Publishing type：Research paper (scientific journal)   Publisher：IOP Publishing  

Abstract

Plant cell walls prevent molecules with high molecular weights from reaching the cell membrane, challenging genome editing in plants. To overcome this challenge, the microplasma method, established as a gene and molecule transfection technology in animal cells, was investigated in tobacco plants. We found that plasma treatment of tobacco leaves and calluses introduced fluorescent molecules into epidermal and callus cells. Scanning electron microscopy revealed that plasma treatment decomposed the cuticula layer on the surface of tobacco leaves and that plasma treatment decomposed the extracellular matrix and caused cracks on the cell wall surface of tobacco callus. These results suggest that when external molecules are introduced into plant cells by plasma treatment, the external molecules’ transport pathway reaches the cell membrane by degradation of the cuticula layer and extracellular matrix. Additionally, the introduction of molecules by plasma treatment was inhibited by an endocytosis inhibitor, indicating that plasma stimulation induces endocytosis. In summary, plasma treatment decomposes the cuticula layer and cellular interstitium, allowing molecules to reach the cell membrane, after which they are introduced into the cell via endocytosis.

DOI： 10.35848/1347-4065/acd3f9

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Other Link： https://iopscience.iop.org/article/10.35848/1347-4065/acd3f9/pdf

Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

An increasing number of plant viruses and viroids have been reported from all over the world due largely to metavirogenomics approaches with technological innovation. Herein, the official changes of virus taxonomy, including the establishment of megataxonomy and amendments of the codes of virus classification and nomenclature, recently made by the International Committee on Taxonomy of Viruses were summarized. The continued efforts of the plant virology community of Japan to index all plant viruses and viroids occurring in Japan, which represent 407 viruses, including 303 virus species and 104 unclassified viruses, and 25 viroids, including 20 species and 5 unclassified viroids, as of October 2021, were also introduced. These viruses and viroids are collectively classified into 81 genera within 26 families of 3 kingdoms (Shotokuvirae, Orthornavirae, Pararnavirae) across 2 realms (Monodnaviria and Riboviria). This review also overviewed how Japan’s plant virus/viroid studies have contributed to advance virus/viroid taxonomy.

DOI： 10.1007/s10327-022-01051-y

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Other Link： https://link.springer.com/article/10.1007/s10327-022-01051-y/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)  

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs-knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.

DOI： 10.3390/ijms23042006

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The CRISPR/Cas9 system is now commonly employed for genome editing in various plants such as Arabidopsis, rice and tobacco. In general, in genome editing of the Arabidopsis genome, the SpCas9 and guide RNA genes are introduced into the genome by the floral dip method. Mutations induced in the target sequence by SpCas9 are confirmed after selecting transformants by screening the T1 seed population. The advantage of this method is that genome-edited plants can be isolated easily. However, mutation efficiency in Arabidopsis using SpCas9 is not as high as that achieved in rice and tobacco, which are subjected to a tissue culture step. In this study, we compared four promoters and found that the parsley UBIQITIN promoter is highly active in Arabidopsis meristem tissue. Furthermore, we examined whether a simple heat treatment could improve mutation efficiency in Arabidopsis. Just one heat treatment at 37°C for 24 h increased the mutation efficiency at all four target sites from 3 to 42%, 43 to 62%, 54 to 75% and 89 to 91%, without detectable off-target mutations. We recommend heat treatment of plate-grown plants at 37°C for 24 h as a simple method to increase the efficiency of CRISPR/Cas9-mediated mutagenesis in Arabidopsis.

DOI： 10.1093/pcp/pcab123

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh.

DOI： 10.3390/ijms22052696

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Language：English   Publishing type：Research paper (scientific journal)  

Loss-of-function mutation of the MILDEW RESISTANCE LOCUS O (Mlo) gene confers durable and broad-spectrum resistance to powdery mildew fungi in various plants, including barley. In combination with the intracellular nucleotide-binding domain and leucine-rich repeat receptor (NLR) genes, which confer the race-specific resistance, the mlo alleles have long been used in barley breeding as genetic resources that confer robust non-race-specific resistance. However, a Japanese Blumeria graminis f. sp. hordei isolate, RACE1, has been reported to have the potential to overcome partially the mlo-mediated penetration resistance, although this is yet uncertain because the putative effects of NLR genes in the tested accessions have not been ruled out. In this study, we examined the reproducibility of the earlier report and found that the infectious ability of RACE1, which partially overcomes the mlo-mediated resistance, is only exerted in the absence of NLR genes recognizing RACE1. Furthermore, using the transient-induced gene silencing technique, we demonstrated that RACE1 can partially overcome the resistance in the host cells with suppressed MLO expression but not in plants possessing the null mutant allele mlo-5.

DOI： 10.1371/journal.pone.0256574

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Language：English   Publisher：Japan International Research Center for Agricultural Sciences  

<p><i>Rice necrosis mosaic virus</i> (RNMV) is a fungus-transmitted bymovirus that leads to losses in rice yield. This research tested ten rice cultivars (cvs) with different levels of resistance to RNMV. The lowest levels of RNMV RNA were found in two high-resistance cvs; the highest levels were found in the two low-resistance cvs. The <i>RNA-dependent RNA polymerase 6 </i>gene in rice (<i>OsRDR6</i>) was found to be the most highly expressed in two high-resistance cvs and the least expressed in two low-resistance cvs although its basal level of constitutive expression was similar among cvs. Plant growth and yields were also tested. The extent of RNMV RNA accumulation affected plant height, panicle/tiller numbers, and seed weight. The RNMV induced <i>OsRDR6 </i>expression level in the rest of cvs was more or less inversely correlated to RNMV RNA accumulation. The observed results suggest a close relationship between RNMV resistance and RNMV induced <i>OsRDR6 </i>expression level in these cvs.</p>

DOI： 10.6090/jarq.55.127

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The authors wish to make the following corrections to this paper [1]: There was a misunderstanding in the annotation of a single gene. The gene, ICS1, has been mentioned several times in the main text and graph labels, but the gene should have been annotated to be SARD1, a regulator of ICS1. Although this mistake does not affect the conclusion of our paper, it is very misleading to any readers of the article. Therefore, the authors would like to publish this correction. The correction includes a change in the labels for the horizontal axis in Figure 1c and Supplementary Figure S1; several changes in the main text in the results, the discussion, and the materials and methods sections; and changes in Supplementary Table S2 and the entirety of Supplementary Tables S4, S5, S8, S9, and S10, in which relationships within the columns had been lost during sorting. The authors would like to apologize for any inconvenience caused to the readers by these changes.

DOI： 10.3390/ijms21228461

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In the present study, we have shown the transcriptional changes in a chlorosis model transgenic tobacco plant, i-amiCHLI, in which an artificial micro RNA is expressed in a chemically inducible manner to silence the expression of CHLI genes encoding a subunit of a chlorophyll biosynthetic enzyme. Comparison to the inducer-treated and untreated control non-transformants and untreated i-amiCHLI revealed that 3568 and 3582 genes were up- and down-regulated, respectively, in the inducer-treated i-amiCHLI plants. Gene Ontology enrichment analysis of these differentially expressed genes indicated the upregulation of the genes related to innate immune responses, and cell death pathways, and the downregulation of genes for photosynthesis, plastid organization, and primary and secondary metabolic pathways in the inducer-treated i-amiCHLI plants. The cell death in the chlorotic tissues with a preceding H2O2 production was observed in the inducer-treated i-amiCHLI plants, confirming the activation of the immune response. The involvement of activated innate immune response in the chlorosis development was supported by the comparative expression analysis between the two transgenic chlorosis model systems, i-amiCHLI and i-hpHSP90C, in which nuclear genes encoding different chloroplast proteins were similarly silenced.

DOI： 10.3390/ijms21197044

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using ‘Mannenboshi’, which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population.

DOI： 10.3390/plants9091153

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up-and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the H2 O2 production was confirmed by diaminobenzidine staining. The results collectively suggest that the reduced levels of HSP90C chaperone lead the plant to develop chlorosis primarily through the global downregulation of chloroplast-and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses, including cell death.

DOI： 10.3390/ijms21124202

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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. High humidity decreases the penetration rate of barley powdery mildew Blumeria graminis f. sp. hordei. However, the mechanism is not well understood. In this study, the morphological and cytochemical analyses revealed that substances containing proteins leaked from the tip of the appressorial germ tube of conidia without the formation of appressorium under a high humidity condition. In addition, exposure to high humidity prior to the formation of appressorium caused the aberrant formation of the appressorial germ tube without appressorium formation, resulting in failure to penetrate the host cell. These findings suggest that the formation and maturation of the appressorium requires a low humidity condition, and will be clues to improve the disease management by humidity control.

DOI： 10.3390/pathogens9010045

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© 2020 The American Phytopathological Society Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a major threat to economically important cucurbit crops worldwide. An attenuated strain (SH33b) derived from a severe strain (SH) of CGMMV caused a reduction in the viral RNA accumulation and the attenuation of symptoms, and it has been successfully used to protect muskmelon plants against severe strains in Japan. In this study, we compared GFP-induced silencing suppression by the 129K protein and the methyltransferase domain plus intervening region (MTIR) of the 129K protein between the SH and SH33b strains, respectively. As a result, silencing suppression activity (SSA) in the GFP-silenced plants was inhibited efficiently by the MTIR and 129K protein of SH strain, and it coincided with drastically reduced accumulation of GFP-specific small interfering RNAs (siRNAs) but not by that of SH33b strain. Furthermore, analyses of siRNA binding capability (SBC) by the MTIR of 129K protein and 129K protein using electrophoretic mobility shift assay revealed that SBC was found with the MTIR and 129K protein of SH but not with that of SH33b, suggesting that a single amino acid mutation (E to G) in the MTIR is responsible for impaired SSA and SBC of SH33b. These data suggest that a single amino acid substitution in the intervening region of 129K protein of CGMMV resulted in attenuated symptoms by affecting RNA silencing suppression.

DOI： 10.1094/PHYTO-12-18-0478-FI

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© 2019 The Mycological Society of Japan We aimed to develop an artificial membrane system to observe the infection process of the obligate biotrophic powdery mildew fungi without the use of living plant cells. The conidia of Blumeria graminis and Erysiphe pisi conidia were inoculated on a formvar membrane laid on an artificial medium. Germinated conidia frequently formed appressoria and then penetrated the membrane to form haustorium-like structures in the artificial medium. Secondary hyphae elongation was also observed after the formation of haustorium-like structures. These results suggested that the formvar membrane laid on artificial medium induced the formation of haustorium-like structures that have roles in the formation of secondary hyphae.

DOI： 10.1016/j.myc.2019.06.006

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© 2018 Elsevier Ltd Plant virus diseases are significant constraints in agricultural production in Bangladesh. The hot and humid environmental conditions are highly favourable for the perpetuation of the viruses as well as vectors round the year. Although, the virus diseases are recorded in many crops, vegetables and pulses are most seriously affected. Several viruses belonging to the genera Begomovirus, Cucumovirus, Potyvirus and Tospovirus have been recorded during the last decade. Whitefly and thrips-transmitted viruses have emerged as major constraints in the horticultural crops. Management of viral diseases largely depends on the control of insect vectors by widespread application of insecticides. The epidemics of leaf curl, yellow vein, yellow mosaic and bud necrosis diseases were witnessed in the recent past. However, the knowledge of identity and diversity of viruses occurring in Bangladesh are largely lacking. This review provides the first comprehensive account of viral disease problems in the cultivation of several important crops and their management in Bangladesh.

DOI： 10.1016/j.cropro.2018.11.023

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© Springer Science+Business Media, LLC, part of Springer Nature 2019. Coexpression of a plant NB-LRR-type resistance (R) gene and corresponding viral avirulent (Avr) gene introduced in Nicotiana benthamiana using Agrobacterium tumefaciens confers hypersensitive response (HR). Such Agrobacterium-mediated transient gene expression methods have contributed to the identification of new plant R genes and facilitated the analysis of their functions. Here we describe a model method, by which several tobamovirus R genes from Solanaceous plants have been successfully identified and characterized molecularly.

DOI： 10.1007/978-1-4939-9635-3_1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer-Verlag Wien  

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.

DOI： 10.1007/s00705-018-3747-4

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© 2018, Indian Virological Society. Tobacco streak virus (TSV, genus Ilarvirus family Bromoviridae) is known to cause stem necrosis disease (SND) in groundnut (Arachis hypogaea) since 2000 in Southern India. The TSV isolate infecting groundnut so far has not been characterized based on the complete genome sequence. In this study, TSV was isolated from a naturally infecting groundnut plant in Kadiri, the hot-spot of the SND in southern India. During the Kharif season of 2014, groundnut plants in an experimental field were affected with chlorosis and necrosis in leaf, stem and buds. The cent percent of the 48 samples with these symptoms collected from the field tested positive for TSV in ELISA samples in this context. One isolate, GN-Kad was established from a single lesion on cowpea cv. C-152 through successive sap inoculation. Cloning and sequencing of coat protein gene (717 nucleotides) of the isolate showed high sequence identity (98–99%) with the TSV isolates reported from different crops in India. The isolate produced local necrotic rings or veinal necrosis following sap inoculation to cowpea (cultivars C-152, Pusa Komal, Pusa Sukomal and Krishi Kanchan), French bean and sunflower; whereas, it produced systemic chlorotic mottling symptoms in Nicotiana benthamiana. The three segments of the virus genome (RNA 1, RNA 2 and RNA 3) contained 3523, 2903 and 2232 nucleotides, respectively. The overall genome sequence (8639 nt) of the present isolate shared 77–99% of nucleotide sequence identity with that of the other seven isolates reported from Australia, India and USA. The GN-Kad shared very close phylogenetic relationship with the okra and pumpkin isolates reported from India. The present report is the first comprehensive study of the molecular characterization of TSV associated with the stem necrosis disease of groundnut.

DOI： 10.1007/s13337-018-0500-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant-pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant-pathogen interactions.

DOI： 10.1007/s10327-017-0712-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer India  

Recent studies with Y satellite RNA (Y-sat) of cucumber mosaic virus have demonstrated that Y-sat modifies the disease symptoms in specific host plants through the silencing of the magnesium protoporphyrin chelatase I subunit (CHLI), which is directed by the Y-sat derived siRNA. Along with the development of peculiar yellow phenotypes, a drastic decrease in CHLI-transcripts and a higher accumulation of Y-sat derived siRNA were observed. To investigate the molecular mechanisms underlying the Y-sat—induced chlorosis, especially whether or not the reduced expression of CHLI causes the chlorosis simply through the reduced production of chlorophyll or it triggers some other mechanisms leading to the chlorosis, we have established a new experimental system with an inducible silencing mechanism. This system involves the expression of artificial microRNAs targeting of Nicotiana tabacum CHLI gene under the control of chemically inducible promoter. The CHLI mRNA levels and total chlorophyll content decreased significantly in 2 days, enabling us to analyze early events in induced chlorosis and temporary changes therein. This study revealed that the silencing of CHLI did not only result in the decreased chlorophyll content but also lead to the downregulation of chloroplast and photosynthesis-related genes expression and the upregulation of defense-related genes. Based on these results, we propose that the reduced expression of CHLI could activate unidentified signaling pathways that lead plants to chlorosis.

DOI： 10.1007/s13337-017-0360-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer India  

Chlorosis is one of the most common symptoms of plant diseases, including those caused by viruses and viroids. Recently, a study has shown that Peach latent mosaic viroid (PLMVd) exploits host RNA silencing machinery to modulate the virus disease symptoms through the silencing of chloroplast-targeted heat shock protein 90 (Hsp90C). To understand the molecular mechanisms of chlorosis in this viroid disease, we established an experimental system suitable for studying the mechanism underlying the chlorosis induced by the RNA silencing of Hsp90C in transgenic tobacco. Hairpin RNA of the Hsp90C-specific region was expressed under the control of a dexamethasone-inducible promoter, resulted in the silencing of Hsp90C gene in 2 days and the chlorosis along with growth suppression phenotypes. Time course study suggests that a sign of chlorosis can be monitored as early as 2 days, suggesting that this experimental model is suitable for studying the molecular events taken place before and after the onset of chlorosis. During the early phase of chlorosis development, the chloroplast- and photosynthesis-related genes were downregulated. It should be noted that some pathogenesis related genes were upregulated during the early phase of chlorosis in spite of the absence of any pathogen-derived molecules in this system.

DOI： 10.1007/s13337-017-0361-0

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Language：English   Publisher：SPRINGER  

The complete nucleotide sequences of Beet pseudoyellows virus (BPYV)-MI (cucumber isolate; Matsuyama, Idai) genomic RNAs 1 and 2 were determined and compared with the previously sequenced Japanese cucumber strain (BPYV-JC) and a strawberry strain (BPYV-S). The RNA 2 of BPYV-MI showed 99 % nucleotide sequence identity with both BPYV-JC and -S having highly conserved eight ORFs. In contrast, the RNA1 of BPYV-MI showed sequence identities of 98 and 86 % with BPYV-JC and -S, respectively. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) coding sequences from three fully sequenced BPYV strains and five partially sequenced cucurbit-infecting BPYV strains from Japan and South Africa has shown that cucurbit-infecting strains are closer to each other than to BPYV-S. In addition, the strawberry strain BPYV-S has an ORF2 in the downstream of RdRp gene in RNA1, but all the cucumber strains, BPYV-JC, -MI, and those from South Africa, lacked the ORF2 of RNA1, highlighting the difference between common BPYV cucumber strains and a unique strawberry strain.

DOI： 10.1007/s11262-016-1376-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

Here we report a novel method to extract nucleic acids for virus detection. Plant tissue in a filter paper sandwich was hit with a hammer, and the crude sap was adsorbed by the filter paper, as in hammer blotting for tissue-print immunodetection of plant viruses. Nucleic acids were extracted from the paper with a guanidine-containing buffer and purified through standard extraction and precipitation. Both viral and cellular RNAs in the extracted RNA were detectable by RT-PCR. The blots can be stored at 4 A degrees C for more than a few days to detect viral RNA, but not cellular RNA.

DOI： 10.1007/s10327-016-0673-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER INDIA  

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.

DOI： 10.1007/s13562-015-0334-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins.

DOI： 10.1104/pp.16.00234

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DOI： 10.1111/pbi.12546

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The presence of high molecular weight double-stranded RNA (dsRNA) within plant cells is an indicator of infection with RNA viruses as these possess genomic or replicative dsRNA. DECS (dsRNA isolation, exhaustive amplification, cloning, and sequencing) analysis has been shown to be capable of detecting unknown viruses. We postulated that a combination of DECS analysis and next-generation sequencing (NGS) would improve detection efficiency and usability of the technique. Here, we describe a model case in which we efficiently detected the presumed genome sequence of Blueberry shoestring virus (BSSV), a member of the genus Sobemovirus, which has not so far been reported. dsRNAs were isolated from BSSV-infected blueberry plants using the dsRNA-binding protein, reverse-transcribed, amplified, and sequenced using NGS. A contig of 4,020 nucleotides (nt) that shared similarities with sequences from other Sobemovirus species was obtained as a candidate of the BSSV genomic sequence. Reverse transcription (RT)-PCR primer sets based on sequences from this contig enabled the detection of BSSV in all BSSV-infected plants tested but not in healthy controls. A recombinant protein encoded by the putative coat protein gene was bound by the BSSV-antibody, indicating that the candidate sequence was that of BSSV itself. Our results suggest that a combination of DECS analysis and NGS, designated here as "DECS-C," is a powerful method for detecting novel plant viruses.

DOI： 10.3390/v8030070

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

We investigated graft transmission of high-temperature tolerance in tomato scions to nontransgenic scions from transgenic rootstocks, where the fatty acid desaturase gene (LeFAD7) was RNA-silenced. Tomato was transformed with a plasmid carrying an inverted repeat of LeFAD7 by Agrobacterium. Several transgenic lines showed the lower amounts of LeFAD7 RNA and unsaturated fatty acids, while nontransgenic control did not, and siRNA was detected in the transgenic lines, but not in control. These lines grew under conditions of high temperature, while nontransgenic control did not. Further, the nontransgenic plants were grafted onto the silenced transgenic plants. The scions showed less of the target gene RNA, and siRNA was detected. Under high-temperature conditions, these grafted plants grew, while control grafted plants did not. Thus, it was shown that high-temperature tolerance was conferred in the nontransgenic scions after grafting onto the silenced rootstocks.

DOI： 10.1111/pbi.12429

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

RNA-dependent RNA polymerases (RDRs) play key roles in gene silencing. The rice RDR6 gene was analyzed in response to viral, bacterial and fungal pathogens, after inoculation of a rice mutant line of OsRDR6, shl2-rol, with Cucumber mosaic virus, Rice necrosis mosaic virus, Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae. Compared with the wild type, the mutant line accumulated more viral RNA after inoculation with the viruses and developed more severe symptoms after inoculation with the bacterium or fungus. Thus, the OsRDR6-mediated RNA silencing pathway seems to participate in defense against not only viruses, but also bacterial and fungal pathogens.

DOI： 10.1007/s10327-015-0630-y

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The complete nucleotide sequence of Rice necrosis mosaic virus (RNMV) RNA1 was determined to be 7178 nt long with one large open reading frame, potentially encoding a polyprotein of 258 kDa with the features of a typical bymovirus. The nucleotide sequence showed 56 % identity with that of Barley mild mosaic virus (BaMMV) and 46 % with those of Oat mosaic virus, Barley yellow mosaic virus and Wheat yellow mosaic virus. RNA2 was 3579 nt long, encoding a 110-kDa polyprotein, and showed 42 and 35/26 % identity with BaMMV and others, respectively. Phylogenetic analysis showed that bymoviruses were classified into two subgroups.

DOI： 10.1007/s10327-015-0618-7

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Impatiens necrotic spot virus (INSV), belonging to the genus Tospovirus, causes severe damage to greenhouse ornamental plants. We conducted population genetic analyses of INSV isolated from various greenhouse flowers in Iwate Prefecture as a model to elucidate how the virus invaded and spread within a local area of Japan. Forty-two and 30 sequences of the nucleocapsid protein gene (NG) and intergenic regions (IGR), respectively, of the small RNA of the virus were generated from 42 isolates collected from seven greenhouses in three districts, and then they were divided into 10 NG haplotypes and 13 IGR haplotypes. A combined haplotype analysis for the two loci revealed the presence of 14 NG/IGR haplotypes in the region. Genetic structure of the INSV population based on the NG sequences was differentiated according to districts and greenhouses, and 3 major NG haplotypes were predominant in each district. Genetic analysis based on IGR sequences showed that the population structure in some greenhouses consisted of 1 major IGR haplotype and some minor haplotypes with the same NG haplotype. Phylogenetic analysis based on NG and IGR sequences illustrated that INSV haplotypes were clustered according to geographic origin, including the isolates previously reported in Japan and other countries. Host species did not seem to influence the genetic structure of the INSV population. These results suggest that some founder isolates were introduced individually from other epidemic regions to each district in the region through different routes and then spread within the local areas and greenhouses.

DOI： 10.1007/s10327-015-0615-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

To study the precise mechanisms underlying the chlorosis caused by plant viruses, we previously established a synchronous experimental system using transgenic plants expressing Cauliflower mosaic virus multifunctional protein, Tav (transactivator/viroplasmin), under the control of an artificially inducible promoter. Shortly after the induction of Tav expression, pathogenesis-related protein (PR) 1a gene expression is upregulated in the transgenic tobacco lines, which show visible chlorosis within a week. The present study showed that the expression of Tav also induces some salicylic acid (SA)- and ethylene-responsive PR genes. In contrast to transiently expressed Tav, which suppressed Agrobacterium-induced and SA-induced PR1a expression, the artificial induction of Tav from the transgene did not affect SA-induced PR1a expression, rather it alone induced PR1a expression. In a deletion analysis, chlorosis and PR1a induction function in transgenic tobacco were mapped to a region in Tav that had been shown to have a role in pathogenesis in a susceptible host, elicitation of the hypersensitive response in a resistant host, suppression of RNA silencing, and the suppression of Tomato bushy stunt virus P19-mediated cell death in tobacco. The results suggest that Tav-induced chlorosis results from a host response, which accompanies PR1a induction, to pathogenic function of Tav.

DOI： 10.1007/s10327-015-0600-4

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HAP (CBF/NF-Y) transcription factors have important functions in regulating plant growth, development, and stress responses. In this study, we examined whether the endogenous gene OsHAP2E and the GUS transgene driven by the promoter of OsHAP2E respond to virus infection. RT-PCR analyses showed OsHAP2E expression was induced after inoculation with Cucumber mosaic virus (CMV) or Rice necrosis mosaic virus (RNMV). After inoculating OsHAP2E::GUS-transgenic plants with either virus, the levels of GUS expression increased significantly. The expression levels of GUS or OsHAP2E reached a plateau 5 days after inoculation of rice with CMV, which paralleled the accumulation of CMV RNA level. Furthermore, transgenically over-expressed lines of OsHAP2E (OsHAP2E-OX) had lower levels of CMV and RNMV RNAs than in in nontransgenic control plants. The OsHAP2E-OX lines developed no significant symptoms from RNMV while control plants had yellowing and stunting. These results suggested that OsHAP2E is induced by virus infection and contributes to resistance against viral pathogens.

DOI： 10.1007/s10327-014-0564-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Heme activator protein (HAP), also known as nuclear factor Y or CCAAT binding factor (HAP/NF-Y/CBF), has important functions in regulating plant growth, development and stress responses. The expression of rice HAP gene (OsHAP2E) was induced by probenazole (PBZ), a chemical inducer of disease resistance. To characterize the gene, the chimeric gene (OsHAP2E::GUS) engineered to carry the structural gene encoding -glucuronidase (GUS) driven by the promoter from OsHAP2E was introduced into rice. The transgenic lines of OsHAP2Ein::GUS with the intron showed high GUS activity in the wounds and surrounding tissues. When treated by salicylic acid (SA), isonicotinic acid (INA), abscisic acid (ABA) and hydrogen peroxide (H2O2), the lines showed GUS activity exclusively in vascular tissues and mesophyll cells. This activity was enhanced after inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae. The OsHAP2E expression level was also induced after inoculation of rice with M. oryzae and X. oryzae pv. oryzae and after treatment with SA, INA, ABA and H2O2, respectively. We further produced transgenic rice overexpressing OsHAP2E. These lines conferred resistance to M. oryzae or X. oryzae pv. oryzae and to salinity and drought. Furthermore, they showed a higher photosynthetic rate and an increased number of tillers. Microarray analysis showed up-regulation of defence-related genes. These results suggest that this gene could contribute to conferring biotic and abiotic resistances and increasing photosynthesis and tiller numbers.

DOI： 10.1111/pbi.12239

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Language：English   Publisher：AMER PHYTOPATHOLOGICAL SOC  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

The underlying molecular mechanism of chlorosis, a typical symptom of plant viral diseases, remains poorly understood. To establish an experimental system to determine the molecular changes during chlorosis, especially in the early phase, we generated transgenic tobacco plants expressing Cauliflower mosaic virus Transactivator/viroplasmin (Tav) under the control of a chemically inducible promoter. Induction of Tav resulted in visible chlorosis in ten days, a statistically significant decrease in chlorophyll content in two days, decreased expression of chloroplast protein genes, and abnormal thylakoid stacks, indicating that this system reproduces the common features of chlorosis in virus-infected plants. (C) 2014 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.pmpp.2014.08.005

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Background: Aspartic protease (APs) plays important roles in plant growth, development and biotic and abiotic stresses. We previously reported that the expression of a rice AP gene (OsAP77, Os10g0537800) was induced by probenazole (PBZ), a chemical inducer of disease resistance. In this study we examined some characteristics of this gene in response to fungal, bacterial and viral pathogens.
Results: To elucidate the spatial and temporal expression of OsAP77, the chimeric gene was constructed carrying the structural gene encoding beta-glucuronidase (GUS) driven by the OsAP77 promoter. This construct was introduced into rice and the transgenic lines were tested to analyze gene expression by fungal, bacterial and viral infections. Inoculation with Magnaporthe oryzae or Xanthomonas oryzae pv. oryzae revealed the enhanced GUS activities in vascular tissues surrounding the symptom sites by each pathogen. Moreover, GUS activity also increased after inoculation with Cucumber mosaic virus (CMV). Transgenic plants immersed in a solution containing salicylic acid (SA), isonicotinic acid (INA), hydrogen peroxide (H2O2) or abscisic acid (ABA) showed an increased level of GUS activity exclusively in vascular tissues. RT-PCR analysis showed that OsAP77 was induced not only by infection with these pathogens, but also after treatment with SA, INA, H2O2 or ABA. A knockout mutant line of OsAP77 by the insertion of Tos17 after inoculation with M. oryzae, X. oryzae pv. oryzae or CMV showed an enhanced susceptibility compared to wild type.
Conclusion: These results suggest that the expression of OsAP77 is induced by pathogen infection and defense related signaling molecules in a vascular tissue specific manner and that this gene has a positive role of defense response against fungal, bacterial and viral infections.

DOI： 10.1186/s12284-014-0009-2

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Cultivars with introgressed natural resistance genes have been widely used for plant disease control, especially in the control of virus diseases, for which no effective chemical control agent is available. However, we often encounter virus mutants that break down or overcome the resistance. In this review, recent studies will be discussed with respect to breakdown of plant virus resistance.

DOI： 10.1007/s10327-014-0527-1

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Microorganisms play an enormously important role in plant disease control. Research on biological control of plant pathogens has received major impetus and attracted many researchers during the past few decades due to the increased public concern on hazards associated with the use of synthetic pesticides. From research on utilizing specific antagonistic microorganisms, many effective biological control agents (BCAs) have been found and are increasingly implemented in integrated pest management strategies to control plant diseases. Here current research results on biological control against plant diseases carried out in Japan are reviewed by focusing on major categories of BCAs: fungi, bacteria and actinomycetes and attenuated viruses.

DOI： 10.1007/s10327-014-0524-4

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In 2007, lisianthus (Eustoma grandiflorum) plants with necrotic ringspots on the leaves were found in Kochi Prefecture, Japan. Tospovirus-like spherical enveloped particles with ca. 160 nm in diameter were observed with electron microscopy. The complete nucleotide sequence of the S RNA segment of the virus was determined, and phylogenetic analysis using deduced amino acid sequences of the nucleocapsid protein and the nonstructural S protein indicated that the virus is phylogenetically distinct from any known tospovirus species. The results suggest that the virus is a new member of the genus Tospovirus, in the family Bunyaviridae. The virus is the fifth distinct tospovirus occurring naturally in lisianthus in Japan. The necrotic symptoms were reproduced on lisianthus seedlings after mechanical inoculation. The host range of the virus isolate on several test plants was also examined.

DOI： 10.1007/s10327-014-0503-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short inframe deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.

DOI： 10.1099/vir.0.057729-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Gentian Kobu-sho-associated virus (GKaV) is a recently discovered novel virus from Kobu-sho (a hyperplastic or tumorous disorder)-affected Japanese gentians. To obtain insight into GKaV transmission and pathogenesis, the genetic diversity of the virus in the putative helicase and RNA-dependent RNA polymerase coding regions was studied. The extent of GKaV sequence diversity within single host plants differed within samples and between viral genomic regions. Phylogenetic analysis of 30 Kobu-sho-affected samples from different production areas and host cultivars revealed that GKaV populations have diverged as they became prevalent in different geographical regions. The diversification of GKaV was shown to be driven by geographical isolation rather than host adaptation; however, no geographical patterns were found. Therefore, it was not feasible to trace the pathway of GKaV spread.

DOI： 10.1099/vir.0.053637-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

We determined the complete nucleotide sequence of a broad bean wilt virus 2 (BBWV-2) isolate from gentian in Japan. The full-length RNA1 and RNA2 sequences, excluding poly(A) tails, were 5955 and 3600 nucleotides long, respectively. Analysis indicated that, in contrast to other BBWV-2 isolates, the 5' end of both RNA1 and RNA2 starts with a GUU sequence. We successfully inoculated Nicotiana benthamiana with BBWV-2 by infiltrating a mixed suspension of two Agrobacterium tumefaciens clones carrying binary vectors with the full-length RNA1 and RNA2 sequences. This is the first report on the efficient, easy and high-throughput use of agroinoculation for generating BBWV-2 infections.

DOI： 10.1007/s00705-013-1625-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

RNA silencing is a mechanism of gene regulation by sequence specific RNA degradation and is involved in controlling endogenous gene expression and defense against invasive nucleic acids such as viruses. RNA silencing has been proven to be transmitted between scions and rootstocks through grafting, mostly using transgenic plants. It has been reported that RNA silencing of tobacco endogenous genes, NtTOM1 and NtTOM3, that are required for tobamovirus multiplication, resulted in high resistance against several tobamoviruses. In the present study, we examined the graft transmission of RNA silencing for conferring virus resistance to non-transgenic scions of the same and different Nicotiana species grafted onto rootstocks in which both NtTOM1 and NtTOM3 were silenced. Non-transgenic Nicotiana tabacum (cvs. Samsun and Xanthi nc) and N. benthamiana were used as scions for grafting onto the rootstocks silenced with both genes. Short interfering RNA (siRNA) of NtTOM1 and NtTOM3 was detected in both the scions and the rootstocks eight weeks after grafting. The leaves were detached from the scions and inoculated with several tobamoviruses. The virus accumulation was tested by ELISA and northern blot analysis. The viruses were detected in grafted scions at extremely low levels, showing that virus resistance was conferred. These results suggest that RNA silencing was induced in and virus resistance was conferred to the non-transgenic scions by grafting onto silenced rootstocks. The effect of low temperature on siRNA accumulation and virus resistance was not significantly observed in the scions.

DOI： 10.1371/journal.pone.0063257

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

A virus-like dsRNA of about 23 kbp was detected in gentian plants showing Kobu-sho syndrome including stunting, shortened internodes, and tumors on stems, nodes and roots. Nucleotide sequence analysis has suggested that this dsRNA is related to Pestivirus species but not to any other plant viruses. It was protected from externally added RNase, suggesting that the dsRNA is encapsidated. The dsRNA was co-extracted in a crude homogenate of glutaraldehyde-fixed tissue with the virus-like particles that have been associated previously with Kobu-sho syndrome in gentian (Usugi et al. Jpn J Phytopathol 76:21-24, 2010). The RNA sequence was detected in more than 99 % of Kobu-sho gentian individuals but in less than 20 % of apparently healthy gentian individuals from fields affected with Kobu-sho. Thus, we propose naming the virus Gentian Kobu-sho-associated virus.

DOI： 10.1007/s10327-012-0423-5

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Small RNA-mediated gene silencing pathways play important roles in the regulation of development, genome stability and various stress responses in many eukaryotes. Recently, a new type of small interfering RNAs (qiRNAs) approximately 20-21 nucleotides long in Neurospora crassa have been shown to mediate gene silencing in the DNA damage response (DDR) pathway. However, the mechanism for RNA silencing in the DDR pathway is largely unknown in plants. Here, we report that a class of small RNAs (qiRNAs) derived from rDNA was markedly induced after treatment by DNA-damaging agents [ ethyl methanesulphonate (EMS and UV-C)], and that aberrant RNAs (aRNAs) as precursors were also highly induced after the DNA damage treatment in rice. However, these RNAs were completely abolished in OsRecQ1 (RecQ DNA helicase homologue) and OsRDR1 (RNA-dependent RNA polymerase homologue) mutant lines where either gene was disrupted by the insertion of rice retrotransposon Tos17 after the same treatment. DNA damage resulted in a more significant increase in cell death and a more severe inhibition of root growth in both mutant lines than in the WT. Together, these results strongly suggest that both OsRecQ1 and OsRDR1 play a pivotal role in the aRNA and qiRNA biogenesis required for the DDR and repair pathway in rice, and it may be a novel mechanism of regulation to the DDR through the production of qiRNA in plants.

DOI： 10.1371/journal.pone.0055252

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In pepper plants (genus Capsicum), the resistance against Tobamovirus spp. is conferred by L gene alleles. The recently identified L variant L1a can recognize coat proteins (CPs) of Tobacco mild green mosaic virus Japanese strain (TMGMV-J) and Paprika mild mottle virus Japanese strain (PaMMV-J), but not of Pepper mild mottle virus (PMMoV), as the elicitor to induce resistance at 24 degrees C. Interestingly, L1a gene-mediated resistance against TMGMV-J, but not PaMMV-J, is retained at 30 degrees C. This observation led us to speculate that L1a can discriminate between CPs of TMGMV-J and PaMMV-J. In this study, we aimed to determine the region(s) in CP by which L1a distinguishes TMGMV-J from PaMMV-J. By using chimeric CPs consisting of TMGMV-J and PaMMV-J, we found that the chimeric TMGMV-J CP, whose residues in the beta-sheet domain were replaced by those of PaMMV-J, lost its ability to induce L1a gene-mediated resistance at 30 degrees C. In contrast, the chimeric PaMMV-J CP with the beta-sheet domain replaced by TMGMV-J CP was able to induce L1a gene-mediated resistance at 30 degrees C. Furthermore, viral particles were not detected in the leaves inoculated with either chimeric virus. These observations indicated that the amino acids within the beta-sheet domain were involved in both the induction of L1a gene-mediated resistance and virion formation. Further analyses using chimeric CPs of TMGMV-J and PMMoV indicated that amino acids within the beta-sheet domain alone were not sufficient for the induction of L1a gene-mediated resistance by TMGMV-J CP. These results suggest that multiple regions in Tobamovirus CP are implicated in the induction of L1a gene-mediated resistance.

DOI： 10.1111/j.1364-3703.2012.00801.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYTOPATHOLOGICAL SOC  

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of A N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L-3 revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L-3 revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.

DOI： 10.1094/MPMI-11-11-0289

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Cucumber green mottle mosaic virus (CGMMV) is a major limiting factor in the production of melon plants worldwide. For effective control of this virus using the transgenic approach, the direct repeat of the movement protein gene of CGMMV was used for transforming melon plants by Agrobacterium tumefaciens. PCR and Southern blot analyses of T-3 confirmed that they carried the transgene. Northern blot analysis with total RNA showed that transgene transcript RNA as well as siRNA was observed in all plants tested. Separate leaves or individual plants were inoculated with CGMMV and subjected to ELISA and RNA blot analysis using the coat protein gene probe of the virus. Compared to nontransgenic control, these plants were shown to have high virus resistance. Furthermore, cytosine of the transgene DNA in the plants was methylated. Thus, these results reveal that the transgenic lines were highly resistant to the virus through RNA silencing.
Key message High virus resistance was obtained in transgenic melon plants with direct repeat of movement protein gene of Cucumber green mottle mosaic tobamovirus through RNA silencing.

DOI： 10.1007/s00299-012-1237-9

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Language：English   Publisher：JAPAN INT RESEARCH CENTER AGRICULTURAL SCIENCES  

Next-generation sequencers have accelerated the advancement of virus hunting or virus detection by metagenomic analysis. Conversely, earlier works enabled virus detection with classic Sanger sequencing by elaborating sample preparation/processing techniques. In this review, we introduce virus hunting technologies both with and without cutting-edge sequencing technology and subsequently discuss the possibility that combining these technologies may extend the application of sequencing-based virus detection from scientific research to everyday diagnostics. Finally, we offer an outlook for another type of technology that leads to pathogen identification using the sequence data obtained in sequencing-based virus hunting.

DOI： 10.6090/jarq.46.123

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Language：English   Publisher：SPRINGER TOKYO  

Attenuated viruses have been isolated and studied not only as a practical means of controlling virus diseases but also to gain a molecular understanding of viral virulence and cross protection. They have been isolated from crop fields and generated through high/low temperature treatment or by mutagens such as nitrous acid and ultraviolet irradiation. Some viruses have been beneficially used in fields and evaluated for one or more decades. Molecular genetic studies on attenuated viruses have revealed that amino acid substitutions are located in replicase and the movement protein in tobamovirus, protein 2b for cucumovirus, and P1 and HC-Pro for potyvirus. In most cases, with a few exceptions, symptom attenuation is positively correlated with a reduced level of RNA silencing suppression. Molecular mechanisms underlying virus attenuation and cross protection and the rationale for practical use of attenuated viruses for effective virus disease control are discussed.

DOI： 10.1007/s10327-011-0318-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator-viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV-TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP-a novel and specific target of TOR/S6K1-in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes. The EMBO Journal (2011) 30, 1343-1356. doi: 10.1038/emboj.2011.39; Published online 22 February 2011

DOI： 10.1038/emboj.2011.39

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYTOPATHOLOGICAL SOC  

The pepper L gene conditions the plant&apos;s resistance to Tobamovirus spp. Alleles L(1), L(2), L(3), and L(4) confer a broadening spectra of resistance to different virus pathotypes. In this study, we report the genetic basis for the hierarchical interaction between L genes and Tobamovirus pathotypes. We cloned L(3) using map-based methods, and L(1), L(1a), L(1c), L(2), L(2b), and L(4) using a homology-based method. L gene alleles encode coiled-coil, nucleotide-binding, leucine-rich repeat (LRR)-type resistance proteins with the ability to induce resistance response to the viral coat protein (CP) avirulence effectors by themselves. Their different recognition spectra in original pepper species were reproduced in an Agrobacterium tuntefaciens-mediated transient expression system in Nicotiana benthamiana. Chimera analysis with L(1), which showed the narrowest recognition spectrum, indicates that the broader recognition spectra conferred by L(2), Lab, L(3), and L(4) require different subregions of the LRR domain. We identified a critical amino acid residue for the determination of recognition spectra but other regions also influenced the L genes&apos; resistance spectra. The results suggest that the hierarchical interactions between L genes and Tobamovirus spp. are determined by the interaction of multiple subregions of the LRR domain of L proteins with different viral CP themselves or some protein complexes including them.

DOI： 10.1094/MPMI-06-10-0127

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

The plant viral re-initiation factor transactivator viroplasmin (TAV) activates translation of polycistronic mRNA by a re-initiation mechanism involving translation initiation factor 3 (eIF3) and the 60S ribosomal subunit (60S). QJ; Here, we report a new plant factor-re-initiation supporting protein (RISP)-that enhances TAV function in re-initiation. RISP interacts physically with TAV in vitro and in vivo. Mutants defective in interaction are less active, or inactive, in transactivation and viral amplification. RISP alone can serve as a scaffold protein, which is able to interact with eIF3 subunits a/c and 60S, apparently through the C-terminus of ribosomal protein L24. RISP pre-bound to eIF3 binds 40S, suggesting that RISP enters the translational machinery at the 43S formation step. RISP, TAV and 60S co-localize in epidermal cells of infected plants, and eIF3-TAV-RISP-L24 complex formation can be shown in vitro. These results suggest that RISP and TAV bridge interactions between eIF3-bound 40S and L24 of 60S after translation termination to ensure 60S recruitment during repetitive initiation events on polycistronic mRNA; RISP can thus be considered as a new component of the cell translation machinery. The EMBO Journal (2009) 28, 3171-3184. doi:10.1038/emboj.2009.256; Published online 10 September 2009

DOI： 10.1038/emboj.2009.256

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In this study, we characterized a Capsicum hypersensitive response (HR)-associated gene, SS52, which encodes a protein that contains an N-terminal C2 domain and a C-terminal XYPPX repeat. Expression analyses revealed that SS52 and its homologue in Arabidopsis were induced by infection with incompatible viruses, indicating the conserved function of this gene. SS52 was not induced by treatment with defense-related hormones, but was induced by abiotic stresses, including wounding. Overexpression of SS52 in tobacco plants suppressed the spread of HR cell death and restricted the spread of an incompatible virus from local lesions. Collectively, the results suggest that SS52 negatively regulates plant HR cell death. (c) 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2009.07.020

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER JAPAN KK  

The isolation of viral replicative form (RF) double-stranded RNA (dsRNA) is a classic technique for plant virus detection when the virus species cannot be predicted from disease symptoms. However, the method has not been very widely used, most likely because dsRNA isolation using CF-11 cellulose is laborious and time-consuming. Here we report an alternative tool, a recombinant plant dsRNA-binding protein, to isolate dsRNA. This tool enables us to isolate viral RF dsRNA in an hour from either extracted nucleic acids or crude detergent extracts. Combining this technique with sequence-non-specific reverse transcription, PCR amplification, cloning, and sequencing, a variety of viruses were efficiently detected using a single set of reagents and procedures.

DOI： 10.1007/s10327-009-0155-3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Capsicum plants harboring the Hk gene (Hk) show resistance to Paprika mild mottle virus (PaMMV) at 32 degrees C but not 24 degrees C. To identify the viral elicitor that activates the Hk-mediated resistance, several chimeric viral genomes were constructed between PaMMV and Tobacco mosaic virus-L Infection patterns of these chimeric viruses in Hk-harboring plants revealed responsibility of PaMMV replicase genes for activation of the Hk-mediated resistance. The comparison of nucleotide sequence of replicase genes between PaMMV and PaHk1, an Hk-resistance-breaking strain of PaMMV, revealed that the adenine-to-uracil substitution at the nucleotide position 721 causes an amino acid change from threonine to serine at the 241 st residue in the methyltransferase domain. Introduction of the A721U mutation into the replicase genes of parental PaMMV overcame the Hk resistance at 32 degrees C. The results indicate that Hk-mediated resistance is induced by PaMMV replicase proteins and that methyltransferase domain has a role in this elicitation. (C) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.virusres.2008.11.011

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The tobamovirus resistance gene L(3) of Capsicum chinense was mapped using an intra-specific F2 population (2,016 individuals) of Capsicum annuum cultivars, into one of which had been introduced the C. chinense L(3) gene, and an inter-specific F2 population (3,391 individuals) between C. chinense and Capsicum frutescence. Analysis of a BAC library with an AFLP marker closely linked to L(3)-resistance revealed the presence of homologs of the tomato disease resistance gene I2. Partial or full-length coding sequences were cloned by degenerate PCR from 35 different pepper I2 homologs and 17 genetic markers were generated in the inter-specific combination. The L(3) gene was mapped between I2 homolog marker IH1-04 and BAC-end marker 189D23M, and located within a region encompassing two different BAC contigs consisting of four and one clones, respectively. DNA fiber FISH analysis revealed that these two contigs are separated from each other by about 30 kb. DNA fiber FISH results and Southern blotting of the BAC clones suggested that the L(3) locus-containing region is rich in highly repetitive sequences. Southern blot analysis indicated that the two BAC contigs contain more than ten copies of the I2 homologs. In contrast to the inter-specific F2 population, no recombinant progeny were identified to have a crossover point within two BAC contigs consisting of seven and two clones in the intra-specific F2 population. Moreover, distribution of the crossover points differed between the two populations, suggesting linkage disequilibrium in the region containing the L locus.

DOI： 10.1007/s00122-008-0848-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Although auxin and ethylene play pivotal roles in leaf abscission, the subsequent signaling molecules are poorly understood. This is mainly because it is difficult to effectively treat the intact abscission zone (AZ) with pharmacological reagents. We developed an in vitro experimental system that reproduces stress-induced leaf abscission in planta. In this system, 1-mm-thick petiole strips, encompassing the AZ, were separated within 4 days of abscission at the AZ through cell wall degradation in an auxin depletion- and ethylene-dependent manner. The system allowed us to show that hydrogen peroxide (H(2)O(2)) is involved in abscission signaling. Microscopic analyses revealed continuous H(2)O(2) production by AZ cells. H(2)O(2) scavengers and diphenylene iodonium, an inhibitor of NADPH oxidase, suppressed in vitro abscission and cellulase expression. Conversely, the application of H(2)O(2) promoted in vitro abscission and expression of cellulase. Ethephon-induced abscission was suppressed by inhibitors of H(2)O(2) production, whereas the expression of ethylene-responsive genes was unaffected by both H(2)O(2) and an H(2)O(2) inhibitor. These results indicated that H(2)O(2) acts downstream from ethylene in in vitro abscission signaling. In planta, salinity stress induced the expression of genes that respond to ethylene and reactive oxygen species, and also induced H(2)O(2) production at the AZ, which preceded leaf abscission. These results indicate that H(2)O(2) has roles in leaf abscission associated with ethylene both in vitro and in planta.

DOI： 10.1111/j.1365-313X.2008.03577.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

One or a few nucleotide changes in the coat protein gene reportedly confer Pepper mild mottle virus (PMMoV) with the ability to overcome L(3) gene-mediated resistance in Capsicum plants. Primer-introduced restriction analysis-PCR (PIRA-PCR) was set up to detect four known resistance-breaking mutations. Each mutation from the L(3)-breaking PMMoV strains was successfully detected by reverse transcription-PCR amplification of the viral coat protein gene, PCR amplification of the regions containing the mutations with restriction site-introducing primers, followed by restriction analysis. Since PIRA-PCR primers were designed such that only PCR products from L(3)-breaking PMMoV strains can be digested by appropriate restriction enzymes, L(3)-breaking strains could be detected when they comprised no more than 20% of the mixture with non-L(3)-breaking strain. Using this PIRA-PCR-based method, L(3)-breaking PMMoV was detected in field soil samples taken from the base of both diseased and non-diseased plants harbouring L(3). The results show that PIRA-PCR is useful to quickly detect L(3)-breaking PMMoV strains not only in Capsicum plants harbouring the L(3) resistance gene but also in field soil, which serves as a reservoir of infectious viruses.

DOI： 10.1111/j.1365-3059.2008.01835.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER TOKYO  

We used SuperSAGE, an improved version of serial analysis of gene expression, to explore transcriptome changes early in the L(3)-mediated resistance response of pepper plants against a tobamovirus. Capsicum chinense plants homozygous for the L(3) resistance gene were infected with virulent and avirulent strains of Pepper mild mottle virus (PMMoV). Plants were maintained at a temperature nonpermissive for the resistance gene to allow the viruses to spread, then transferred to a permissive temperature for 3 h and subsequently analyzed. In the incompatible reaction, we selected 152 SuperSAGE tags (each 26 nucleotides long) possibly corresponding to upregulated genes, and 84 tags for downregulated genes. Approximately 70% of tags had matching ESTs in the genus Capsicum, other genera within the Solanaceae and/or other families of plants. More than 90% of tags with EST matches could be annotated with either functionally characterized or uncharacterized proteins. We compared genes annotated by SuperSAGE tags and those annotated by partial cDNA that was obtained using the SuperSAGE tag sequences as rapid amplification of the cDNA ends-PCR primers. Of genes annotated by SuperSAGE tags, c. 90% were consistent with those annotated by longer cDNA sequences. We cloned 17 full-length cDNAs from different SuperSAGE tags and confirmed that these genes were upregulated during normal infection in the incompatible interaction. We identified several early resistance response genes including a Ran/TC4 protein and a beta-oxidation multifunctional protein, indicating that SuperSAGE is a powerful tool for investigating plant-pathogen interactions.

DOI： 10.1007/s10327-008-0106-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER WIEN  

Tobamovirus resistance in Capsicum plants, which is mediated by L genes (L-1, L-2, L-3 or L-4), is known to be temperature sensitive. However, the L-1a gene, a newly identified tobamovirus resistance gene that is mapped to the L locus, confers temperature-insensitive resistance against the tobamovirus P-0 pathotype. To identify the viral elicitor that activates the L-1a-gene-mediated resistance, several chimeric viral genomes were constructed between tobacco mosaic virus-L (P-0 pathotype), paprika mild mottle virus-J (P-1 pathotype) and pepper mild mottle virus-J (P-1,P-2 pathotype). Infection patterns of these chimeric viruses in L-1a-harboring plants revealed that the L-1a-gene-mediated resistance was activated by the CP of a particular pathotype of tobamovirus, like other L-gene-mediated resistances, but the L-1a-gene-mediated resistance differs from those conferred by other L genes in terms of temperature sensitivity.

DOI： 10.1007/s00705-008-0032-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Landes Bioscience  

Reactive oxygen species (ROS) are produced in response to many environmental stresses, such as UV, chilling, salt and pathogen attack. These stresses also accompany leaf abscission in some plants, however, the relationship between these stresses and abscission is poorly understood. In our recent report, we developed an in vitro abscission system that reproduces stress-induced pepper leaf abscission in planta. Using this system, we demonstrated that continuous production of hydrogen peroxide (H 2O2) is involved in leaf abscission signaling. Continuous H2O2 production is required to induce expression of the cell wall-degrading enzyme, cellulase and functions downstream of ethylene in abscission signaling. Furthermore, enhanced production of H2O 2 occurs at the execution phase of abscission, suggesting that H 2O2 also plays a role in the cell-wall degradation process. These data suggest that H2O2 has several roles in leaf abscission signaling. Here, we propose a model for these roles. ©2008 Landes Bioscience.

DOI： 10.4161/psb.6737

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Language：Japanese  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

We found that an L-3 resistance-breaking field isolate of Pepper mild mottle virus (PMMoV), designated PMMoV-Is, had two amino acid changes in its coat protein (CP), namely leucine to phenylalanine at position 13 (L13F) and glycine to valine at position 66 (G66V), as compared with PMMoV-J, which induces a resistance response in L-3-harboring Capsicum plants. The mutations were located to a CP domain corresponding to the outer surface of PMMoV particles in computational molecular modeling. Analyses of PMMoV CP mutants containing either or both of these amino acid changes revealed that both changes were required to efficiently overcome L-3-mediated resistance with systemic necrosis induction. Although CP mutants containing either L13F or G66V could not efficiently overcome L-3-mediated resistance, these amino acid changes had different effects on the elicitor activity of PMMoV CP. L13F caused a slight reduction in the elicitor activity, resulting in virus restriction to necrotic local lesions that were apparently larger than those induced by wild-type PMMoV, while G66V rendered wild-type PMMoV the ability to overcome L-3-mediated resistance, albeit with a lower efficiency than PMMoV with both changes. These results suggest that a cooperative effect of the L13F and G66V mutations on the elicitor activity of CP is responsible for overcoming the L-3-mediated resistance.

DOI： 10.1007/s11262-006-0049-9

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Language：Japanese   Publisher：Japanese Society of Breeding  

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Other Link： https://agriknowledge.affrc.go.jp/RN/2010734523

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

To establish a simple and an efficient system to minimize the environmental risk of genetically modified plants, we tested the applicability of the barnase/barstar system in conferring bisexual sterility; that is, in preventing plants setting seeds by self-fertilization and out-crossing. Transgenic tobacco plants were generated to express barnase, a cell death inducing ribonuclease, under the control of the gamete-specific AtDMC1 promoter, and barstar, a specific inhibitor of barnase, under the control of the ACT2 promoter, which is constitutively active in almost all tissues except gametes. In contrast to control plants harboring the barstar expression unit only, which set seeds normally with self-pollination, all transformants harboring both barnase and barstar were bisexually sterile. They produced aberrant anthers containing no detectable pollen and failed to set seeds even after pollination with wild-type tobacco pollen.

DOI： 10.1007/s00299-006-0206-6

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Language：Japanese   Publisher：Japanese Society of Breeding  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

The Cauliflower mosaic virus (CaMV) open reading frame VI product (136) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle.

DOI： 10.1105/tpc.104.029017

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：INST PURE APPLIED PHYSICS  

Scanning force microscopy (SFM) was used to image ribosomes and ribosomal subunits isolated from Escherichia coli. Topographic images of ribosomal particles adsorbed on a mica surface were obtained directly. At 80 nM, the ribosomes aggregated. At less than or equal to8 nM, a sparser coverage was achieved, with single ribosomes isolated from one another. Although the obtained height was significantly lower than the diameter measured by X-ray diffraction, the height histogram revealed that the observed ribosomal particles belong to three height distributions: approximately 4, 9 and 11 nm. These three distributions correspond to the 70 S ribosome, and the 50 S and 30 S ribosomal subunits, respectively. This work demonstrates that SFM is useful in the discrimination of very small amounts of ribosomal particles on a solid substrate surface.

DOI： 10.1143/JJAP.43.4599

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Phytopathological Society  

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is a multifunctional protein essential for basic replication of CaMV. It also plays a role in viral pathogenesis in crucifer and solanaceous host plants. Deletion mutagenesis revealed that N- and C-terminal parts of Tav are not essential for CaMV replication in transfected protoplasts. Two deletion mutants having only minimal defects in basic replication were infectious in turnips but only with highly attenuated virulence. This was shown to be due to delayed virus spread within the inoculated leaves and to the upper leaves. Unlike the wild-type virus, the mutant viruses successfully spread locally without inducing a host defense response in inoculated Datura stramonium leaves, but did not spread systemically. These results provide the first evidence that a Tav domain required for avirulence function in solanaceous plants is not essential for CaMV infectivity but has a role in viral virulence in susceptible hosts.

DOI： 10.1094/MPMI.2004.17.5.475

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Cauliflower mosaic virus (CaMV) transactivator/viroplasmin (Tav) is an essential multifunctional viral protein. Dissection of Tav by deletion mutagenesis revealed that the central region is essential for CaMV replication in single cells but that the N- and C-terminal parts are not. Strains with mutations in the central region were defective in the translational transactivator function and could be complemented by coexpressing Gag (capsid protein precursor) and Pol (polyprotein with protease, reverse transcriptase, and RNase H activity) from separate monocistronic plasmids. In contrast, total omission of Tav was only partially complemented by Gag and Pol overexpression from separate plasmids. These results indicate that CaMV basic replication requires both Tav-activated polycistronic translation and some posttranslational function(s) of Tav that is not affected by the deletions in the central region of Tav.

DOI： 10.1128/JVI.77.15.8577-8583.2003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Cauliflower mosaic virus (CaMV) open reading frame III (ORF III) codes for a virion-associated protein (Vap), which is one of two viral proteins essential for aphid transmission. However, unlike the aphid transmission factor encoded by CaMV ORF II, Vap is also essential for systemic infection, suggesting that it is a multifunctional protein. To elucidate the additional function or functions of Vap, we tested the replication of noninfectious ORF III-defective mutants in transfected turnip protoplasts. PCR and Western blot analyses revealed that CaMV replication had occurred with an efficiency similar to that of wild-type virus and without leading to reversions. Electron microscopic examination revealed that an ORF III frameshift mutant formed normally structured virions. These results demonstrate that Vap is dispensable for replication in single cells and is not essential for virion morphogenesis. Analysis of inoculated turnip leaves showed that the ORF III frameshift mutant does not cause any detectable local infection. These results are strongly indicative of a role for Vap in virus movement.

DOI： 10.1128/JVI.76.18.9457-9464.2002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC PLANT BIOLOGISTS  

Numerous studies have shown that transcription factors are important in regulating plant responses to environmental stress. However, specific functions for most of the genes encoding transcription factors are unclear. In this study, we used mRNA profiles generated from microarray experiments to deduce the functions of genes encoding known and putative Arabidopsis transcription factors. The mRNA levels of 402 distinct transcription factor genes were examined at different developmental stages and under various stress conditions. Transcription factors potentially controlling downstream gene expression in stress signal transduction pathways were identified by observed activation and repression of the genes after certain stress treatments. The mRNA levels of a number of previously characterized transcription factor genes were changed significantly in connection with other regulatory pathways, suggesting their multifunctional nature. The expression of 74 transcription factor genes responsive to bacterial pathogen infection was reduced or abolished in mutants that have defects in salicylic acid, jasmonic acid, or ethylene signaling. This observation indicates that the regulation of these genes is mediated at least partly by these plant hormones and suggests that the transcription factor genes are involved in the regulation of additional downstream responses mediated by these hormones. Among the 43 transcription factor genes that are induced during senescence, 28 of them also are induced by stress treatment, suggesting extensive overlap responses to these stresses. Statistical analysis of the promoter regions of the genes responsive to cold stress indicated unambiguous enrichment of known conserved transcription factor binding sites for the responses. A highly conserved novel promoter motif was identified in genes responding to a broad set of pathogen infection treatments. This observation strongly suggests that the corresponding transcription factors play general and crucial roles in the coordinated regulation of these specific regulons. Although further validation is needed, these correlative results provide a vast amount of information that can guide hypothesis-driven research to elucidate the molecular mechanisms involved in transcriptional regulation and signaling networks in plants.

DOI： 10.1105/tpc.010410

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COLD SPRING HARBOR LAB PRESS  

DOI： 10.1101/sqb.2001.66.269

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MARY ANN LIEBERT INC PUBL  

To investigate the role of cytokines in the pathogenesis of acute herpetic keratitis (HK), we examined the kinetics of cytokine expression in the corneas and the trigeminal ganglia (TG) of C57BL/6Cr (B6) mice after herpes simplex virus type 1 (HSV-1) infection and observed the influence of the targeted disruption of interferon-gamma (IFN-gamma) gene on the clinical course of HK and/or viral clearance. Following corneal infection with HSV-1 Amakata strain, all corneas developed a typical dendritic keratitis, Quantitative analysis using enzyme-linked immunosorbent assay (ELISA) revealed that the expression of interleukin-1 alpha (IL-1 alpha), IL-5, IL-6, and IFN-gamma in corneas and TGs significantly elevated immediately after infection, peaked between days 2 and 7 postinfection (p.i.), and then diminished. One exception was IFN-gamma, whose expression significantly persisted in the TGs until day 30 p.i, An additional experiment using IFN-gamma(-/-) (gko) mice revealed that there was no significant difference in the peak level of viral replication in corneas and TGs between gko and B6 mice, although gko mice showed a significant delay of virus clearance in both corneas and TGs (p &lt; 0.005) and higher mortality rate than B6 mice after HSV-1 infection (p &lt; 0.01), These data suggest that the production of proinflammatory cytokines closely correlates with the pathogenesis of HK, and that IFN-gamma plays an important role in enhancing viral clearance from the cornea and TG.

DOI： 10.1089/107999099313749

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Human cytomegalovirus (HCMV) reportedly induces the expression of a 70 kDa heat shock protein (hsp70) with no known function in the virus replication cycle. We report here a remarkably specific translocation pattern of hsp70 during HCMV infection of human diploid fibroblasts. Immunofluorescent observation and Western blotting of subcellular fractions revealed nuclear localization of hsp70 early in infection and predominantly cytoplasmic localization of hsp70 late in infection. Treatment of HCMV-infected cells with cycloheximide followed by treatment with actinomycin D allowed virus immediate-early gene expression but inhibited hsp70 nuclear localization. Phosphonoacetic acid and tunicamycin, both of which reportedly inhibit HCMV DNA replication, did not inhibit HCMV-induced nuclear localization of hsp70 but inhibited hsp70 translocation from the nucleus to the cytoplasm. These results indicate a correlation between HCMV multiplication and hsp70 localization, suggesting that hsp70 may play a role in HCMV multiplication.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

In an effort to obtain a better signal-to-noise ratio and ultrastructural preservation, we sought to improve electron microscopic in situ hybridization technique. In our method, protease treatment was omitted and visualization of the digoxigenin (DIG)-labelled deoxyribonucleic acid (DNA)-probe was enhanced using a three-step procedure. These improvements allowed us to localize viral DNA with good signal-to-noise ratio. DNA specific to herpes simplex virus 1 (HSV-1) was localized by this method to HSV-I infected cultured cells; DNA was not observed in the empty-cored HSV-1. Using this method and the immunogold cytochemical method, we co-localized viral DNA and capsid protein ICP35 on Lowicryl-embedded sections of HSV-1 infected cells. Interestingly labelling for both DNA and ICP was observed on some HSV-1 particles in cell nucleus. This finding is consistent with the notion that ICP35 is necessary for assembly of viral DNA. Combination of in situ hybridization and immunocytochemical techniques is a powerful tool for examination of the functional relationship between viral DNA and proteins and help us to study protein function in viral multiplication.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

Cauliflower mosaic virus (CaMV) open reading frame (ORF) III encodes a 15 kDa protein; the function of which is as yet unknown, This protein has non-sequence-specific DNA binding activity and is associated with viral particles, suggesting that the ORF III product (PS) is involved in the folding of CaMV DNA during encapsidation, In this study, we demonstrated that P3 forms a tetramer in CaMV-infected plants. A P3-related protein with an apparent molecular weight of 60 kDa was detected by Western blotting analysis using anti-P3 antiserum under non-reducing conditions, while only 15 kDa P3 was detected under reducing conditions. Analysis of P3 using viable mutants with a 27-bp insertion in either ORF III or TV revealed that the 60 kDa protein was a tetramer of P3, The P3 tetramer co-sedimented with viral coat protein in multiple fractions on sucrose gradient centrifugation, suggesting that P3 tetramer binds to mature and immature virions. These results strongly suggested that CaMV P3 forms a tetramer in planta and that disulfide bonds are involved in its formation and/or stabilization. The finding of P3 tetramer in planta suggested that viral DNA. would be folded compactly by the interaction with multiple P3 molecules, which would form tetramers, while being packaged into the capsid shell.

DOI： 10.1111/j.1348-0421.1999.tb02469.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

Cauliflower mosaic virus (CaMV) open reading frame (ORF) VI product (P6) has been shown to be the major constituent of viral inclusion body, to function as a post-transcriptional transactivator, and to be essential for infectivity on whole plants. Although these findings suggest that P6 has an important role in viral multiplication, it is unknown whether P6 is required for viral multiplication in a single cell. To address this question, we transfected turnip protoplasts with an ORF VI frame-shift (4 bp deletion) mutant (pCaFS6) of an infectious CaMV DNA clone (pCa122). The mutant was uninfectious. Co-transfection of plasmids expressing P6 complemented the mutant. Overexpression of P6 elevated the infection rate in co-transfection experiments with either pCa122 or pCaFS6. This would have been achieved by elevating the level of pregenomic 35S RNA, a putative polycistronic mRNA for ORFs I, II, III, IV and V, and by enhancing the accumulation of these five viral gene products. When CaMV ORFs I, II, III, TV and V were expressed from monocistronic constructs in which each of the ORFs was placed just downstream of the 35S promoter, the accumulation of ORF III, IV and V products depended an the co-expression of P6. The accumulation of ORF I and II products was not detected, even in the presence of P6. These results suggest that P6 is involved in the stabilization of other viral gene products as well as in the activation of viral gene expression, and thus, is a prerequisite for CaMV multiplication.

DOI： 10.1111/j.1348-0421.1998.tb02298.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

The expression of cauliflower mosaic virus (CaMV) genes was studied in a turnip protoplast system. Six CaMV-encoded gene products were detected in infected turnip protoplasts by means of Western blotting. The infected turnip protoplasts showed different patterns of protein accumulation; e.g. an open reading frame (ORF) I-encoded movement protein, an ORF V-encoded reverse transcriptase and an ORF VI-encoded posttranscriptional transactivator representing the early accumulated proteins, an ORF II-encoded aphid transmission factor and an ORF IV-encoded coat protein the late accumulated proteins and an ORF III-encoded DNA binding protein the intermediate protein. The results suggest that the expression of CaMV genes is differentially regulated.

DOI： 10.1111/j.1348-0421.1998.tb01972.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

The expression and subcellular localization of cellular heat-shock protein hsp70 were examined in varicella-zoster virus (VZV)-infected human diploid fibroblasts. Infection with VZV elevated the steady-state levels of hsp70 mRNA by 24 hr post-infection (hpi), Western blotting analysis revealed an increase in accumulation of hsp70 from 24 hpi, Subcellular localization of the hsp70 in VZV-infected cells was examined by indirect immunofluorescence, In most VZV-infected cells, hsp70 was localized to inclusion bodies induced in the cell nucleus by infection with VZV, In some cells, however, the remaining parts of the cell nucleus and the cytoplasm were also stained with anti-hsp70 antibody. These results indicate that infection with VZV induces the expression of hsp70 and its localization to VZV-specific inclusion bodies, which suggests the involvement of hsp70 in molecular events within inclusion bodies.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CENTER ACADEMIC PUBL JAPAN  

Studies have indicated that cauliflower mosaic virus (CaMV) gene expression is mediated by the translation of polycistronic 35S pregenomic RNA, but the involvement of some minor subgenomic RNA species is also suspected. We examined the involvement of the 35S promoter in the expression of CaMV open reading frames (ORFs) I and TV using both 35S RNA-driven and promoter-less ORF I- and ORF IV-beta-glucuronidase (GUS) fusion constructs. In addition to the 35S promoter-dependent expression of both ORF I- and IV-GUS fusions, rye detected the 35S promoter-independent expression of both fusion genes via subgenomic mRNAs, which were detected by Northern blotting in the protoplasts transfected with the 35S promoter-driven constructs as well as in those transfected with the promoter-less constructs. These results suggest the involvement of subgenomic RNAs in the expression of CaMV ORFs I and IV: and the operation of a dual strategy in the expression of two viral genes.

DOI： 10.1111/j.1348-0421.1998.tb02291.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS UNITED KINGDOM  

The heat shock proteins (HSPs) are believed To act as molecular chaperones which appear to play some roles in regulation of normal protein folding and also in preventing damage to protein structures under various conditions of environmental stress. We examined the expression of the major HSP families, HSP6O, 70 and 90 families and small HSP32, at the mRNA level in ocular development. Expression of HSP32, HSP60, HSP70, HSP84, HSP86 and heat shock cognate protein (HSC)70 mRNAs was examined by in situ hybridization. HSC70, HSP84, HSP86 and HSP60 mRNAs were expressed strongly in all ocular tissues during early stages 3 to 5, corresponding to embryonic day (E)11.5 to E14.5. At stages 6 to 7 (E15.5 to E18.5), the expression of these four mRNA species was decreased markedly in most ocular tissue, while in the retina strong HSC70 and HSP86 mRNA expression was still detected. HSP32 and HSP70 mRNAs were not detected at any stage. These results suggest that the expression of HSPs is developmentally regulated through ocular organogenesis, and the proteins may play some important roles in ocular development.

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Language：Japanese   Publisher：The Phytopathological Society of Japan (PSJ)  

DOI： 10.3186/jjphytopath.60.496

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Language：English   Publisher：CENTER ACADEMIC PUBL JAPAN  

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.

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DOI： 10.3186/jjphytopath.60.27

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Language：English   Publisher：SOC GENERAL MICROBIOLOGY  

The synthesis and accumulation of aphid transmission factor protein (p18) in cauliflower mosaic virus (CaMV)-infected turnip protoplasts were examined in time course and pulse-labelling experiments, comparing an aphid-non-transmissible isolate (CM1841) with an in vitro recombinant aphid-transmissible CaMV (CMBX) generated from the CM1841 isolate. There was little difference in the synthesis and accumulation of p18 between CM1841- and CMBX-infected protoplasts. When the accumulation of p18 in infected leaves was monitored from 3 to 28 days post-symptom emergence (p.e.) by Western blotting, the amount of p18 accumulated in CM 1 84 1 -infected leaves continuously decreased from 3 days p.e. throughout the experimental period, whereas the amount of p18 in CMBX-infected leaves was lowest at 3 days p.e. and increased thereafter. These results suggested that CM 1 841 differed from CMBX not in the synthesis of p18 but in the stability of p18 in infected leaves.

DOI： 10.1099/0022-1317-74-11-2469

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DOI： 10.3186/jjphytopath.58.773

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Other Link： https://jlc.jst.go.jp/DN/JALC/00079237444?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYTOPATHOLOGICAL SOC  

We constructed a cosmid vector pKV-beta-for isolating genes by complementation of mutations in Colletotrichum lagenarium. pKV-beta-contains the bacteriophage lambda-cos site and the benomyl-resistant C. lagenarium beta-tubulin gene as a selective marker for Colletotrichum transformation. A genomic DNA library of wild-type C. lagenarium was constructed in pKV-beta. An albino mutant strain 79215 was transformed with DNA from this library and benomyl-resistant transformants were obtained at frequencies of approximately 20 transformants per microgram of DNA. Seven melanin-restored transformants were obtained from approximately 10,000 benomyl-resistant transformants. Albino mutants of C. lagenarium form nonmelanized appressoria and possess little penetrating ability. However, the transformants formed melanized appressoria with the ability to penetrate as efficiently as in the wild-type strain. From genomic DNA of a melanin-restored transformant integrated cosmid sequences (pAC7) were recovered by transduction of Escherichia coli to ampicillin resistance following treatment in vitro with lambda packaging extract. pAC7 transformed albino mutant 79215 to a melanin-restored wild phenotype with high frequency. From structural analysis of pAC7, an 8.4-kb BamHI fragment of pAC7 contains a wild-type copy of the gene involved in albino phenotype.

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Language：English   Publisher：The Phytopathological Society of Japan  

Aphid transmissible Japanese isolate of caulifiower mosaic virus M (CaMV-M) was cloned into pUC19 and its ORF II region was sequenced. Comparison of this sequence with that of CM1841 (non-aphid transmissible isolate of CaMV) showed that there were nine base changes, four of which gave rise to alterations in the predicted amino acid sequence. Characterization of <i>in vitro</i> recombined hybrid viruses between CaMV-M and CM1841 showed that 5' half of ORF II was responsible for the lack of aphid transmissibility in CM1841 and this region influenced the amount of accumulation of ORF II product in the infected leaves.

DOI： 10.3186/jjphytopath.57.634

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Full-length biologically active cDNAs of brome mosaic virus genomic RNAs 1, 2 and 3 were constructed by joining cDNA fragments. The cDNAs were constructed so that, at the 5' ends, unique SnaBI sites were present at the site of initiation of transcription. The cDNAs were inserted between a modified cauliflower mosaic virus (CaMV) 35S RNA promoter and terminator regions derived from CaMV DNA, and cloned into pUC18. When a mixture of the plasmid DNAs was inoculated onto Chenopodium hybridum leaves, local lesions appeared 5 to 6 days later. However, no symptoms appeared in similarly inoculated barley plants. Plasmid cDNAs with extra sequences at the 5' end were infectious but RNAs transcribed from cDNAs with similar sequences were not.

DOI： 10.1099/0022-1317-72-2-243

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© Springer Science+Business Media, LLC, part of Springer Nature 2019. RNA silencing is a sequence-specific suppression of gene expression conserved in eukaryotes including fungi, plants, and animals. Based on this mechanism, crop improvements have been made to confer pathogen resistance and abiotic stress tolerance. Here we have applied this technique to produce virus resistant tomato plants using host genes involved in viral replication. Tomato homologs of Arabidopsis TOM1 involved in tobamovirus replication has been isolated and used to construct the plasmids that carried inverted repeats of the genes for induction of RNA silencing. Tomato plants were transformed by the plasmids via Agrobacterium, and tested for virus resistance. Actually, the T2 and T3 plants showed resistance to tomato mosaic virus. Here we describe the method to construct RNA silencing-inducing plasmids, to transform tomato plants and to check the introduction of transgenes and virus resistance.

DOI： 10.1007/978-1-4939-9635-3_14

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© Springer Science+Business Media, LLC, part of Springer Nature 2019. NB-LRR class plant virus resistance gene is a one of the key players that shape the plant–virus interaction. Evolutionary arms race between plants and viruses often results in the breakdown of virus resistance in plants, which leads to a disastrous outcome in agricultural production. Although studies have analyzed the nature of plant virus resistance breakdown, it is still difficult to foresee the breakdown of a given virus resistance gene. In this chapter, we provide a protocol for evaluating the durability of plant virus resistance gene, which comprises the random mutagenesis of a virus gene, the introduction of the mutagenized gene into a virus context with highly efficient inoculation system, and the efficient screening of virus mutants that can overcome or escape a virus resistance.

DOI： 10.1007/978-1-4939-9635-3_5

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© Springer Science+Business Media, LLC, part of Springer Nature 2019. It is prerequisite to detect plant disease resistance proteins for studying the function of the proteins. Numerous studies have used epitope tags fused to either N- or C-terminus for the detection of resistance proteins. However, some resistance proteins do not tolerate the terminal fusions of epitope tags. In this chapter, we provide a protocol for searching the protein regions in which the inserted epitope tag does not affect the protein function. In the protocol, we first perform an in silico search to select the insertion site candidates and then insert there a short sequence containing restriction sites to find out the sites, in which the insertion does not affect the protein function. Epitope tags are inserted into the experimentally selected sites to produce a functional protein with an epitope tag.

DOI： 10.1007/978-1-4939-9635-3_2

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© Springer Science+Business Media New York 2015. When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double- stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a signifi cant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate dsRNA from many samples at the same time. To overcome these problems, we developed a novel dsRNA isolation method involving a recombinant dsRNA-binding protein. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously. Purifi ed dsRNA can be used as a template for cDNA synthesis and sequencing, enabling detection of both known and unknown viruses.

DOI： 10.1007/978-1-4939-1743-3_3

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Language：Japanese   Publisher：The Phytopathological Society of Japan  

DOI： 10.3186/jjphytopath.80.S179

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Language：Japanese   Publisher：The Phytopathological Society of Japan  

DOI： 10.3186/jjphytopath.80.S165

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Other Link： https://agriknowledge.affrc.go.jp/RN/2010890311

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DOI： 10.1007/s00299-012-1296-y

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DOI： 10.1007/s00299-006-0227-1

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Objective. To realize local selective gene expression in grafted chondrocytes for cartilage defect, we investigated the usefulness of an ex vivo gene delivery method using an adenovirus vector. Methods. β- galactosidase gene (LacZ) was transfected using an adenovirus vector to chondrocytes isolated from rat joints. The cells were then embedded into collagen gel, and LacZ expression in the gel was examined using 5-bromo-4- chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining
β-galactosidase activity was also measured. The collagen gel containing transfected chondrocytes was grafted to the experimental cartilage defects, and the expression of delivered gene was histologically examined after X-gal staining of the tissue containing the grafted area. Results. X-gal positive chondrocytes in the gel accounted for 82% at one week and 55% at 8 weeks after gene delivery. β-galactosidase activity decreased with time, but its expression was maintained even at 8 weeks after gene delivery. Chondrocytes used in the allograft maintained their morphology, and the expression of delivered gene continued during the 8 week period. Conclusion. In this ex vivo method, delivered gene can be expressed efficiently for a long time
this method would be useful in allografts for cartilage defects.

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Language：Japanese   Publisher：日本医師会  

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Language：English   Publisher：J RHEUMATOL PUBL CO  

Objective, To investigate the effects of adenovirus vector-mediated gene transduction of E. coli beta-galactosidase (LacZ), transforming growth factor-beta 1 (TGF-beta 1), and heat shock protein 70 (HSP 70) on human chondrocyte-like cell line (HCS-2/8).
Methods. We examined expression of transduced genes and their expression periods by 5 bromo-4-chloroindolyl-beta-D-galactoside (X-gal) staining, Northern blotting, ELISA? and Western blotting. To assess the influence of TGF-beta 1 and HSP 70 gene transduction, the expression of mRNA of type II collagen, proteoglycan core protein, matrix metalloproteinase 3 (MMP3) and tissue inhibitor of matrix metalloproteinase 1 were examined by Northern blotting.
Results. Staining with X-gal indicated that the genes were transduced into a majority of the cells. Expression of the transduced genes in the cells was continued for at least 21 days. Transduction of TGF-beta 1 gene enhanced mRNA expression of type II collagen and proteoglycan core protein, but suppressed MMP3 mRNA expression in the cells. Expression of HSP 70 was also high. Enhanced expression of HSP 70 elevated mRNA expression of proteoglycan core protein.
Conclusion. These results indicate adenovirus vector is useful in chondrocyte gene therapy, and it could be an efficient mediator of TGF-beta 1 and HSP 70 gene transduction.

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The 47-kDa heat shock protein (HSP47) is assumed to be a molecular chaperone specific to collagen. We examined and compared the expression patterns of HSP47 and alpha 1(I) collagen in the developing mouse cornea. Expression of HSP47 and alpha 1(I) collagen mRNAs was assessed by Northern blot analysis and in situ hybridization, and that of HSP47 protein was determined by Western immunoblotting and immunohistochemistry. HSP47 mRNA and protein were expressed strongly in early embryonic ocular tissues and were developmentally down-regulated during ocular development. Both were distributed in the mesenchymal cells (presumptive corneal stroma, sclera and choroid) and hyaloid blood vessels until post-natal day 14. The level of alpha 1(I) collagen mRNA increased during ocular development, was maximally expressed in the three days after birth, and was detected in mesenchymal cells. HSP47 expression is regulated developmentally toward completion and plays an important role during ocular development especially in corneal morphogenesis. We discuss here the relation between expression of HSP47 and alpha 1(I) collagen. (C) 1996 Academic Press Limited

DOI： 10.1006/exer.1996.0128

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT-RAVEN PUBL  

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Language：Japanese   Publisher：医歯薬出版  

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Other Link： http://search.jamas.or.jp/link/ui/1995192489

Language：English   Publishing type：Research paper, summary (international conference)   Publisher：LIPPINCOTT-RAVEN PUBL  

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