[Show abstract][Hide abstract] ABSTRACT: Background
Capsaicinoids, including capsaicin and its analogs, are responsible for the pungency of pepper (Capsicum species) fruits. Even though capsaicin is familiar and used daily by humans, the genes involved in the capsaicin biosynthesis pathway have not been well characterized. The putative aminotransferase (pAMT) and Pungent gene 1 (Pun1) proteins are believed to catalyze the second to last and the last steps in the pathway, respectively, making the Pun1 protein the putative capsaicin synthase. However, there is no direct evidence that Pun1 has capsaicin synthase activity.
To verify that the Pun1 protein actually plays a role in capsaicin production, we generated anti-Pun1 antibodies against an Escherichia coli-synthesized Pun1 protein and used them to antagonize endogenous Pun1 activity. To confirm the anti-Pun1 antibodies’ specificity, we targeted Pun1 mRNA using virus-induced gene silencing. In the Pun1-down-regulated placental tissues, the accumulated levels of the Pun1 protein, which was identified on a western blot using the anti-Pun1 antibodies, were reduced, and simultaneously, capsaicin accumulations were reduced in the same tissues. In the de novo capsaicin synthesis in vitro cell-free assay, which uses protoplasts isolated from placental tissues, capsaicin synthesis was inhibited by the addition of anti-Pun1 antibodies. We next analyzed the expression profiles of pAMT and Pun1 in various pepper cultivars and found that high levels of capsaicin accumulation always accompanied high expression levels of both pAMT and Pun1, indicating that both genes are important for capsaicin synthesis. However, comparisons of the accumulated levels of vanillylamine (a precursor of capsaicin) and capsaicin between pungent and nonpungent cultivars revealed that vanillylamine levels in the pungent cultivars were very low, probably owing to its rapid conversion to capsaicin by Pun1 soon after synthesis, and that in nonpungent cultivars, vanillylamine accumulated to quite high levels owing to the lack of Pun1.
Using a newly developed protoplast-based assay for de novo capsaicin synthesis and the anti-Pun1 antibodies, we successfully demonstrated that the Pun1 gene and its gene product are involved in capsaicin synthesis. The analysis of the vanillylamine accumulation relative to that of capsaicin indicated that Pun1 was the primary determinant of their accumulation levels.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0476-7) contains supplementary material, which is available to authorized users.
Full-text · Article · Dec 2015 · BMC Plant Biology
[Show abstract][Hide abstract] ABSTRACT: White lupin (Lupinus albus L.) plants develop cluster roots and have strong resistance to phosphorus starvation. Although many expressed sequences have been identified to explain the mechanisms used by white lupin to acquire phosphorus, the lack of a stable transformation technique has made it challenging to evaluate the functions of these expressed sequences. Virus-induced gene silencing (VIGS) is an attractive method for assaying gene function in species that are difficult to stably transform. Here, we show that the Peanut stunt virus (PSV) vector effectively induces silencing of endogenous genes in white lupin. It is unknown whether PSV is useful for VIGS; therefore, we first inoculated Nicotiana benthamiana plants with PSV harbouring fragments of the N. benthamiana phytoene desaturase gene (NbPDS). Two out of four distinct sequence fragments of NbPDS induced photo-bleaching in N. benthamiana, indicating that PSV can be used to knockdown endogenous gene sequences in a sequence-dependent manner. White lupin plants inoculated with PSV harbouring fragments of the L. albus PDS gene (LaPDS) developed photo-bleaching that was associated with a significant reduction in LaPDS mRNA accumulation. PSV spread systemically in leaves, roots, and cluster roots, and small interfering RNA of LaPDS was detected in these organs. This is the first study to demonstrate the use of VIGS by PSV, suggesting that this vector can be applied to supress endogenous gene expression in shoots and roots of white lupin and to clarify the mechanisms of phosphorus starvation resistance.
Preview · Article · Oct 2015 · Plant Biotechnology
[Show abstract][Hide abstract] ABSTRACT: We previously observed that Turnip mosaic virus (TuMV) induces systemic necrosis in Arabidopsis ecotype Ler but not in Col-0. Sharing most of the common features of the hypersensitive reaction, this systemic necrosis has been found to be a result of a gene-for-gene interaction between the host TuNI gene and viral P3 gene. We here analyzed the TuNI locus in detail and identified three very similar candidates for TuNI gene(s). Our functional and expression analyses using transgenic plants expressing each of the candidates and a protoplast transient expression assay suggested that the expression of the best candidate, named RGX, is responsible for the systemic necrosis although concomitant expression with the other two candidates may be necessary. To understand the expression profile of RGX, we analyzed promoter activity by producing transgenic Col-0 plants that express the reporter GFP gene under the control of the 500-1400-bp upstream regions of RGX. The transgenic plants reproduced the expression patterns of RGX; GFP expression increased after TuMV infection but decreased in the shade treatment. The expression of two other candidates was also upregulated by TuMV infection, suggesting that transcriptional activation of the TuNI candidate genes has a role in controlling TuMV-mediated systemic necrosis.
No preview · Article · May 2015 · Journal of General Plant Pathology
[Show abstract][Hide abstract] ABSTRACT: Fungal viruses (mycoviruses) often have a significant impact not only on phenotypic expression of the host fungus but also on higher order biological interactions, e.g., conferring plant stress tolerance via an endophytic host fungus. Arbuscular mycorrhizal (AM) fungi in the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. So far, little information about mycoviruses has been obtained in the fungi due to their obligate biotrophic nature. Here we provide a technical breakthrough, "two-step strategy" in combination with deep-sequencing, for virological study in AM fungi; dsRNA is first extracted and sequenced using material obtained from highly productive open pot culture, and then the presence of viruses is verified using pure material produced in the in vitro monoxenic culture. This approach enabled us to demonstrate the presence of several viruses for the first time from a glomeromycotan fungus.
No preview · Article · Jan 2015 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The protocol for a simple, sensitive, and specific method using a cDNA macroarray to detect multiple viruses is provided. The method can be used even at the production sites for crops, which need a reliable routine diagnosis for mixed infection of plant viruses. The method consists of three steps: RNA extraction, duplex RT-PCR, and "microtube hybridization" (MTH). Biotinylated cDNA probes are prepared using RT-PCR and used to hybridize a nylon membrane containing target viral cDNAs by MTH. Positive signals can be visualized by colorimetric reaction and judged by eyes. We here demonstrate this method to detect asparagus viruses (Asparagus virus 1 and Asparagus virus 2) from latently infected asparagus plants.
No preview · Article · Jan 2015 · Methods in Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: GmPT7 was originally identified as an arbuscular mycorrhiza-inducible gene of soybean that encodes a member of subfamily I in the
PHOSPHATE TRANSPORTER 1 family. In the present study, we established conditions under which a number of dwarf soybean plants complete their life
cycles in a growth chamber. Using this system, we grew transgenic soybean with a GmPT7 promoter–β-glucuronidase fusion gene and evaluated GmPT7 expression in detail. GmPT7 was highly expressed in mature, but not in collapsed, arbuscule-containing cortical cells, suggesting its importance in the
absorption of fungus-derived phosphate and/or arbuscule development. GmPT7 was also expressed in the columella cells of root caps and in the lateral root primordia of non-mycorrhizal roots. The expression
of GmPT7 occurred only in the late stage of phosphorus translocation from leaves to seeds, after water evaporation from the leaves
ceased, and later than the expression of GmUPS1-2, GmNRT1.7a and GmNRT1.7b, which are possibly involved in nitrogen export. GmPT7 expression was localized in a pair of tracheid elements at the tips of vein endings of senescent leaves. Transmission electron
microscopy revealed that the tip tracheid elements in yellow leaves were still viable and had intact plasma membranes. Thus,
we think that GmPT7 on the plasma membranes transports phosphate from the apoplast into the tip elements. GmPT7 knockdown resulted in no significant effects, the function of GmPT7 remaining to be clarified. We propose a working model
in which phosphate incorporated in vein endings moves to seeds via xylem to phloem transfer.
No preview · Article · Oct 2014 · Plant and Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT Asparagus virus 2 (AV-2) is a member of the genus Ilarvirus and thought to induce the asparagus decline syndrome. AV-2 is known to be transmitted by seed, and the possibility of pollen transmission was proposed 25 years ago but not verified. In AV-2 sequence analyses, we have unexpectedly found mixed infection by two distinct AV-2 isolates in two asparagus plants. Because mixed infections by two related viruses are normally prevented by cross protection, we suspected that pollen transmission of AV-2 is involved in mixed infection. Immunohistochemical analyses and in situ hybridization using AV-2-infected tobacco plants revealed that AV-2 was localized in the meristem and associated with pollen grains. To experimentally produce a mixed infection via pollen transmission, two Nicotiana benthamiana plants that were infected with each of two AV-2 isolates were crossed. Derived cleaved-amplified polymorphic sequence analysis identified each AV-2 isolate in the progeny seedlings, suggesting that pollen transmission could indeed result in a mixed infection, at least in N. benthamiana.
[Show abstract][Hide abstract] ABSTRACT: To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host-pathogen interactions. By analogy of the steps in virus-host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus-host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus-plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response.
No preview · Article · May 2014 · Current Opinion in Plant Biology
[Show abstract][Hide abstract] ABSTRACT: Arbuscular mycorrhizal (AM) fungi that belong to the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. One of the four viral segments found by deep-sequencing of dsRNA in the AM fungus Rhizophagus clarus strain RF1 showed similarity to mitoviruses and is characterized in this report. The genome segment is 2,895 nucleotides in length, and the largest ORF was predicted by applying either the mold mitochondrial or the universal genetic code. The ORF encodes a polypeptide of 820 amino acids with a molecular mass of 91.2 kDa and conserves the domain of the mitovirus RdRp superfamily. Accordingly, the dsRNA was designated as R. clarus mitovirus 1 strain RF1 (RcMV1-RF1). Mitoviruses are localized exclusively in mitochondria and thus generally employ the mold mitochondrial genetic code. The distinct codon usage of RcMV1-RF1, however, suggests that the virus is potentially able to replicate not only in mitochondria but also in the cytoplasm. RcMV1-RF1 RdRp showed the highest similarity to the putative RdRp of a mitovirus-like ssRNA found in another AM fungus, followed by RdRp of a mitovirus in an ascomycotan ectomycorrhizal fungus. The three mitoviruses found in the three mycorrhizal fungi formed a deeply branching clade that is distinct from the two major clades in the genus Mitovirus.
No preview · Article · Feb 2014 · Archives of Virology
[Show abstract][Hide abstract] ABSTRACT: To elucidate events in the molecular arms race between the host and pathogen in evaluating plant immunity, a zigzag model is useful for uncovering aspects common to different host–pathogen interactions. By analogy of the steps in virus–host interactions with the steps in the standard zigzag model outlined in recent papers, we may regard RNA silencing as pattern-triggered immunity (PTI) against viruses, RNA silencing suppressors (RSSs) as effectors to overcome host RNA silencing and resistance gene (R-gene)-mediated defense as effector-triggered immunity (ETI) recognizing RSSs as avirulence proteins. However, because the standard zigzag model does not fully apply to some unique aspects in the interactions between a plant host and virus, we here defined a model especially designed for viruses. Although we simplified the phenomena involved in the virus–host interactions in the model, certain specific interactive steps can be explained by integrating additional host factors into the model. These host factors are thought to play an important role in maintaining the efficacy of the various steps in the main pathway of defense against viruses in this model for virus–plant interactions. For example, we propose candidates that may interact with viral RSSs to induce the resistance response.
[Show abstract][Hide abstract] ABSTRACT: Key message:
Soybean expressing the Cucumber mosaic virus 2b gene manifests seed coat pigmentation due to suppression of endogenous RNA silencing but no other morphological abnormality. This gene may help prevent transgene silencing. RNA silencing is an important mechanism for gene regulation and antiviral defense in plants. It is also responsible for transgene silencing, however, and thus hinders the establishment of transgenic plants. The 2b protein of Cucumber mosaic virus (CMV) functions as a suppressor of RNA silencing and therefore might prove beneficial for stabilization of transgene expression. We have now generated transgenic soybean that harbors the 2b gene of a CMV-soybean strain under the control of a constitutive promoter to investigate the effects of 2b expression. No growth abnormality was apparent in 2b transgenic plants, although the seed coat was pigmented in several of the transgenic lines. Genes for chalcone synthase (CHS), a key enzyme of the flavonoid pathway, are posttranscriptionally silenced by the inhibitor (I) locus in nonpigmented (yellow) soybean seeds. The levels of CHS mRNA and CHS small interfering RNA in strongly pigmented 2b transgenic seed coats were higher and lower, respectively, than those in the seed coat of a control transgenic line. The expression level of 2b also correlated with the extent of seed coat pigmentation. On the other hand, introduction of the 2b gene together with the DsRed2 gene into somatic embryos prevented the time-dependent decrease in transient DsRed2 expression. Our results indicate that the 2b gene alone is able to suppress RNA silencing of endogenous CHS genes regulated by the I locus, and that 2b is of potential utility for stabilization of transgene expression in soybean without detrimental effects other than seed coat pigmentation.
[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.
No preview · Article · Jul 2013 · Journal of General Plant Pathology
[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: 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.
No preview · Article · May 2013 · Journal of General Plant Pathology
[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.
Preview · Article · Apr 2013 · Frontiers in Genetics
[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.