[show abstract][hide abstract] ABSTRACT: The Arabidopsis gene AINTEGUMENTA (At-ANT) functions in cell proliferation and organ growth. The ANT protein has two copies of the AP2 domains,
R1 and R2. Recently, a partial cDNA sequence of the At-ANT homolog in Antirrhinum majus (Am-ANT) was reported (Delgado-Benarroch et al., 2009). Here, we used virus-induced gene silencing (VIGS) to analyze the
function of the reported Am-ANT. We then determined the open reading frame (ORF) of Am-ANT and its predicted amino acid sequence.
We induced VIGS using the Cucumber mosaic virus vector (CMV-A1) that contained partial sequence of Am-ANT (A1:ANT) and suppressed the level of Am-ANT mRNA and noted for
any phenotypic changes. The function of Am-ANT was very similar to that of At-ANT. The A1:ANT-infected Antirrhinum plants had smaller floral organs and leaves, even though cell sizes were unchanged in flowers and larger in leaves. The CMV-based
VIGS showed that the isolated Am-ANT gene was indeed functional in cell proliferation and organ growth as observed for At-ANT.
In conclusion, we found that the CMV vector had the advantage of systemically infecting A. majus without severe symptoms for functional analysis of A. majus genes.
Horticulture, Environment, and Biotechnology. 08/2013; 52(2).
[show abstract][hide abstract] ABSTRACT: Plants have developed RNA silencing as an antiviral defense mechanism. To escape from the plant host’s defenses, viruses have countered their host’s antiviral silencing by producing RNA silencing suppressor proteins (RSSs). Although the mode of action of the majority of viral RSSs has been found to be through double-stranded RNA-binding, viruses have different strategies to counteract the host’s antiviral silencing pathways. The 2b protein of Cucumber mosaic virus, which is one of the most extensively studied viral RSSs, is reviewed here to provide insights on the molecular arms race between viruses and their host plants.
Journal of General Plant Pathology 08/2013; 79(4). · 0.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plants may activate posttranscriptional gene silencing (PTGS) as an immunity system when they are infected with viruses. Viruses may in turn interfere with this system by producing RNA silencing suppressor (RSS) proteins; most RSSs bind to viral small interfering RNAs (siRNAs). We previously reported that ascorbic acid (AsA) has the ability to interfere with the binding between viral siRNAs and viral RSSs in vitro. We thus expected that AsA-treated plants would show some tolerance to virus infection because the host PTGS will be strengthened by AsA. Brassica rapa subsp. rapa was inoculated with Turnip mosaic virus and treated with the AsA derivatives, l(+)-ascorbic acid 2-sulfate disodium salt dihydrate (AsA–SO4), l(+)-ascorbyl palmitate (AsA–Pal) and dehydroascorbic acid (DHA) at 1 h postinoculation. The number of infection sites on inoculated leaves decreased by around 40 % after AsA–SO4 and AsA–Pal treatments and by 80 % after DHA treatment compared with the untreated control. As evidenced by an enzyme-linked immunosorbent assay, viral accumulation was significantly reduced after regular sprays with the AsA derivatives and DHA. In a detached leaf assay, AsA clearly functioned as a viral inhibitor in cells. Additionally, we confirmed that DHA also worked in the silencing pathway because its antiviral effect was not observed in the silencing-defective double mutant dcl2/dcl4 of Arabidopsis thaliana. On the basis of these results, we concluded that the AsA derivatives and DHA can significantly reduce viral infection and accumulation and that we can develop those compounds as a practical antiviral agent in the future.
Journal of General Plant Pathology 08/2013; 79(3). · 0.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus in the family Bromoviridae. We cloned the coat protein (CP) and the 2b protein (2b) genes of AV-2 isolates from asparagus plants from various regions and found that the sequence for CP and for 2b was highly conserved among the isolates, suggesting that AV-2 from around the world is almost identical. We then made an AV-2 infectious clone by simultaneous inoculation with in vitro transcripts of RNAs 1-3 of AV-2 and in vitro-synthesized CP, which is necessary for initial infection. Because 2b of cucumoviruses in Bromoviridae can suppress systemic silencing as well as local silencing, we analyzed whether there is functional synteny of 2b between AV-2 and cucumovirus. Using the AV-2 infectious clone, we here provided first evidence that Ilarvirus 2b functions as an RNA silencing suppressor; AV-2 2b has suppressor activity against systemic silencing but not local silencing.
[show abstract][hide abstract] ABSTRACT: Cytosine methylation can be induced by double-stranded RNAs through the RNA-directed DNA methylation (RdDM) pathway. A DNA glycosylase REPRESSOR OF SILENCING 1 (ROS1) participates in DNA demethylation in Arabidopsis and may possibly counteract RdDM. Here, we isolated an ortholog of ROS1 (NbROS1) from Nicotiana benthamiana and examined the antagonistic activity of NbROS1 against virus-induced RdDM by simultaneously inducing RdDM and NbROS1 knockdown using a vector based on Cucumber mosaic virus. Plants were inoculated with a virus that contained a portion of the Cauliflower mosaic virus 35S promoter, which induced RdDM of the promoter integrated in the plant genome and transcriptional silencing of the green fluorescent protein gene driven by the promoter. Plants were also inoculated with a virus that contained a portion of NbROS1, which induced downregulation of NbROS1. Simultaneous induction of RdDM and NbROS1 knockdown resulted in an increase in the level of cytosine methylation of the target promoter. These results provide evidence for the presence of antagonistic activity of NbROS1 against virus-induced RdDM and suggest that the simultaneous induction of promoter-targeting RdDM and NbROS1 knockdown by a virus vector is useful as a tool to enhance targeted DNA methylation.
[show abstract][hide abstract] ABSTRACT: Plants and animals can recognize the invasion of pathogens through their perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Plant PRRs identified have been exclusively receptor-like kinases/proteins (RLK/Ps), and no RLK/P that can detect viruses has been identified to date. RNA silencing (RNA interference, RNAi) is regarded as an antiviral basal immunity because the majority of plant viruses has RNA as their genomes and encode RNA silencing suppressor (RSS) proteins to counterattack antiviral RNAi. Many RSSs were reported to bind to double-stranded RNAs (dsRNAs), which are regarded as viral PAMPs. We have recently identified a tobacco calmodulin (CaM)-like protein, rgs-CaM, as a PRR that binds to diverse viral RSSs through its affinity for the dsRNA-binding domains. Because rgs-CaM seems to target RSSs for autophagic degradation with self-sacrifice, the expression level of rgs-CaM is important for antiviral activity. Here, we found that the rgs-CaM expression was induced immediately (within 1 h) after wounding at a wound site on tobacco leaves. Since the invasion of plant viruses is usually associated with wounding, and several hours are required for viruses to replicate to a detectable level in invaded cells, the wound-induced expression of rgs-CaM seems to be linked to its antiviral function, which should be ready before the virus establishes infection. CaMs and CaM-like proteins usually transduce calcium signals through their binding to endogenous targets. Therefore, rgs-CaM is a unique CaM-like protein in terms of binding to exogenous targets and functioning as an antiviral PRR.
[show abstract][hide abstract] ABSTRACT: RCY1, which encodes a coiled coil nucleotide-binding site leucine-rich repeat (LRR) class R protein, confers the hypersensitive response (HR) to a yellow strain of Cucumber mosaic virus (CMV[Y]) in Arabidopsis thaliana. Nicotiana benthamiana transformed with hemagglutinin (HA) epitope-tagged RCY1 (RCY1-HA) also exhibited a defense response accompanied by HR cell death and induction of defense-related gene expression in response to CMV(Y). Following transient expression of RCY1-HA by agroinfiltration, the defense reaction was induced in N. benthamiana leaves infected with CMV(Y) but not in virulent CMV(B2)-infected N. benthamiana leaves transiently expressing RCY1-HA or CMV(Y)-infected N. benthamiana leaves transiently expressing HA-tagged RPP8 (RPP8-HA), which is allelic to RCY1. This result suggests that Arabidopsis RCY1-conferred resistance to CMV(Y) could be reproduced in N. benthamiana leaves in a gene-for-gene manner. Expression of a series of chimeric constructs between RCY1-HA and RPP8-HA in CMV(Y)-infected N. benthamiana indicated that induction of defense responses to CMV(Y) is regulated by the LRR domain of RCY1. Interestingly, in CMV(Y)-infected N. benthamiana manifesting the defense response, the levels of both RCY1 and chimeric proteins harboring the RCY1 LRR domain were significantly reduced. Taken together, these data indicate that the RCY1-conferred resistance response to CMV(Y) is regulated by an LRR domain-mediated interaction with CMV(Y) and seems to be tightly associated with the degradation of RCY1 in response to CMV(Y).
[show abstract][hide abstract] ABSTRACT: In plants, jasmonic acid (JA) and its derivatives are thought to be involved in mobile forms of defense against biotic and abiotic stresses. In this study, the distal transport of JA-isoleucine (JA-Ile) that is synthesized de novo in response to leaf wounding in tomato (Solanum lycopersicum) plants was investigated. JA-[(13)C(6)]Ile was recovered in distal untreated leaves after wounded leaves were treated with [(13)C(6)]Ile. However, as [(13)C(6)]Ile was also recovered in the distal untreated leaves, whether JA-Ile was synthesized in the wounded or in the untreated leaves was unclear. Hence, stem exudates were analyzed to obtain more detailed information. When [(13)C(6)]Ile and [(2)H(6)]JA were applied separately into the wounds on two different leaves, JA-[(13)C(6)]Ile and [(2)H(6)]JA-Ile were detected in the stem exudates but [(2)H(6)]JA-[(13)C(6)]Ile was not, indicating that JA was conjugated with Ile in the wounded leaf and that the resulting JA-Ile was then transported into systemic tissues. The [(2)H(3)]JA-Ile that was applied exogenously to the wounded tissues reached distal untreated leaves within 10min. Additionally, applying [(2)H(3)]JA-Ile to the wounded leaves at concentrations of 10 and 60nmol/two leaves induced the accumulation of PIN II, LAP A, and JAZ3 mRNA in the distal untreated leaves of the spr2 mutant S. lycopersicum plants. These results demonstrate the transportation of de novo synthesized JA-Ile and suggest that JA-Ile may be a mobile signal.
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 06/2012; 109(25):10113-8. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: RNA silencing plays an important role in plant resistance against viruses. As a counter-defense against RNA silencing, plant viruses have evolved RNA silencing suppressors (RSSs). RNA silencing is likely to play a major role in disease development. For example, RSSs have been found to disturb the gene expression controlled by miRNAs in plant tissue and organ development, resulting in plant malformation. Mosaic symptoms, which are typical in virus-infected plants, are actually a consequence of local arms race between host RNA silencing and viral RSSs. In addition, recent studies revealed that viral siRNAs could induce RNA silencing even against a certain host gene and thus a disease symptom through a complementary (homologous) sequence coincidentally found between virus and host gene. RNA silencing is the principal mediator of viral pathogenicity and disease induction and therefore should be exploited as a powerful tool for engineering virus resistance in plants as well as in animals.
[show abstract][hide abstract] ABSTRACT: Jasmonic acid (JA) and salicylic acid (SA) are plant hormones involved in plant growth and development. Recent studies demonstrated
that presence of a complex interplay between JA and SA signaling pathways to response to pathogenesis attack and biotic stresses.
To our best knowledge, no method has existed for simultaneous analyses of JA, SA, and their related compounds. Especially,
the glucosides are thought to be the storages or the inactivated compounds, but their contribution should be considered for
elucidating the amount of the aglycons. It is also valuable for measuring the endogenous amount of phenylalanine, cinnamic
acid, and benzoic acid that are the biosynthetic intermediates of SA due to the existence of isochorismate pathway to synthesize
SA. We established this method using deuterium labeled compounds as internal standards. This is the first report of simultaneous
analysis of endogenous JA, SA, and their related compounds. Measuring the endogenous JA, SA, and their related compounds that
had been accumulated in tobacco plants proved the practicality of the newly developed method. It was demonstrated that accumulation
of JA, SA and their related compounds were induced in both case of TMV infection and abiotic stresses.
[show abstract][hide abstract] ABSTRACT: RNA silencing is a sequence-specific defense mechanism against viruses. As a counterdefense, viruses evolved silencing suppressors
to interfere with host silencing. In analyses using protoplasts prepared from cultured cells (BY-2) and mesophyll cells of
Nicotiana tabacum and N. benthamiana, viral suppressors differentially functioned in different cell types. This phenomenon has not been discussed in earlier papers
on protoplast systems and RNA silencing. In investigations of the cellular activities of viral suppressors and their role
in the RNA-silencing pathway, assays with host protoplasts offer many advantages and can complement other in planta assays
such as Agrobacterium-mediated transient expression.
Journal of General Plant Pathology 04/2012; 74(4):326-330. · 0.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Small interfering RNAs (siRNAs) are key molecules in RNA silencing, which includes posttranscriptional gene silencing, cosuppression,
quelling, and RNA interference. The presence of siRNAs indicates RNA silencing in cells. We present a method of detecting
siRNAs using nonradioactive probes that involves isolating the small RNA fraction, separating siRNAs using denaturing gel
electrophoresis, and performing a Northern blot analysis under low-stringency hybridization conditions. We used digoxigenin-labeled
DNA probes for hybridization and detected siRNAs in petunia and rice plants exhibiting silenced phenotypes. This method is
a simple and rapid way to detect siRNAs without using radioisotopes.
[show abstract][hide abstract] ABSTRACT: Arbuscular mycorrhizal (AM) fungi form mutualistic associations with most land plants and enhance phosphorus uptake of the host plants. Fungal viruses (mycoviruses) that possess a double-stranded RNA (dsRNA) genome often affect plant-fungal interactions via altering phenotypic expression of their host fungi. The present study demonstrates, for the first time, the presence of dsRNAs, which are highly likely to be mycoviruses, in AM fungi. dsRNA was extracted from mycelia of Glomus sp. strain RF1, purified, and subjected to electrophoresis. The fungus was found to harbor various dsRNA segments that differed in size. Among them, a 4.5-kbp segment was termed Glomus sp. strain RF1 virus-like medium dsRNA (GRF1V-M) and characterized in detail. The GRF1V-M genome segment was 4,557 nucleotides in length and encoded RNA-dependent RNA polymerase and a structural protein. GRF1V-M was phylogenetically distinct and could not be assigned to known genera of mycovirus. The GRF1V-M-free culture line of Glomus sp. strain RF1, which was raised by single-spore isolation, produced twofold greater number of spores and promoted plant growth more efficiently than the GRF1V-M-positive lines. These observations suggest that mycoviruses in AM fungi, at least some of them, have evolved under unique selection pressures and are a biologically active component in the symbiosis.
[show abstract][hide abstract] ABSTRACT: Many plant viruses encode suppressors of RNA silencing, including the helper component-proteinase (HC-Pro) of potyviruses. Our previous studies showed that a D-to-Y mutation at amino acid position 193 in HC-Pro (HC-Pro-D193Y) drastically attenuated the virulence of clover yellow vein virus (ClYVV) in legume plants. Furthermore, RNA-silencing suppression (RSS) activity of HC-Pro-D193Y was significantly reduced in Nicotiana benthamiana. Here, we examine the effect of expression of heterologous suppressors of RNA silencing, i.e., tomato bushy stunt virus p19, cucumber mosaic virus 2b, and their mutants, on the virulence of the ClYVV point mutant with D193Y (Cl-D193Y) in pea. P19 and 2b fully and partially complemented Cl-D193Y multiplication and virulence, including lethal systemic HR in pea, respectively, but the P19 and 2b mutants with defects in their RSS activity did not. Our findings strongly suggest that the D193Y mutation exclusively affects RSS activity of HC-Pro and that RSS activity is necessary for ClYVV multiplication and virulence in pea.
Archives of Virology 03/2012; 157(6):1019-28. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plant viral symptoms are rarely explained by direct molecular interaction between a viral protein and a host factor, but rather understood as a consequence of arms race between host RNA silencing and viral silencing suppressors. However, we have recently demonstrated that the 2b protein (2b) of Cucumber mosaic virus (CMV) HL strain could bind to Arabidopsis catalase that is important in scavenging cellular hydrogen peroxide, leading to the induction of distinct necrosis on Arabidopsis. Because we previously used virulent strains of subgroup I CMV in the study, we here further analyzed mild strains of subgroup II CMV, which share 70 to 80% sequence homology with subgroup I, to understand whether the necrosis induction is a general phenomenon to compromise host defense system mediated by catalase in the pathosystem of any CMV strains and Arabidopsis. Based on the results, we concluded that 2bs of subgroup II could also bind to catalase, resulting in decrease in catalase activity and weak necrosis on Arabidopsis. Because the 2b-catalase interaction did not prevent CMVs from spreading, it may eventually operate in favor of CMV.
[show abstract][hide abstract] ABSTRACT: Viroids and satellite RNAs, which are the smallest infectious agents in plants, have noncoding RNA genomes and characteristic secondary structures. Some satellite RNAs (satRNAs) cause disease symptoms that are different from those induced by their helper virus. This phenomenon has been implicated in RNA silencing of host gene(s) as a result of sequence identity or complementarity between satRNAs and host RNAs. To investigate the effects of satRNA sequence on direct coincident interference with host gene expression, we developed a transient RNA silencing assay using protoplasts. With this protoplast system, we can induce various forms and lengths of silencing inducers at various concentrations to uniform cells without viral infection, and then we can use the satRNA-treated protoplasts in further analyses such as real-time RT-PCR and northern blot hybridization analyses to investigate whether the satRNA-induced symptoms are due to down-regulation of the target gene expression.
Methods in molecular biology (Clifton, N.J.) 01/2012; 894:273-86.
[show abstract][hide abstract] ABSTRACT: The expression of transgenes in plant genomes can be inhibited by either transcriptional gene silencing or posttranscriptional gene silencing (PTGS). Overexpression of the chalcone synthase-A (CHS-A) transgene triggers PTGS of CHS-A and thus results in loss of flower pigmentation in petunia. We previously demonstrated that epigenetic inactivation of CHS-A transgene transcription leads to a reversion of the PTGS phenotype. Although neomycin phosphotransferase II (nptII), a marker gene co-introduced into the genome with the CHS-A transgene, is not normally silenced in petunia, even when CHS-A is silenced, here we found that nptII was silenced in a petunia line in which CHS-A PTGS was induced, but not in the revertant plants that had no PTGS of CHS-A. Transcriptional activity, accumulation of short interfering RNAs, and restoration of mRNA level after infection with viruses that had suppressor proteins of gene silencing indicated that the mechanism for nptII silencing was posttranscriptional. Read-through transcripts of the CHS-A gene toward the nptII gene were detected. Deep-sequencing analysis revealed a striking difference between the predominant size class of small RNAs produced from the read-through transcripts (22 nt) and that from the CHS-A RNAs (21 nt). These results implicate the involvement of read-through transcription and distinct phases of RNA degradation in the coincident PTGS of linked transgenes and provide new insights into the destabilization of transgene expression.
[show abstract][hide abstract] ABSTRACT: Recently we reported that rice salicylic acid (SA) glucosyltransferase (OsSGT) is active toward 12-hydroxyjasmonic acid (tuberonic acid, TA) and that OsSGT gene expression is induced by wounding stress. Here we report that tobacco SA glucosyltransferase (NtSGT), which is thought to be an ortholog of OsSGT, is also active toward TA. Although NtSGT expression is known to be induced by biotrophic stress, it was also induced by wounding stress in the same manner as OsSGT. These results indicate that this glucosyltransferase is important not only in biotrophic stress but also for wounding stress. It was found that this enzyme is dually functional, with activity both toward TA and SA.
Bioscience Biotechnology and Biochemistry 12/2011; 75(12):2316-20. · 1.27 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nucleotide sequences from the P1 gene and the 5' untranslated region of leek yellow stripe virus (LYSV), collected from several locations, were used to refine the phylogenetic relationships among the isolates. Multiple alignments revealed three distinct regions of insertions and deletions to classify LYSVs. In our phylogenetic analyses, the LYSV isolates separated into two major groups (N-type and S-type). S-type viruses had two large deletions compared to N-type viruses. Considering that the outgroup, onion yellow dwarf virus (OYDV) also has the sequences corresponding to the deletions in the S-type viruses, our study shows that the sequences missing in the S-type were present in the common ancestor of the N-type and S-type. In the phylogenetic trees, we found three distinct clades of isolates, from Uruguay (U), Okinawa (O) and Spain (Sp), suggesting that LYSVs have unique evolutionary histories depending on their garlic origins. The P1 gene of LYSV is thus quite suited to reflecting viral evolution, as recently suggested for other potyviruses.
Archives of Virology 10/2011; 157(1):147-53. · 2.03 Impact Factor