Nobuyuki Yoshikawa

Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing among a broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes

Apple latent spherical virus (ALSV) vectors were evaluated for virus-induced gene silencing (VIGS) of endogenous genes among a broad range of plant species. ALSV vectors carrying partial sequences of a subunit of magnesium chelatase (SU) and phytoene desaturase (PDS) genes induced highly uniform knockout phenotypes typical of SU and PDS inhibition on model plants such as tobacco and Arabidopsis thaliana, and economically important crops such as tomato, legume, and cucurbit species. The silencing phenotypes persisted throughout plant growth in these plants. In addition, ALSV vectors could be successfully used to silence a meristem gene, proliferating cell nuclear antigen and disease resistant N gene in tobacco and RCY1 gene in A. thaliana. As ALSV infects most host plants symptomlessly and effectively induces stable VIGS for long periods, the ALSV vector is a valuable tool to determine the functions of interested genes among a broad range of plant species.

Virus-induced gene silencing in soybean seeds and the emergence stage of soybean plants with Apple latent spherical virus vectors

Virus-induced gene silencing (VIGS) has great potential as a reverse-genetics tool in plant genomics. In this study, we examined the potential of VIGS in soybean seeds and the emergence stage of soybean plants using Apple latent spherical virus (ALSV) vectors. Inoculation of an ALSV vector (soyPDS-ALSV) carrying a fragment of the soybean phytoene desaturase (soyPDS) gene into soybean seedlings resulted in a highly uniform photo-bleached phenotype, typical of PDS inhibition, on the upper leaves throughout plant growth. The photo-bleached phenotype was also found on all immature pods, all seed coats, and about 50% embryos of seeds on soybean plants infected with soyPDS-ALSV. Infection with an ALSV vector (soyIFS2-ALSV) having a fragment of soybean isoflavone synthase 2 (soyIFS2) gene also led to a reduction of the levels of both soyIFS2- and soyIFS1- mRNAs and an isoflavone content in the cotyledons of about 36% mature seeds of infected soybean plants. Furthermore, VIGS of soyPDS was induced in the next generation plants by the seed transmission of soyPDS-ALSV. Thus ALSV vectors will be useful for studying gene functions in the reproductive stages and early growth stages, such as emergence and cotyledon stages, in addition to the vegetative stages of soybean plants.

Efficient virus-induced gene silencing in apple, pear and Japanese pear using Apple latent spherical virus vectors

BackgroundVirus-induced gene silencing (VIGS) is an effective technology for the analysis of gene functions in plants. Though there are many reports on virus vectors for VIGS in plants, no VIGS vectors available for Rosaceae fruit trees were reported so far. We present an effective VIGS system in apple, pear, and Japanese pear using Apple latent spherical virus (ALSV) vectors.ResultsInoculation of ALSV vectors carrying a partial sequence of endogenous genes from apple [ribulose-1, 5-bisphosphate carboxylase small subunit (rbcS), alpha subunit of chloroplast chaperonin (CPN60a), elongation factor 1 alpha (EF-1a), or actin] to the cotyledons of seeds by a particle bombardment induced highly uniform knock-down phenotypes of each gene on the true leaves of seedlings from 2~3 weeks after inoculation. These silencing phenotypes continued for several months. Northern blot and RT-PCR analyses of leaves infected with ALSV containing a fragment of rbcS gene showed that the levels of rbcS-mRNA drastically decreased in the infected apple and pear leaves, and, in reverse, rbcS- siRNAs were generated in the infected leaves. In addition, some of apple seedlings inoculated with ALSV vector carrying a partial sequence of a TERMINAL FLOWER 1 gene of apple (MdTFL1) showed precocious flowering which is expected as a knock-down phenotype of the silencing of MdTFL1 gene.ConclusionsThe ALSV-based VIGS system developed have provides a valuable new addition to the tool box for functional genomics in apple, pear, and Japanese pear.

The LeATL6-associated ubiquitin/proteasome system may contribute to fungal elicitor-activated defense response via the jasmonic acid-dependent signaling pathway in tomato.

The expression of LeATL6, an ortholog of Arabidopsis ATL6 that encodes a RING-H2 finger protein, was induced in tomato roots treated with a cell wall protein fraction (CWP) elicitor of the biocontrol agent Pythium oligandrum. The LeATL6 protein was expressed as a fusion protein with a maltose-binding protein (MBP) in Escherichia coli, and it catalyzed the transfer of ubiquitin to the MBP moiety on incubation with ubiquitin, the ubiquitin-activating enzyme E1, and the ubiquitin-conjugating enzyme E2; this indicated that LeATL6 represents ubiquitin ligase E3. LeATL6 expression also was induced by elicitor treatment of jail-1 mutant tomato cells in which the jasmonic acid (JA)-mediated signaling pathway was impaired; however, JA-dependent expression of the basic PR-6 and TPI-1 genes that encode proteinase inhibitor II and I, respectively, was not induced in elicitor-treated jail-1 mutants. Furthermore, transient overexpression of LeATL6 under the control of the Cauliflower mosaic virus 35S promoter induced the basic PR6 and TPI-1 expression in wild tomato but not in the jail-1 mutant. In contrast, LeATL6 overexpression did not activate salicylic acid-responsive acidic PR-1 and PR-2 promoters in wild tomato. These results indicated that elicitor-responsive LeATL6 probably regulates JA-dependent basic PR6 and TPI-1 gene expression in tomato. The LeATL6-associated ubiquitin/proteasome system may contribute to elicitor-activated defense responses via a JA-dependent signaling pathway in plants.

Seed and pollen transmission of Apple latent spherical virus in apple

To examine whether Apple latent spherical virus (ALSV) has spread among apple trees in an orchard, we surveyed 21 apple trees surrounding two ALSV-infected trees for virus infection using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). None of the 21 trees were infected, indicating that ALSV has not spread from the infected trees to the neighboring apple trees since it was first detected in 1984. We analyzed seed embryos and seedlings derived from infected trees and detected ALSV in 10 of 223 seed embryos (4.5%) and 10 of 227 seedlings (4.4%). From these results, we conclude that ALSV is seed-transmitted at a rate of ca. 4.5% in apple. We also analyzed seed embryos and seedlings from uninfected apple trees that were hand-pollinated with pollen from infected trees. We detected ALSV in only 1 of 260 seed embryos and in none of the 227 apple seedlings. This result indicated that the seed transmission rate via infected pollen is only 0–0.38%. In situ hybridization analysis of ALSV-infected apple flower buds showed that ALSV was present inside almost all pollen grains and in all ovary and ovule tissues, including the embryo sac and inner integument.

Histochemical detection of Blueberry latent virus in highbush blueberry plant

Blueberry latent virus (BBLV) was detected in 27 of 95 asymptomatic highbush blueberry trees in a blueberry field in Japan. In situ hybridization showed that the viral RNAs were detected in the palisade mesophyll, spongy mesophyll and vascular bundle of leaves. In western blot analysis, an antibody to the viral protein encoded by open reading frame 1 (ORF1) reacted with a 48-kDa protein specific for blueberry trees in which BBLV was detected. Immunogold electron microscopy revealed that amorphous bodies in the cytoplasm of blueberry cells were labeled with antibodies to the ORF1 protein.

Identification and characterization of blueberry latent spherical virus, a new member of subgroup C in the genus Nepovirus.

A new member of the genus Nepovirus was isolated from blueberry in Japan. The virus was associated with latent infection of blueberry trees and provisionally named blueberry latent spherical virus (BLSV). BLSV was found to have isometric particles approximately 30xa0nm in diameter, which were composed of a single coat protein (CP) of 55xa0kDa. The viral genome consisted of two positive-sense single-stranded RNA species (RNA1 and RNA2), which were 7,960 and 6,344 nucleotides (nt) long, respectively. The organization of RNA1 and RNA2 was similar to that of nepoviruses. The 3′ non-coding regions of RNA1 and RNA2 were 1,379xa0nt and 1,392xa0nt long, respectively. The amino acid sequences of the BLSV polymerase and CP shared the highest amino acid sequence similarities with those of the subgroup C nepoviruses (57% and 43%, respectively). Additionally, the BLSV genome, in contrast to other nepovirus genomes, was predicted to encode a serine protease.

Virus-Induced Gene Silencing of Endogenous Genes and Promotion of Flowering in Soybean by Apple latent spherical virus-Based Vectors

Soybean, which has been traditional food in Asian countries, is one of the most important crops due to its high quality protein and oil. Various functional components derived from secondary metabolite also have received significant attention in terms of human health. Elucidation of the gene function associated with the biosynthesis of various seed components at molecular level will provide valuable information for improvement of soybean. A great deal of information at the molecular level of soybean, including expression sequence tag (EST) libraries, microarray data, genome sequences, genetic linkage map, comparative genomics, and DNA markers has been reported (Alkharouf et al., 2004; Cheng et al., 2008; Choi et al., 2007; Grant et al., 2010; Haerizadeh et al., 2009; Hecht et al., 2005; Hisano et al., 2007; Nelson and shoemaker., 2006; Schmutz et al., 2010; Shoemaker et al., 2004; Wong et al., 2009; Zhu et al., 2005). This information can provide the candidate gene sequences controlling important traits of soybean, and the functional gene analysis tool for soybean is very important to identify the genes associated with important traits, soybean management, and efficient genetic improvements of soybean. Agrobacteriumand biolistic-mediated technologies have been developed for the introduction of foreign genes into plants (Klein et al., 1987; Zambryski et al., 1983). As another approach, a virus vector-mediated gene delivery system has been developed for several plants. Plant virus vectors could be used for both the expression of the foreign genes and the suppression of the target genes by virus-induced gene silencing (VIGS) in infected plants (Gleba et al., 2004; Purkayastha and Dasgupta., 2009). A virus-mediated gene delivery system is quick and does not require transformation and regeneration techniques, and is widely used for functional gene analysis in plants. Especially, it is an attractive tool for major crop plants including soybean, in which efficient transformation is not established for various cultivars. In soybean, five virus vectors were constructed from Clover yellow vein virus (CIYVV), Soybean mosaic virus (SMV), Bean pod mottle virus (BPMV), Cucumber mosaic virus (CMV), and Apple latent spherical virus (ALSV) (Igarashi et al., 2009; Masuta et al., 2000; Nagamatsu et al., 2007; Wang et al., 2006; Zhang and Ghabiral., 2006). CIYVV and SMV have been developed for foreign gene expression by a fusion polyprotein expression strategy, and BPMVand ALSV-based vectors have been developed for both the expression of foreign genes and VIGS

Complete nucleotide sequence and latency of a novel blueberry-infecting closterovirus

A novel latent closterovirus was detected from highbush blueberry in Japan and provisionally named blueberry virus A (BVA). The BVA genome (17,798 nucleotides) contains 10 open reading frames, but no minor coat protein could be identified in the virus genome. The BVA RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (HSP70h), and major coat protein (CP) shared the highest amino acid sequence identities with those of viruses in the genus Closterovirus (61.2, 27.6, and 20.9xa0%, respectively). In a phylogenetic analysis of the RdRp, HSP70h, and CP, BVA did not cluster with any genus in the family Closteroviridae.

First report of raspberry yellows disease caused by raspberry bushy dwarf virus in Japan

Severe yellowing of leaves was observed on red raspberry in Akita Prefecture. When inoculated with sap from symptomatic raspberry leaves, Chenopodium quinoa plants developed chlorotic ringspot and mottling that are typical of raspberry bushy dwarf virus (RBDV) infection. In western blot analysis, an antibody to the coat protein (CP) of RBDV reacted against ca. 30-kDa protein specific to the diseased trees. In RT-PCR testing for RBDV, single DNA fragments were amplified from total RNA samples of the diseased trees. The nucleotide sequences of the DNA fragments covering the entire CP region revealed 87–97xa0% identities with those of RBDV isolates.