Ichiro Uyeda

Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10

The proteins encoded by rice black streaked dwarf fijivirus (RBSDV) genomic segments 7-10 (S7-S10) were characterized. Open reading frames (ORFs) from these segments were expressed as fusion proteins in Escherichia coli. Antibodies raised against the expressed products were used as probes to determine whether the viral ORFs encode structural proteins. In Western blots, antibodies to the expressed S8 and S10 products reacted with a core capsid (65 kDa) and a major outer capsid (56 kDa) protein, respectively, while none of the antibodies to S7 and S9 products reacted with structural proteins. Antisera to RBSDV S7 ORF1 and S9 ORF1 each detected a single protein of the predicted size in total protein extracts from infected rice plants and viruliferous Laodelphax striatellus. Immunoelectron microscopy revealed that antibodies to RBSDV S7 ORF1 and RBSDV S9 ORF1 reacted with tubular structures and viroplasm, respectively, in sections of both infected maize plants and viruliferous L. striatellus. Antisera to ORF2 of S7 and S9 failed to detect any proteins in the infected tissue using either Western blotting or immuno-electron microscopic techniques.

Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses

Arabidopsis thaliana plants with mutations in the genes encoding eukaryotic initiation factor (eIF4E) or isoform of eIF4E (eIF(iso)4E) were tested for susceptibility to Clover yellow vein virus (ClYVV), a member of the genus Potyvirus. ClYVV accumulated in both inoculated and upper uninoculated leaves of mutant plants lacking eIF(iso)4E, but not in mutant plants lacking eIF4E. In contrast, Turnip mosaic virus (TuMV), another member of the genus Potyvirus, multiplied in mutant plants lacking eIF4E but not in mutant plants lacking eIF(iso)4E. These results suggest the selective involvement of members of the eIF4E family in infection by potyviruses.

Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors

RNA silencing (RNAi) induced by virus-derived double-stranded RNA (dsRNA), which is in a sense regarded as a pathogen-associated molecular pattern (PAMP) of viruses, is a general plant defense mechanism. To counteract this defense, plant viruses express RNA silencing suppressors (RSSs), many of which bind to dsRNA and attenuate RNAi. We showed that the tobacco calmodulin-like protein, rgs-CaM, counterattacked viral RSSs by binding to their dsRNA-binding domains and sequestering them from inhibiting RNAi. Autophagy-like protein degradation seemed to operate to degrade RSSs with the sacrifice of rgs-CaM. These RSSs could thus be regarded as secondary viral PAMPs. This study uncovered a unique defense system in which an rgs-CaM–mediated countermeasure against viral RSSs enhanced host antiviral RNAi in tobacco.

Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution.

BackgroundPlant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV)-like sequences (ERTBVs) have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response.ResultsWe have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome.ConclusionsThese ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease.

A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR

A simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. Five distinct citrus viroids (CVds), chrysanthemum stunt viroid (CSVd), hop stunt viroid (HSVd), hop latent viroid (HLVd) and potato spindle tuber viroid (PSTVd) were detected in their natural host plants by hybridization using cRNA probes and reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acids (NA) were liberated from tissues by incubation in a buffer containing potassium ethyl xanthogenate (PEX) without tissue homogenization, and then precipitated with ethanol (NA-PEX). All the viroids except CVd-IV could be detected clearly in NA-PEX by hybridization. HSVd, HLVd and PSTVd could also be detected in NA-PEX by RT-PCR. Although CVds and CSVd could not be detected in NA-PEX by RT-PCR, they were detected after further purification: differential precipitation with 2-butoxyethanol and HCl treatment followed by ethanol-precipitation. In addition, PCR in the presence of tetramethylammonium chloride specifically amplified the cDNA of all five distinct CVds under the same temperature and cycle conditions. Since all the viroids could be detected in NA liberated by PEX, the amount of NA extracted by the method described here is sufficient for detecting viroids, enabling the processing of a large number of samples.

Close similarity between genome structures of rice black-streaked dwarf and maize rough dwarf viruses

The complete nucleotide sequences of rice black-streaked dwarf virus (RBSDV) genome segments 8 (S8) and 7 (S7) were determined, and were found to have high sequence identities to the corresponding maize rough dwarf virus (MRDV) genome segments. RBSDV S8 and S7 consisted of 1927 and 2193 nucleotides, respectively. RBSDV S8 had a single long open reading frame (ORF) encoding 591 amino acids. RBSDV S7 had two nonoverlapping ORFs encoding 362 (ORF1) and 309 (ORF2) amino acids. The two ORFs of RBSDV S7 were inserted separately into an Escherichia coli expression vector (pKK223-3). When they were expressed in E. coli cells, the products of both ORFs migrated identically at an apparent M(r) of 40K. High nucleotide sequence identity was observed between RBSDV S7 and MRDV S6 (85%), and between the terminal regions of RBSDV S8 and MRDV S7. In addition, RBSDV S7 and MRDV S6 showed 91% (ORF1 product) and 85% (ORF2 product) amino acid sequence identities.

Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean

SummaryHelper component protease (HC-Pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. Here, we show the involvement of clover yellow vein virus (ClYVV) HC-Pro in necrotic symptom expression in broad bean (Vicia faba cv. Wase). In this host, lethal necrosis was induced by ClYVV no. 30, from which a spontaneous, mosaic-inducing mutant (MM) was obtained. Mapping with chimeric viruses between ClYVV no. 30 and MM attributed the symptom attenuation to two mutations at the HC-Pro positions 27 (threonine to isoleucine) and 193 (aspartic acid to tyrosine). Although neither mutant with the single amino acid substitution at position 27 or 193 (ClYVV/T27I or D193Y) induced the lethal necrosis, ClYVV/T27I still retained the ability to induce necrotic symptoms, but ClYVV/D193Y scarcely did so. The virus accumulation of ClYVV/D193Y was also lower than that of ClYVV no. 30. The mutations, T27I and D193Y, are located in a putative zinc finger domain and in one (N-terminal) of the two RNA binding domains, respectively, of HC-Pro. RNA-silencing suppression (RSS) activity of P1/HC-Pro in Nicotiana benthamiana was weakened by both mutations. Our results suggest a correlation between viral virulence and RSS function and the importance of the two domains in HC-Pro.

Activation of the salicylic acid signaling pathway enhances Clover yellow vein virus virulence in susceptible pea cultivars.

The wild-type strain (Cl-WT) of Clover yellow vein virus (ClYVV) systemically induces cell death in pea cv. Plant introduction (PI) 118501 but not in PI 226564. A single incompletely dominant gene, Cyn1, controls systemic cell death in PI 118501. Here, we show that activation of the salicylic acid (SA) signaling pathway enhances ClYVV virulence in susceptible pea cultivars. The kinetics of virus accumulation was not significantly different between PI 118501 (Cyn1) and PI 226564 (cyn1); however, the SA-responsive chitinase gene (SA-CHI) and the hypersensitive response (HR)-related gene homologous to tobacco HSR203J were induced only in PI 118501 (Cyn1). Two mutant viruses with mutations in P1/HCPro, which is an RNA-silencing suppressor, reduced the ability to induce cell death and SA-CHI expression. The application of SA and of its analog benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH) partially complemented the reduced virulence of mutant viruses. These results suggest that high activation of the SA signaling pathway is required for ClYVV virulence. Interestingly, BTH could enhance Cl-WT symptoms in PI 226564 (cyn1). However, it could not enhance symptoms induced by White clover mosaic virus and Bean yellow mosaic virus. Our report suggests that the SA signaling pathway has opposing functions in compatible interactions, depending on the virus-host combination.

Involvement of the P1 Cistron in Overcoming eIF4E-Mediated Recessive Resistance Against Clover yellow vein virus in Pea

Two recessive genes (cyv1 and cyv2) are known to confer resistance against Clover yellow vein virus (ClYVV) in pea. cyv2 has recently been revealed to encode eukaryotic translation initiation factor 4E (eIF4E) and is the same allele as sbm1 and wlm against other potyviruses. Although mechanical inoculation with crude sap is rarely able to cause infection of a cyv2 pea, biolistic inoculation of the infectious ClYVV cDNA clone does. At the infection foci, the breaking virus frequently emerges, resulting in systemic infection. Here, a derived cleaved-amplified polymorphic sequence analysis showed that the breakings were associated with a single nonsynonymous mutation on the ClYVV genome, corresponding to an amino-acid substitution at position 24 (isoleucine to valine) on the P1 cistron. ClYVV with the point mutation was able to break the resistance. This is a first report demonstrating that P1 is involved in eIF4E-mediated recessive resistance.

Molecular characterization of Hop latent virus and phylogenetic relationships among viruses closely related to carlaviruses

Summary. The complete nucleotide sequence of the hop latent virus (HpLV) genome was determined. The viral RNA genome is 8,612 nucleotides long, excluding the poly(A) tail, and contains six open reading frames (ORFs), which encode putative proteins of 224-kDa (ORF 1), 25-kDa (ORF 2), 11-kDa (ORF 3), 7-kDa (ORF 4), 34-kDa (ORF 5), and 12-kDa (ORF 6). ORF 5 encodes the coat protein as demonstrated by N-terminal sequencing of three proteolytic peptides derived from the virus particle. The genome organization of HpLV is similar to that of other species in the genus Carlavirus, and the overall sequence of HpLV is more similar to that of Potato virus M than to sequences of other carlaviruses reported to date. The amino acid sequences of the putative methyltransferase, RNA helicase, and RNA-dependent RNA polymerase encoded in ORF 1 and an ‘accessory’ helicase encoded in ORF 2 of the HpLV genome were compared with those of viruses in the ‘tymo’ lineage: the genera Carlavirus, Potexvirus, Allexivirus, Foveavirus, Trichovirus, Capillovirus, Vitivirus, and Tymovirus. The phylogenetic relationships among the viruses in these genera are discussed. This is the first molecular characterization of a carlavirus infecting hop plants.