Publications by authors named "Tetsuro Okuno"

81 Publications

CoGRIM19 is required for invasive hyphal growth of Colletotrichum orbiculare inside epidermal cells of cucumber cotyledons.

Microb Pathog 2021 Mar 11;154:104847. Epub 2021 Mar 11.

Department of Plant Life Science, Ryukoku University, Seta, Shiga, 520-2194, Japan.

Colletotrichum orbiculare, an anthracnose disease fungus of cucurbit plants, extends penetration hyphae inside the epidermal cells of host plants. Unlike vegetative hyphae formed on a nutrient rich medium, this pathogen initially develops biotrophic penetration hyphae, which acquire nutrient resources from living host cells and secret effector proteins to suppress host defense responses. Subsequently, the nature of penetration hyphae changes from biotrophy to necrotrophy in response to the interaction with a host plant. Hence, controlling the extension of penetration hyphae is crucial for C. orbiculare infection. Here, we identified CoGRIM19 encoding Nadh-ubiquinone oxidoreductase subunit as a pathogenicity gene. Pathogenicity assays showed that the cogrim19 mutant caused no visible symptoms on cucumber cotyledons. Microscopic observations revealed that the cogrim19 mutant developed an appressorium and penetration hyphae under artificial conditions such as on coverslips or cellulose membranes, but the penetration hyphae of the mutant were retarded in the cucumber cotyledons. Microscopic observations of biotrophy-specific expression fluorescent signals revealed that the biotrophic stage was maintained in the retarded penetration hyphae of the cogrim19 mutant as the penetration of the wild type. In addition to cytological observations, pathogenicity assays using wounded leaves showed that the cogrim19 mutant had an attenuated pathogenesis. Taking our results together, CoGRIM19 is required for invasive hyphal growth inside the epidermal cells of cucumber cotyledons in C. orbiculare.
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http://dx.doi.org/10.1016/j.micpath.2021.104847DOI Listing
March 2021

Trade-Off Relation between Fungicide Sensitivity and Melanin Biosynthesis in Plant Pathogenic Fungi.

iScience 2020 Nov 8;23(11):101660. Epub 2020 Oct 8.

Department of Plant Life Science, Ryukoku University, Seta, Shiga 520-2194, Japan.

Circumventing the emergence of fungicide-resistant strains is a crucial issue for robust disease management in agriculture. The agricultural fungicide ferimzone has been used for the control of rice diseases including rice blast. The emergence of ferimzone-resistant strains in rice fields has not been reported. Here, we identified the copper transport gene as the ferimzone sensitivity gene in and the rice blast fungus . Genetic and cytological analyses showed that functional defects in the copper transport pathways, consisting of CoIct1 and P-type ATPase CoCcc2, led to the low sensitivity to ferimzone and the pathogenicity defect due to attenuated melanization in the appressorium. Importantly, the presence of CuSO induced high sensitivity to ferimzone even in the mutant. Our study shows that there is a trade-off relation between the sensitivity to ferimzone and fungal pathogenicity.
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http://dx.doi.org/10.1016/j.isci.2020.101660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582099PMC
November 2020

Hijacking of host cellular components as proviral factors by plant-infecting viruses.

Adv Virus Res 2020 25;107:37-86. Epub 2020 May 25.

Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga, Japan.

Plant viruses are important pathogens that cause serious crop losses worldwide. They are obligate intracellular parasites that commandeer a wide array of proteins, as well as metabolic resources, from infected host cells. In the past two decades, our knowledge of plant-virus interactions at the molecular level has exploded, which provides insights into how plant-infecting viruses co-opt host cellular machineries to accomplish their infection. Here, we review recent advances in our understanding of how plant viruses divert cellular components from their original roles to proviral functions. One emerging theme is that plant viruses have versatile strategies that integrate a host factor that is normally engaged in plant defense against invading pathogens into a viral protein complex that facilitates viral infection. We also highlight viral manipulation of cellular key regulatory systems for successful virus infection: posttranslational protein modifications for fine control of viral and cellular protein dynamics; glycolysis and fermentation pathways to usurp host resources, and ion homeostasis to create a cellular environment that is beneficial for viral genome replication. A deeper understanding of viral-infection strategies will pave the way for the development of novel antiviral strategies.
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http://dx.doi.org/10.1016/bs.aivir.2020.04.002DOI Listing
April 2021

Complete genome sequence of a novel partitivirus from a wild brassicaceous plant, Arabidopsis halleri.

Arch Virol 2020 Sep 12;165(9):2091-2094. Epub 2020 Jun 12.

Center for Ecological Research, Kyoto University, Hirano 2-509-3, Otsu, Shiga, 520-2113, Japan.

Two contigs with high similarity to partitivirus sequences were identified by de novo assembly of sequences obtained by RNA-Seq from a wild brassicaceous plant, Arabidopsis halleri subsp. gemmifera. Here, we report the complete genome sequence of a putative novel partitivirus. Excluding the poly-A tail, it consists of two RNA genome segments of 1912 and 1769 bp, which are predicted to encode a 585-amino-acid-long putative RNA-dependent RNA polymerase (RdRp) and a 487-amino-acid-long putative capsid protein (CP), respectively. Phylogenetically, this virus belongs to the genus Alphapartitivirus and is most closely related to Raphanus sativus partitivirus 1 from radish. We propose the name "Arabidopsis halleri partitivirus 1" (AhPV1) for this novel virus.
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http://dx.doi.org/10.1007/s00705-020-04670-yDOI Listing
September 2020

Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare.

Microb Pathog 2019 Dec 13;137:103746. Epub 2019 Sep 13.

Department of Plant Life Science, Ryukoku University, Seta, Shiga, 520-2194, Japan.

Upon recognition of host plants, Colletotrichum orbiculare, an anthracnose disease fungus of cucurbitaceous plants, initiates morphological differentiation, including conidial germination and appressorium formation on the cuticle layer. The series of infection processes of C. orbiculare requires enormous nutrient and energy, but the surface of the cucurbitaceous hosts is hardly nutrient-rich. Hence, C. orbiculare must exert tight management of its intracellular nutrients in order to properly induce infection-related morphogenesis. Here, we carried out a large-scale insertional mutagenesis screen using Agrobacterium tumefaciens-mediated transformation to identify novel genes involved in the pathogenicity of C. orbiculare and found that CoTHR4-encoded threonine synthase, a homolog of Saccharomyces cerevisiae THR4, is required for pathogenicity and conidiation in C. orbiculare. Threonine supplementation allowed the cothr4 mutant to produce conidia to a level equivalent to that of the wild-type. The conidia produced from the threonine-treated cothr4 mutant failed to germinate in the absence of threonine, but retained the ability to germinate and to form appressoria in the presence of threonine. However, the conidia produced from the threonine-treated cothr4 mutant remained attenuated in pathogenicity on cucumber cotyledons even in the presence of threonine. Cytorrhysis assays revealed that appressoria of the cothr4 mutant induced by exogenous threonine treatment showed low turgor generation. Taken together, these results showed that threonine synthase CoThr4 plays a pivotal role in infection-related morphogenesis during the pre-penetration stage of C. orbiculare.
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http://dx.doi.org/10.1016/j.micpath.2019.103746DOI Listing
December 2019

5'-Terminal stem-loop of carnation ringspot virus RNA1 is required for the efficient amplification of viral RNAs.

Virus Res 2019 05 16;265:138-142. Epub 2019 Mar 16.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Carnation ringspot virus (CRSV) is the prototype virus of the genus Dianthovirus. Full-length cDNAs of CRSV strainsPV-0097 and PV-21 were constructed and the infectivity of in vitro transcripts was analyzed. Infectivity of PV-0097 and PV-21 to several plants was markedly higher than that of 1.30, a previously reported infectious CRSV clone. Overall RNA sequences of these viruses were similar, but PV-0097 and PV-21 contained additional nucleotides at the 5' end of RNA1. Stem-loop structures were predicted in the 5'-terminal region of PV-0097 and PV-21 RNA1 but not in 1.30 RNA1. Mutant CRSV 1.30 RNA1 that contains the terminal 4 nucleotides of PV-0097, predicted to fold a 5'-terminal stem-loop structure, recovered higher level accumulation of viral RNAs in the inoculated protoplasts and leaves of Nicotiana benthamiana. These results suggest that the 5'-terminal stem-loop structure of CRSV RNA1 plays an important role in efficient amplification of the virus.
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http://dx.doi.org/10.1016/j.virusres.2019.03.009DOI Listing
May 2019

Hijacking a host scaffold protein, RACK1, for replication of a plant RNA virus.

New Phytol 2019 01 31;221(2):935-945. Epub 2018 Aug 31.

Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga, 520-2194, Japan.

Receptor for activated C kinase 1 (RACK1) is strictly conserved across eukaryotes and acts as a versatile scaffold protein involved in various signaling pathways. Plant RACK1 is known to exert important functions in innate immunity against fungal and bacterial pathogens. However, the role of the RACK1 in plant-virus interactions remains unknown. Here, we addressed the role of RACK1 of Nicotiana benthamiana during infection by red clover necrotic mosaic virus (RCNMV), a plant positive-stranded RNA virus. NbRACK1 was shown to be recruited by the p27 viral replication protein into endoplasmic reticulum-derived aggregated structures (possible replication sites). Downregulation of NbRACK1 by virus-induced gene silencing inhibited viral cap-independent translation and p27-mediated reactive oxygen species (ROS) accumulation, which are prerequisite for RCNMV replication. We also found that NbRACK1 interacted with a host calcium-dependent protein kinase (NbCDPKiso2) that activated a ROS-generating enzyme. Interestingly, NbRACK1 was required for the interaction of p27 with NbCDPKiso2, suggesting that NbRACK1 acts as a bridge between the p27 viral replication protein and NbCDPKiso2. Collectively, our findings provide an example of a viral strategy in which a host multifaceted scaffold protein RACK1 is highjacked for promoting viral protein-triggered ROS production necessary for robust viral replication.
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http://dx.doi.org/10.1111/nph.15412DOI Listing
January 2019

Dual function of a cis-acting RNA element that acts as a replication enhancer and a translation repressor in a plant positive-stranded RNA virus.

Virology 2017 12 21;512:74-82. Epub 2017 Sep 21.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga 520-2194, Japan. Electronic address:

The genome of red clover necrotic mosaic virus is divided into two positive-stranded RNA molecules of RNA1 and RNA2, which have no 5' cap structure and no 3' poly(A) tail. Previously, we showed that any mutations in the cis-acting RNA replication elements of RNA2 abolished its cap-independent translational activity, suggesting a strong link between RNA replication and translation. Here, we investigated the functions of the 5' untranslated region (UTR) of RNA2 and revealed that the basal stem-structure (5'BS) predicted in the 5' UTR is essential for robust RNA replication. Interestingly, RNA2 mutants with substitution or deletion in the right side of the 5'BS showed strong translational activity, despite their impaired replication competency. Furthermore, nucleotide sequences other than the 5'BS of the 5' UTR were essential to facilitate the replication-associated translation. Overall, these cis-acting RNA elements seem to coordinately regulate the balance between RNA replication and replication-associated translation.
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http://dx.doi.org/10.1016/j.virol.2017.09.008DOI Listing
December 2017

Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus.

Virology 2017 09 21;509:152-158. Epub 2017 Jun 21.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan; Department of Plant Life Science, Faculty of Agriculture, Ryukoku University, Otsu, Shiga 520-2194, Japan. Electronic address:

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4f1 mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.
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http://dx.doi.org/10.1016/j.virol.2017.06.015DOI Listing
September 2017

Roles of superoxide anion and hydrogen peroxide during replication of two unrelated plant RNA viruses in Nicotiana benthamiana.

Plant Signal Behav 2017 06 8;12(6):e1338223. Epub 2017 Jun 8.

b Laboratory of Plant Pathology , Graduate School of Agriculture, Kyoto University , Sakyo-ku , Kyoto , Japan.

Reactive oxygen species (ROS), including superoxide anion (O), hydrogen peroxide (HO), and hydroxyl radical, act as signaling molecules to transduce biotic and abiotic stimuli into stress adaptations in plants. A respiratory burst oxidase homolog B of Nicotiana benthamiana (NbRBOHB) is responsible for O production to inhibit pathogen infection during plant innate immunity. RBOH-derived O can be immediately converted into HO by the action of superoxide dismutase. Interestingly, we recently showed that red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, hijacks the host's ROS-generating machinery during infection. An RCNMV replication protein associates with NbRBOHB and triggers intracellular ROS bursts. These bursts are required for robust viral RNA replication. However, what types of ROS are required for viral replication is currently unknown. Here, we found that RCNMV replication was sensitive to an O scavenger but insensitive to an HO scavenger. Interestingly, replication of another plant (+)RNA virus, brome mosaic virus, was sensitive to both types of scavengers. These results indicate a virus-specific pattern requirement of O and HO for (+)RNA virus replication and suggest a conserved nature of the roles of ROS in (+)RNA virus replication.
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http://dx.doi.org/10.1080/15592324.2017.1338223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566351PMC
June 2017

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus.

Proc Natl Acad Sci U S A 2017 02 1;114(7):E1282-E1290. Epub 2017 Feb 1.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan;

As sessile organisms, plants have to accommodate to rapid changes in their surrounding environment. Reactive oxygen species (ROS) act as signaling molecules to transduce biotic and abiotic stimuli into plant stress adaptations. It is established that a respiratory burst oxidase homolog B of (NbRBOHB) produces ROS in response to microbe-associated molecular patterns to inhibit pathogen infection. Plant viruses are also known as causative agents of ROS induction in infected plants; however, the function of ROS in plant-virus interactions remains obscure. Here, we show that the replication of red clover necrotic mosaic virus (RCNMV), a plant positive-strand RNA [(+)RNA] virus, requires NbRBOHB-mediated ROS production. The RCNMV replication protein p27 plays a pivotal role in this process, redirecting the subcellular localization of NbRBOHB and a subgroup II calcium-dependent protein kinase of (NbCDPKiso2) from the plasma membrane to the p27-containing intracellular aggregate structures. p27 also induces an intracellular ROS burst in an RBOH-dependent manner. NbCDPKiso2 was shown to be an activator of the p27-triggered ROS accumulations and to be required for RCNMV replication. Importantly, this RBOH-derived ROS is essential for robust viral RNA replication. The need for RBOH-derived ROS was demonstrated for the replication of another (+)RNA virus, brome mosaic virus, suggesting that this characteristic is true for plant (+)RNA viruses. Collectively, our findings revealed a hitherto unknown viral strategy whereby the host ROS-generating machinery is diverted for robust viral RNA replication.
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http://dx.doi.org/10.1073/pnas.1610212114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320965PMC
February 2017

Pathogenesis mediated by proviral host factors involved in translation and replication of plant positive-strand RNA viruses.

Curr Opin Virol 2016 04 30;17:11-18. Epub 2015 Nov 30.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address:

Viral pathogenesis comes from complex interactions between viruses and hosts. All the processes of viral infection, including translation of viral factors and replication of viral genomes, define viral pathogenesis; therefore, molecular insights into the mechanisms underlying viral replication strategies unambiguously pave the way for our comprehensive understanding of viral pathogenesis and disease outcome, as well as for developing new antiviral strategies against plant virus disease. Recent studies of plant positive-strand RNA [(+)RNA] viruses have advanced our understanding of co-opted host factors and their roles in viral translation and replication. It is becoming clear that plant (+)RNA viruses harness host factors involved in membrane trafficking and lipid metabolism to establish the viral replication complex (VRC). In this review, we aim to discuss the contribution of co-opted host factors in translation and genome replication of plant (+)RNA viruses mainly focusing on those involved in the biogenesis of the VRC, which may act as a central hub in almost all the processes of viral infection as well as viral pathogenesis.
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http://dx.doi.org/10.1016/j.coviro.2015.11.004DOI Listing
April 2016

Phosphatidic acid produced by phospholipase D promotes RNA replication of a plant RNA virus.

PLoS Pathog 2015 May 28;11(5):e1004909. Epub 2015 May 28.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.
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http://dx.doi.org/10.1371/journal.ppat.1004909DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4447390PMC
May 2015

GAPDH--a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement.

PLoS Pathog 2014 Nov 20;10(11):e1004505. Epub 2014 Nov 20.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process.
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http://dx.doi.org/10.1371/journal.ppat.1004505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4239097PMC
November 2014

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes.

Front Plant Sci 2014 7;5:321. Epub 2014 Jul 7.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto Japan.

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes.
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http://dx.doi.org/10.3389/fpls.2014.00321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083346PMC
July 2014

Base-paired structure in the 5' untranslated region is required for the efficient amplification of negative-strand RNA3 in the bromovirus melandrium yellow fleck virus.

Virus Res 2014 Aug 25;188:162-9. Epub 2014 Apr 25.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Electronic address:

Melandrium yellow fleck virus belongs to the genus Bromovirus, which is a group of tripartite plant RNA viruses. This virus has an approximately 200-nucleotide direct repeat sequence in the 5' untranslated region (UTR) of RNA3 that encodes the 3a movement protein. In the present study, protoplast assays suggested that the duplicated region contains amplification-enhancing elements. Deletion analyses of the 5' UTR of RNA3 showed that mutations in the short base-paired region, which is located dozens of bases upstream of the initiation codon of the 3a gene, greatly reduced the accumulation of RNA3. Disruption and restoration of the base-paired structure caused the accumulation of RNA3 to be decreased and restored, respectively. In vitro translation/replication assays demonstrated that the base-paired structure is important for the efficient amplification of negative-stand RNA3. A similar base-paired structure in RNA3 of another bromovirus, brome mosaic virus (BMV), also facilitated the efficient amplification of BMV RNA3, but only in combination with melandrium yellow fleck virus (MYFV) replicase and not with BMV replicase, thereby suggesting specific interactions between base-paired structures and MYFV replicase.
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http://dx.doi.org/10.1016/j.virusres.2014.04.002DOI Listing
August 2014

Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus.

Plant Signal Behav 2014 8;9(3):e28644. Epub 2014 Apr 8.

Graduate School of Agriculture; Kyoto University; Kyoto, Japan.

Although positive-strand RNA [(+)RNA] viruses have a limited coding capacity, they can replicate efficiently in host cells because of their ability to use host-derived proteins, membranes, lipids, and metabolites, and to rewire cellular trafficking pathways. Previously, we showed that a plant RNA virus, the Red clover necrotic mosaic virus (RCNMV), hijacked Arf1 and Sar1, which are small GTPases that regulate the biogenesis of COPI and COPII vesicles, respectively, for viral RNA replication. These small GTPases are relocated from appropriate subcellular compartments to the viral RNA replication sites by p27 replication protein, which raises the possibility that RCNMV interferes with the cellular secretory pathway. Here, we examined this possibility by using green fluorescent protein-fused rice SCAMP1 and Arabidopsis LRR84A as secretory pathway marker proteins and showed that p27 inhibited the trafficking of these proteins. RCNMV-mediated inhibition of the host secretion pathway and its possible impact on plant-virus interaction are discussed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091560PMC
http://dx.doi.org/10.4161/psb.28644DOI Listing
April 2015

Functional characterization of the mutations in Pepper mild mottle virus overcoming tomato tm-1-mediated resistance.

Mol Plant Pathol 2014 Jun 19;15(5):479-87. Epub 2014 Jan 19.

Laboratory of Plant Pathology and Biotechnology, Kochi University, Nankoku, Kochi, 783-8502, Japan.

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm-1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants. We found that two mutants, PMMoV-899R,F976Y and PMMoV-899R,F976Y,D1098N, were able to replicate in tomato protoplasts, but only PMMoV-899R,F976Y,D1098N was able to multiply in tomato plants. Further analysis showed that the D1098N mutation of the replication proteins weakened the inhibitory effect of the tm-1 protein and enhanced the replication efficiency of PMMoV-899R,F976Y,D1098N. We also observed that the infectivity of the viruses decreased in the order wild-type PMMoV > PMMoV-899R,F976Y > PMMoV-899R,F976Y,D1098N in original host plants, pepper and tobacco plants. On the contrary, the single mutation D1098N abolished PMMoV replication in tobacco protoplasts. On the basis of these observations, it is likely that the deleterious side-effects of mutations in replication proteins prevent the emergence of PMMoV mutants that can overcome tm-1-mediated resistance.
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http://dx.doi.org/10.1111/mpp.12107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6638807PMC
June 2014

Molecular biology and epidemiology of dianthoviruses.

Adv Virus Res 2013 ;87:37-74

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.

The genus Dianthovirus is one of eight genera in the family Tombusviridae. All the genera have monopartite positive-stranded RNA genomes, except the dianthoviruses which have bipartite genomes. The dianthoviruses are distributed worldwide. Although they share common structural features with the other Tombusviridae viruses in their virions and the terminal structure of the genomic RNAs, the bipartite nature of the dianthovirus genome offers an ideal experimental system with which to study basic issues of virology. The two genomic RNAs seem to use distinct strategies to regulate their translation, transcription, genome replication, genome packaging, and cell-to-cell movement during infection. This review summarizes the current state of our knowledge of the dianthoviruses, with its main emphasis on the molecular biology of the virus, including the viral and host factors required for its infection of host plants. The epidemiology of the virus and the possible viral impacts on agriculture and the environment are also discussed.
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http://dx.doi.org/10.1016/B978-0-12-407698-3.00002-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150339PMC
October 2013

ADP ribosylation factor 1 plays an essential role in the replication of a plant RNA virus.

J Virol 2013 Jan 24;87(1):163-76. Epub 2012 Oct 24.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.

Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.
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http://dx.doi.org/10.1128/JVI.02383-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536388PMC
January 2013

Composition of plant virus RNA replicase complexes.

Curr Opin Virol 2012 Dec 18;2(6):669-75. Epub 2012 Oct 18.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

The replication of the genome of positive-strand RNA viruses depends on their own RNA replicase complexes. Substantial advances in the experimental approaches used to determine the composition of the viral replicase complexes revealed that the replicase complexes of eukaryotic positive-strand RNA viruses are assembled in a host-membrane-derived microenvironment and that this process is regulated by orchestrated interactions between viral proteins, viral genomic RNAs, and co-opted host factors, including molecular chaperones, RNA-binding proteins, and proteins associated with membrane remodeling and lipid synthesis. This review focuses on recent progress in our understanding of how plant RNA viruses organize viral and host factors to form their replicase complexes.
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http://dx.doi.org/10.1016/j.coviro.2012.09.014DOI Listing
December 2012

LAC2 encoding a secreted laccase is involved in appressorial melanization and conidial pigmentation in Colletotrichum orbiculare.

Mol Plant Microbe Interact 2012 Dec;25(12):1552-61

Kyoto University, Kyoto, Japan.

Both Colletotrichum and Magnaporthe spp. develop appressoria pigmented with melanin, which is essential for fungal pathogenicity. 1,8-Dihydroxynaphthalene (1,8-DHN) is believed to be polymerized to yield melanin around the appresorial cell wall through the oxidative activity of laccases. However, no 1,8-DHN laccase has yet been identified in either Colletotrichum or Magnaporthe spp. Here, we report a laccase gene, LAC2, that is involved in the appressorial melanization of Colletotrichum orbiculare, which causes cucumber anthracnose. LAC2 encodes a protein with a signal peptide and has high homology to fungal laccases. The conidial color of lac2 mutants is distinct from that of the C. orbiculare wild type, and the mutants are nonpathogenic. Notably, the mutant appressoria are defective in melanization, and a host invasion assay showed that the appressoria are nonfunctional. LAC2 was induced during appressorial melanization. These results suggest that LAC2 oxidizes 1,8-DHN in the appressoria. The LAC2 homologues of other fungi located in the same phylogenetic clade as LAC2 fully complemented the lac2 mutants. Interestingly, a LAC2 homologue, located in a different clade, complemented the conidial pigmentation but not appressorial melanization of the mutants, suggesting that the LAC2 function in appressorial melanization might only be conserved in laccases of the LAC2 clade.
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http://dx.doi.org/10.1094/MPMI-05-12-0131-RDOI Listing
December 2012

Differential roles of Hsp70 and Hsp90 in the assembly of the replicase complex of a positive-strand RNA plant virus.

J Virol 2012 Nov 29;86(22):12091-104. Epub 2012 Aug 29.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

Assembly of viral replicase complexes of eukaryotic positive-strand RNA viruses is a regulated process: multiple viral and host components must be assembled on intracellular membranes and ordered into quaternary complexes capable of synthesizing viral RNAs. However, the molecular mechanisms underlying this process are poorly understood. In this study, we used a model virus, Red clover necrotic mosaic virus (RCNMV), whose replicase complex can be detected readily as the 480-kDa functional protein complex. We found that host heat shock proteins Hsp70 and Hsp90 are required for RCNMV RNA replication and that they interact with p27, a virus-encoded component of the 480-kDa replicase complex, on the endoplasmic reticulum membrane. Using a cell-free viral translation/replication system in combination with specific inhibitors of Hsp70 and Hsp90, we found that inhibition of p27-Hsp70 interaction inhibits the formation of the 480-kDa complex but instead induces the accumulation of large complexes that are nonfunctional in viral RNA synthesis. In contrast, inhibition of p27-Hsp90 interaction did not induce such large complexes but rendered p27 incapable of binding to a specific viral RNA element, which is a critical step for the assembly of the 480-kDa replicase complex and viral RNA replication. Together, our results suggest that Hsp70 and Hsp90 regulate different steps in the assembly of the RCNMV replicase complex.
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http://dx.doi.org/10.1128/JVI.01659-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3486462PMC
November 2012

Identification of domains in p27 auxiliary replicase protein essential for its association with the endoplasmic reticulum membranes in Red clover necrotic mosaic virus.

Virology 2012 Nov 14;433(1):131-41. Epub 2012 Aug 14.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

Positive-strand RNA viruses require host intracellular membranes for replicating their genomic RNAs. In this study, we determined the domains and critical amino acids in p27 of Red clover necrotic mosaic virus (RCNMV) required for its association with and targeting of ER membranes in Nicotiana benthamiana plants using a C-terminally GFP-fused and biologically functional p27. Confocal microscopy and membrane-flotation assays using an Agrobacterium-mediated expression system showed that a stretch of 20 amino acids in the N-terminal region of p27 is essential for the association of p27 with membranes. We identified the amino acids in this domain required for the association of p27 with membranes using alanine-scanning mutagenesis. We also found that this domain contains amino acids not critical for the membrane association but required for the formation of viral RNA replication complexes and negative-strand RNA synthesis. Our results extend our understanding of the multifunctional role of p27 in RCNMV replication.
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http://dx.doi.org/10.1016/j.virol.2012.07.017DOI Listing
November 2012

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region.

J Virol 2012 Aug 16;86(15):7836-49. Epub 2012 May 16.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.
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http://dx.doi.org/10.1128/JVI.00538-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421650PMC
August 2012

Cell death of Nicotiana benthamiana is induced by secreted protein NIS1 of Colletotrichum orbiculare and is suppressed by a homologue of CgDN3.

Mol Plant Microbe Interact 2012 May;25(5):625-36

Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein-based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.
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http://dx.doi.org/10.1094/MPMI-12-11-0316DOI Listing
May 2012

A long-distance RNA-RNA interaction plays an important role in programmed -1 ribosomal frameshifting in the translation of p88 replicase protein of Red clover necrotic mosaic virus.

Virology 2011 Aug 23;417(1):169-78. Epub 2011 Jun 23.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, 606-8502, Japan.

Programmed -1 ribosomal frameshifting (-1 PRF) is one viral translation strategy to express overlapping genes in positive-strand RNA viruses. Red clover necrotic mosaic virus (RCNMV) uses this strategy to express its replicase component protein p88. In this study, we used a cell-free translation system to map cis-acting RNA elements required for -1 PRF. Our results show that a small stem-loop structure adjacent to the cap-independent translation element in the 3' untranslated region (UTR) of RCNMV RNA1 is required for -1 PRF. Site-directed mutagenesis experiments suggested that this stem-loop regulates -1 PRF via base-pairing with complementary sequences in a bulged stem-loop adjacent to the shifty site. The existence of RNA elements responsible for -1 PRF and the cap-independent translation of replicase proteins in the 3' UTR of RNA1 might be important for switching translation to replication and for regulating the ratio of p88 to p27.
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http://dx.doi.org/10.1016/j.virol.2011.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111920PMC
August 2011

Arabidopsis ENHANCED DISEASE RESISTANCE 1 is required for pathogen-induced expression of plant defensins in nonhost resistance, and acts through interference of MYC2-mediated repressor function.

Plant J 2011 Sep 6;67(6):980-92. Epub 2011 Jul 6.

Graduate School of Agriculture, Kyoto University, Kyoto, Japan.

Arabidopsis thaliana exhibits durable resistance, called nonhost resistance, against non-adapted fungal pathogens that typically terminates pathogen entry. The PEN2-dependent indole glucosinolate metabolism pathway is involved in preventing the entry of a range of non-adapted fungi. Here, we report that ENHANCED DISEASE RESISTANCE 1 (EDR1) functions in pre-invasive nonhost resistance. Plants lacking EDR1 exhibit impaired entry resistance to the non-adapted hemibiotrophic Colletotrichum gloeosporioides, in contrast to the enhanced resistance of edr1 against biotrophic infection of a host-adapted powdery mildew fungus. Analysis of the edr1 pen2 double mutant indicates that EDR1 acts in a defense pathway independent from the PEN2 indole glucosinolate pathway. The edr1 mutant also exhibited enhanced susceptibility to host-adapted pathogens, including Colletotrichum higginsianum and necrotrophic Alternaria brassicicola. Comparative transcript profiling revealed that upon C. gloeosporioides inoculation, the expression of four plant defensin genes was severely impaired in edr1, indicating that EDR1 is required for the induced expression of these antifungal proteins. Inactivation of the MYC2-encoded transcription factor fully restored defensin expression in edr1, implying that EDR1 interferes with MYC2 function to abrogate repression of defensin expression. Furthermore, constitutive expression of plant defensin PDF1.2b largely rescued pre-invasive resistance responses in edr1 plants. These results indicate that EDR1 exerts a positive and critical role in resistance responses to hemibiotrophic/necrotrophic fungi, in part by inducing antifungal protein expression through derepression of MYC2 function.
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http://dx.doi.org/10.1111/j.1365-313X.2011.04651.xDOI Listing
September 2011

Identification of amino acids in auxiliary replicase protein p27 critical for its RNA-binding activity and the assembly of the replicase complex in Red clover necrotic mosaic virus.

Virology 2011 May 26;413(2):300-9. Epub 2011 Mar 26.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606–8502, Japan.

The specific recognition of genomic RNAs by viral replicase proteins is a key regulatory step during the early replication process in positive-strand RNA viruses. In this study, we characterized the RNA-binding activity of the auxiliary replicase protein p27 of Red clover necrotic mosaic virus (RCNMV), which has a bipartite genome consisting of RNA1 and RNA2. Aptamer pull-down assays identified the amino acid residues of p27 involved in its specific interaction with RNA2. The RNA-binding activity of p27 correlated with its activity in recruiting RNA2 to membranes. We also identified the amino acids required for the formation of the 480-kDa replicase complex, a key player of RCNMV RNA replication. These amino acids are not involved in the functions of p27 that bind viral RNA or replicase proteins, suggesting an additional role for p27 in the assembly of the replicase complex. Our results demonstrate that p27 has multiple functions in RCNMV replication.
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http://dx.doi.org/10.1016/j.virol.2011.02.017DOI Listing
May 2011

Viral cell-to-cell movement requires formation of cortical punctate structures containing Red clover necrotic mosaic virus movement protein.

Virology 2011 May 4;413(2):205-15. Epub 2011 Mar 4.

Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.

Movement protein (MP) of Red clover necrotic mosaic virus (RCNMV) forms punctate structures on the cortical endoplasmic reticulum (ER) of Nicotiana benthamiana cells, which are associated with viral RNA1 replication (Kaido et al., Virology 395, 232-242. 2009). We investigated the significance of ER-targeting by MP during virus movement from cell to cell, by analyzing the function of a series of MPs with varying length deletions at their C-terminus, either fused or not fused with green fluorescent protein (GFP). The C-terminal 70 amino acids were crucial to ER-localization of MP-GFP and cell-to-cell movement of the recombinant virus encoding it. However, C-terminal deletion did not affect MP functions, such as increasing the size exclusion limit of plasmodesmata, single-stranded RNA binding in vitro, and MP interacting in vivo. We discuss the possible role of this MP region in virus movement from cell to cell.
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http://dx.doi.org/10.1016/j.virol.2011.02.008DOI Listing
May 2011