resulted from the competition in the interactions of 39 kDa protein and intact virus with the virus receptors on the insect cells. These results suggest that the spike proteins of the plant reoviruses are essential for the virus infection in the interactions of virus, insect vectors and host plants. These results are also useful in the practical applications.

Evidence for a Phytoreovirus Associated with Tobacco Exhibiting Leaf Curl Symptoms in South Africa

ABSTRACT Three forms of tobacco leaf curl (termed classes I, II, and III, based on symptomatology) recently have been described in southern Africa. Numerous attempts to isolate virus particles responsible for a nongeminivirus-induced leaf curl disease (class I) of tobacco in South Africa have been unsuccessful. Recently, 12 dsRNA segments were isolated from tobacco exhibiting class I leaf curl symptoms, suggesting a possible reovirus genome. The objective of our study was to confirm whether the dsRNA segments are associated with a reovirus. Isolation of icosahedral particles with an outer core 60 to 65 nm in diameter and an inner core 40 to 45 nm in diameter was achieved. Twelve distinct nonpolyadenylated dsRNAs were isolated from purified virions, and the total molecular masses of the dsRNAs ranged from 17.86 to 18.40 x 10(6) Da in polyacrylamide and agarose gels, respectively. Using hybridization analysis, dsRNAs were identified as non-homologous distinct segments. Comparisons with other known reoviruses revealed a unique banding pattern that was most similar to the wound tumor virus (WTV), the type species of the genus Phytoreovirus. Hybridizations of WTV cloned DNA probes (segments S4 and S6 to S9) and dsRNAs from infected tobacco indicated no significant sequence similarity, whereas indirect enzyme-linked immunosorbent assay with a polyclonal antiserum to WTV showed strong positive cross-reactivity to tobacco virions. Our results indicate a virus with features consistent with those of phytoreoviruses. This is the first report of a plant reovirus in tobacco, the first record in Africa, and the second example of a field-isolated dicot phytoreovirus.

Preliminary investigation of the causal agent(s) of a disease causing leaf curl of tobacco in South Africa

A high incidence (86%) of potyvirus infection was noted in tobacco plants exhibiting a form of leaf curl in South Africa. Despite leaf curl being reported in the literature to be of geminiviral aetiology, no geminiviruses were detected. Furthermore, no other virus particles were detected by virus purification, TEM and serology. Twelve species of dsRNA were consistently isolated from these tobacco plants, but were absent from other forms of leaf curl‐affected and healthy tobacco. Aphid and mechanical inoculation demonstrated that the purified potyvirus(es) did not cause leaf curl symptoms, but rather mild mottle and mosaic symptoms in tobacco. Partial characterization of the potyvirus preparation showed a possible relationship to a South African strain of potato virus Y. Because potyvirus‐inoculated plants did not manifest leaf curl symptoms, and because leaf curl symptoms were noted in some plants not infected with a potyvirus, it was concluded that the potyvirus is not involved in the leaf curl aetiology, but causes a latent infection, the symptoms of which are masked. The pattern of the dsRNA banding, induction of enations and lack of mechanical and seed transmission are common to plant reoviruses. The possibility of a phytoreovirus involvement in this form of leaf curl is currently being investigated. The results from this study suggest that tobacco leaf curl disease worldwide, with regard to geminiviruses, be re‐evaluated.

High Resolution Genome Typing and Genomic Reassortment Events of Rice Dwarf Phytoreovirus

Genomic reassortment of rice dwarf Phytoreovirus (RDV) was experimentally demonstrated for the first time in plant reoviruses. Combinations of two genomic variants, most of the genomic segments of which could be distinguished by a high resolution polyacrylamide gel electrophoresis, were used to produce genomic reassortants. After artificial mixed injection of two of three isolates (RDV-S, RDV-Al, and RDV-AN] into the insect vector Nephotettix cincticeps , rice seedlings were sequentially inoculated and the genomic origin of the viruses present in the infected plants was examined by electrophoresis. The progeny virus population contained either one or both of the respective genomic segments from the parents. Genomic segments reassorted randomly except for genome segment 1 (S1) and S9. S9 of RDV-S was mostly excluded in the reassortants in both file insects and the infected plants when it was mixed with RDV-Al or RDV-AN. On the other hand, S9 reassorted randomly in most of the virus populations in infected plants when RDV-A1 and RDV-AN were co-injected into insects. When RDV-S and RDV-Al were mixed. S1 from RDV-S was present more frequently in the infected plants although both parental S1's were present in equimolar amounts in insects.

Rice ragged stunt Oryzavirus genome segment 9 encodes a 38 600 Mr structural protein.

The complete nucleotide sequence of rice ragged stunt virus genome segment 9 (S9) was determined. The S9 segment is 1132 nucleotides long and has a long open reading frame starting from the first AUG codon at nucleotide position 14-16 and terminating at a UAG codon located at 1028-1030, which could encode a polypeptide with an Mr of 38 600 (P9). The encoded polypeptide has no sequence homology to polypeptides of any other plant reoviruses published previously. An immunological study demonstrated that P9 was the smallest of the structural proteins. The P9 polypeptide was expressed as a fusion protein with maltose binding protein in Escherichia coli. Antisera to purified virions and to the fusion protein reacted with both the bacterially expressed polypeptide and the smallest polypeptide of the purified virus in immunoblotting analyses.

Characterization of Genome Structure And Establishment of Vector Cell Lines for Plant Reoviruses

Publisher Summary Plant reoviruses are a unique group among plant pathogenic viruses. They are members of a family that includes viruses of both vertebrates and invertebrates, and all members share characteristic features: (1) they are leafhopper and planthopper borne in a persistent manner, (2) they contain spherical particles 60-70 nm in diameter, and (3) their genomes consist of 10-12 segmented double-stranded RNAs (dsRNAs). The classical study of plant reoviruses that attracted the attention of researchers focuses chiefly on the multiplication of the plant pathogenic viruses in their insect vectors, starting with Fukushi's finding in 1940, based on his spectacular experimental evidence that rice dwarf virus (RDV) multiplied in its insect vector; this finding later promoted “controversial discussions”. Subsequently, further evidence based on biological and electron microscopic studies fully supported Fukushi's discovery. Among the plant reoviruses, RDV and wound tumor virus (WTV) have been most extensively studied, with respect to their insect transmission, transovarial passage, virus particles, localization of the viruses in their plant and insect hosts, establishment of cell lines from embryonic tissue culture of the respective insect vectors, and genome characterization. This chapter reviews the recent findings concerning dsRNA genomes of plant reoviruses, especially the detailed characterization of all 12 genomic segments of RDV achieved in Japan. This is the first report of a successful genomic analysis of the 12 segmented dsRNAs of a virus belonging to the plant reoviruses.

進む植物レオウイルスのゲノム解析

進む植物レオウイルスのゲノム解析