Apple Latent Spherical Virus Vector Research Articles

Novel Functional Analysis for Pathogenic Proteins of Bursaphelenchus xylophilus in Pine Seed Embryos Using a Virus Vector.

Pine wilt disease (PWD), which is caused by the pine wood nematode Bursaphelenchus xylophilus, is among the most serious tree diseases worldwide. PWD is thought to be initiated by sequential excessive hypersensitive responses to B. xylophilus. Previous studies have reported candidate pathogenic molecules inducing hypersensitive responses in pine trees susceptible to B. xylophilus. The functions of some of these molecules have been analyzed in model plants using transient overexpression; however, whether they can induce hypersensitive responses in natural host pines remains unclear due to the lack of a suitable functional analysis method. In this study, we established a novel functional analysis method for susceptible black pine (Pinus thunbergii) seed embryos using transient overexpression by the Apple latent spherical virus vector and investigated five secreted proteins of B. xylophilus causing cell death in tobacco to determine whether they induce hypersensitive responses in pine. We found that three of five molecules induced significantly higher expression in pathogenesis-related genes ( p < 0.05), indicating hypersensitive response in pine seed embryos compared with mock and green fluorescence protein controls. This result suggests that tobacco-based screening may detect false positives. This study is the first to analyze the function of pathogenic candidate molecules of B. xylophilus in natural host pines using exogenous gene expression, which is anticipated to be a powerful tool for investigating the PWD mechanism.

Efficient virus-induced gene silencing system in pumpkin (Cucurbita maxima) using apple latent spherical virus vector

Crude-sap of apple latent spherical virus (ALSV)-infected Chenopodium quinoa leaves was rub-inoculated on the expanded cotyledons of various Cucurbitaceae plants. Most of the species were systemically infected with the virus without obvious symptoms, except pumpkin (Cucurbita maxima). In pumpkin, the ALSV infection was restricted to inoculated cotyledons; it did not spread to the upper true leaves. In situ hybridization showed that the ALSV was confined to part of the cotyledon tissues and it did not invade the phloem tissue, when inoculated at the expanded cotyledon stage. However, when total RNAs from ALSV-infected C. quinoa leaves were inoculated into the cotyledons immediately after germination (folded cotyledon stage) using particle bombardment, ALSV efficiently caused systemic infection. Systemic infection of pumpkin seedlings occurred only when the cotyledons were inoculated within a few days after germination. No systemic infection was observed in the seedlings 4 days after germination. In the grafting test, ALSV was not transmitted from the infected rootstocks to the healthy scions of pumpkins. An efficient virus-induced gene silencing system for pumpkins was established, in which infection with ALSV vectors harboring the phytoene desaturase or sulfur gene fragments resulted in a uniform phenotype in the true leaves of pumpkin seedlings.

Development and application of a virus-induced gene silencing protocol for the study of gene function in narrow-leafed lupin

BackgroundLupins are promising protein crops with an increasing amount of genomic and transcriptomic resources. The new resources facilitate the in silico identification of candidate genes controlling important agronomic traits. However, a major bottleneck for lupin research and crop improvement is the in planta characterization of gene function. Here, we present an efficient protocol for virus-induced gene silencing (VIGS) to down-regulate endogenous genes in narrow-leafed lupin (NLL) using the apple latent spherical virus (ALSV).ResultsWe identified ALSV as an appropriate VIGS vector able to infect NLL without causing a discernible phenotype. We created improved ALSV vectors to allow for efficient cloning of gene fragments into the viral genome and for easier viral propagation via agroinfiltration of Nicotiana benthamiana. Using this system, we silenced the visual marker gene phytoene desaturase (PDS), which resulted in systemic, homogenous silencing as indicated by bleaching of newly produced tissues. Furthermore, by silencing lysine decarboxylase (LaLDC)—a gene likely to be involved in toxic alkaloid biosynthesis—we demonstrate the applicability of our VIGS method to silence a target gene alone or alongside PDS in a ‘PDS co-silencing’ approach. The co-silencing approach allows the visual identification of tissues where silencing is actively occurring, which eases tissue harvesting and downstream analysis, and is useful where the trait under study is not affected by PDS silencing. Silencing LaLDC resulted in a ~ 61% or ~ 67% decrease in transcript level, depending on whether LaLDC was silenced alone or alongside PDS. Overall, the silencing of LaLDC resulted in reduced alkaloid levels, providing direct evidence of its involvement in alkaloid biosynthesis in NLL.ConclusionsWe provide a rapid and efficient VIGS method for validating gene function in NLL. This will accelerate the research and improvement of this underutilized crop.

Virus-Induced Flowering by Apple Latent Spherical Virus Vector: Effective Use to Accelerate Breeding of Grapevine.

Apple latent spherical virus (ALSV) was successfully used in promoting flowering (virus-induced flowering, VIF) in apple and pear seedlings. In this paper, we report the use of ALSV vectors for VIF in seedlings and in vitro cultures of grapevine. After adjusting experimental conditions for biolistic inoculation of virus RNA, ALSV efficiently infected not only progeny seedlings of Vitis spp. ‘Koshu,’ but also in vitro cultures of V. vinifera ‘Neo Muscat’ without inducing viral symptoms. The grapevine seedlings and in vitro cultures inoculated with an ALSV vector expressing the ‘florigen’ gene (Arabidopsis Flowering locus T, AtFT) started to set floral buds 20–30 days after inoculation. This VIF technology was successfully used to promote flowering and produce grapes with viable seeds in in vitro cultures of F1 hybrids from crosses between V. ficifolia and V. vinifera and made it possible to analyze the quality of fruits within a year after germination. High-temperature (37 °C) treatment of ALSV-infected grapevine disabled virus movement to newly growing tissue to obtain ALSV-free shoots. Thus, the VIF using ALSV vectors can be used to shorten the generation time of grapevine seedlings and accelerate breeding of grapevines with desired traits.

Estimation of the functions of viral RNA silencing suppressors by apple latent spherical virus vector.

Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs) from eight plant viruses: P19 of carnation Italian ring spot virus (tombusvirus), 2b of peanut stunt virus (cucumovirus), NSs of tomato spotted wilt virus (tospovirus), HC-Pro of bean yellow mosaic virus (potyvirus), γb of barley stripe mosaic virus (hordeivirus), P15 of peanut clump virus (pecluvirus), P1 of rice yellow mottle virus (sobemovirus), or P21 of beet yellows virus (closterovirus). These vectors were inoculated to Nicotiana benthamiana to investigate the effects of RSSs on the virulence and accumulation of ALSV. Among the vectors, ALSV expressing NSs (ALSV-NSs) developed severe mosaic symptoms in newly developed leaves followed by plant death. Infection of ALSV-γb induced characteristic concentric ringspot symptoms on leaves, and plants infected with ALSV-HC-Pro showed mosaic and dwarf symptoms. Infection of the other five ALSV vectors did not show symptoms. ELISA and immunoblot assay indicated that virus titer increased in leaves infected with ALSV-NSs, γb, HC-Pro, or P19. RT-qPCR indicated that the amount of ALSV in plants infected with ALSV-NSs was increased by approximately 45 times compared with that of wtALSV without expression of any RSS. When ALSV-P19, NSs, or HC-Pro was inoculated to Cucumis sativus plants, none of these ALSV vectors induced symptoms, but accumulation of ALSV in plants infected with ALSV-NSs was increased, suggesting that functions of RSSs on virulence and accumulation of ALSV depend on host species.

Virus-induced gene silencing in chili pepper by apple latent spherical virus vector

Apple latent spherical virus (ALSV) can infect a variety of crops, usually without inducing symptoms. Partial gene sequences can be introduced into ALSV vectors for the induction of virus-induced gene silencing (VIGS). These features are beneficial for the estimation of gene functions in plants, with relatively concise experimental manipulations. Given that the infectability of chili peppers (Capsicum spp.) by ALSV was unknown, an ALSV infectivity test was performed on the highly pungent Capsicum chinense cultivar ‘Habanero’. The chili pepper plants were not infected after rub-inoculation with a crude homogenate of ALSV-infected Chenopodium quinoa leaves, whereas inoculating them with a concentrated ALSV virus preparation caused an infection. Inoculation with an ALSV RNA preparation by gold particle bombardment resulted in high infection rates (about 90%). The infection was systemic and the infected plants were symptomless. For the induction of VIGS, 201-nucleotide fragments of the putative aminotransferase (pAMT) gene were introduced into the ALSV vector. These ALSV vectors infected 80–90% of RNA-inoculated chili pepper seedlings. Expression of pAMT-mRNA was repressed in the placenta of immature fruit of infected plants. The silencing of pAMT in the infected plants caused a substantial decrease in capsaicin content and a concomitant moderate accumulation of the non-pungent bioactive metabolite capsiate in these plants. These results showed that ALSV could be used to study gene functions by VIGS and to enhance capsiate accumulation in chili pepper through genetic modification.

RNA Silencing-Mediated Apple Latent Spherical Virus Vaccine in Plants.

The apple latent spherical virus (ALSV), originally isolated from an apple tree in Japan, is a small spherical virus with a diameter of 25 nm and comprises a bisegmented, single-stranded RNA genome (RNA1 and RNA2) and three different capsid proteins (Vp25, Vp20, and Vp24). The virus can experimentally infect a broad range of plants including, not only model plants (Arabidopsis thaliana and Nicotiana species) but also economically important crops such as cucumber, soybean, tomato, fruit trees, and flowers. ALSV has been used as an effective plant virus vector for virus-induced gene silencing (VIGS) to assess gene functions because the virus infects most of the host plants without showing any symptoms and induces a uniform knockout phenotype in infected plants. Moreover, the VIGS persists throughout plant growth in infected plants. Here, we show that genetically engineered ALSV vectors (ALSV vaccines) containing a partial genome sequence of pathogenic viruses display a high degree of cross-protection against the challenge inoculation of the corresponding pathogenic viruses. Treatment effects can also be expected in virus-infected plants by subsequent inoculation with ALSV vaccine.

Gentian (Gentiana triflora) prevents transmission of apple latent spherical virus (ALSV) vector to progeny seeds.

Gentian plants ( Gentiana triflora ) severely restrict apple latent spherical virus (ALSV) invasion to the gametes (pollens and ovules) and block seed transmission to progeny plants. Early flowering of horticultural plants can be induced by infection of ALSV vector expressing Flowering Locus T (FT) gene. In the present study, flowering of gentian plants was induced by infection with an ALSV vector expressing a gentian FT gene and the patterns of seed transmission of ALSV in gentian were compared with those in apple and Nicotiana benthamiana. Infection of gentian progeny plants with ALSV was examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), reverse transcription-loop-mediated isothermal amplification (RT-LAMP), and enzyme-linked immunosorbent assay (ELISA). ALSV was not transmitted to the progeny gentian plants, whereas small proportions of apple and N. benthamiana progeny plants are infected with ALSV. The in situ hybridization analyses indicated that ALSVs are not present in gentian pollen and ovules, but detected in most of gametes in apple and N. benthamiana. Collectively, these results suggest that seed transmission of ALSV is blocked in gentian plants through the unknown barriers present in their gametes. On the other hand, apple and N. benthamiana seem to minimize ALSV seed transmission by inhibiting viral propagation in embryos.

Apple latent spherical virus vector-induced flowering for shortening the juvenile phase in Japanese gentian and lisianthus plants.

Infection by apple latent spherical virus (ALSV) vectors that promote the expression of Arabidopsis thaliana FLOWERING LOCUS T ( AtFT ) or Gentiana triflora GtFT s accelerates flowering in gentian and lisianthus plants. Apple latent spherical virus (ALSV) has isometric virus particles (25nm in diameter) that contain two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). ALSV vectors are used for foreign gene expression and virus-induced gene silencing in a broad range of plant species. Here, we report the infection by ALSV vectors that express FLOWERING LOCUS T (AtFT) from Arabidopsis thaliana or its homolog GtFT1 from Gentiana triflora in three gentian cultivars ('Iwate Yume Aoi' [early flowering], 'Iwate' [medium flowering], and 'Alta' [late flowering]), and two lisianthus cultivars ('Newlination Pink ver. 2' and 'Torukogikyou daburu mikkusu') promotes flowering within 90days post-inoculation using particle bombardment. Additionally, seedlings from the progeny of the early-flowering plants were tested by tissue blot hybridization, and the results showed that ALSV was not transmitted to the next generation. The promotion of flowering in the family Gentianaceae by ALSV vectors shortened the juvenile phase from 1-3years to 3-5months, and thus, it could be considered as a new plant breeding technique in ornamental gentian and lisianthus plants.

Promotion of Flowering by Apple Latent Spherical Virus Vector and Virus Elimination at High Temperature Allow Accelerated Breeding of Apple and Pear.

Plant viral vectors are superior tools for genetic manipulation, allowing rapid induction or suppression of expression of a target gene in plants. This is a particularly effective technology for use in breeding fruit trees, which are difficult to manipulate using recombinant DNA technologies. We reported previously that if apple seed embryos (cotyledons) are infected with an Apple latent spherical virus (ALSV) vector (ALSV-AtFT/MdTFL1) concurrently expressing the Arabidopsis thaliana florigen (AtFT) gene and suppressing the expression of the apple MdTFL1-1 gene, the period prior to initial flowering (generally lasts 5–12 years) will be reduced to about 2 months. In this study, we examined whether or not ALSV vector technology can be used to promote flowering in pear, which undergoes a very long juvenile period (germination to flowering) similar to that of apple. The MdTFL1 sequence in ALSV-AtFT/MdTFL1 was replaced with a portion of the pear PcTFL1-1 gene. The resulting virus (ALSV-AtFT/PcTFL1) and ALSV-AtFT/MdTFL1 were used individually for inoculation to pear cotyledons immediately after germination in two inoculation groups. Those inoculated with ALSV-AtFT/MdTFL1 and ALSV-AtFT/PcTFL1 then initiated flower bud formation starting one to 3 months after inoculation, and subsequently exhibited continuous flowering and fruition by pollination. Conversely, Japanese pear exhibited extremely low systemic infection rates when inoculated with ALSV-AtFT/MdTFL1, and failed to exhibit any induction of flowering. We also developed a simple method for eliminating ALSV vectors from infected plants. An evaluation of the method for eliminating the ALSV vectors from infected apple and pear seedlings revealed that a 4-week high-temperature (37°C) incubation of ALSV-infected apples and pears disabled the movement of ALSV to new growing tissues. This demonstrates that only high-temperature treatment can easily eliminate ALSV from infected fruit trees. A method combining the promotion of flowering in apple and pear by ALSV vector with an ALSV elimination technique is expected to see future application as a new plant breeding technique that can significantly shorten the breeding periods of apple and pear.

Virus-induced gene silencing in various Prunus species with the Apple latent spherical virus vector

Virus-induced gene silencing (VIGS) has been used as a rapid and effective tool for functional analysis of genes in various plants, including woody fruit tree species. We previously reported the successful induction of VIGS of the endogenous PHYTOENE DESATURASE (PDS) gene in apricot (Prunus armeniaca L.) using Apple latent spherical virus (ALSV) vectors. In contrast, our attempts to infect Japanese apricot (Prunus mume Siebold & Zucc.) with ALSV vectors was unsuccessful, suggesting that species- and/or cultivar-dependent differences of ALSV susceptibility may exist in Prunus. In this study, we investigated whether this VIGS-based gene evaluation system using ALSV vectors was applicable to seven Prunus species, including apricot, sweet cherry (Prunus avium L.), almond [Prunus dulcis (Mill.) D. A. Webb.], peach (Prunus persica Batsch), Japanese apricot, Japanese plum (Prunus salicina Lindl.), and European plum (Prunus domestica L.). ALSV vectors carrying part of the apricot PDS gene sequence were amplified in Nicotiana benthamiana, and inoculated into the cotyledons of Prunus seedlings by particle bombardment. Typical PDS-silenced phenotypes, characterized by uniform discoloration of the upper leaves, were observed in sweet cherry and some cultivars of apricot and almond several weeks after inoculation. The amounts of PDS mRNA in the infected leaves were significantly reduced, while a 21nt antisense small RNA, which was assumed to play a central role as a guide RNA in PDS mRNA degradation, was highly accumulated. However, ALSV infection of Japanese apricot, Japanese plum, European plum, and the other cultivars of apricot and almond was unsuccessful. Furthermore, although the infection rate of ALSV in peach was high, severe pale spots (a viral infection symptom) were observed in the infected leaves. These results collectively suggested that the efficiency of ALSV infection and VIGS could vary depending on species and/or cultivar in Prunus. The possible use of the ALSV-mediated VIGS system for functional analysis of genes in Prunus is discussed.

Isolation and characterization of the C-class MADS-box gene involved in the formation of double flowers in Japanese gentian.

BackgroundGenerally, double-flowered varieties are more attractive than single-flowered varieties in ornamental plants. Japanese gentian is one of the most popular floricultural plants in Japan, and it is desirable to breed elite double-flowered cultivars. In this study, we attempted to characterize a doubled-flower mutant of Japanese gentian. To identify the gene that causes the double-flowered phenotype in Japanese gentian, we isolated and characterized MADS-box genes.ResultsFourteen MADS-box genes were isolated, and two of them were C-class MADS-box genes (GsAG1 and GsAG2). Both GsAG1 and GsAG2 were categorized into the PLE/SHP subgroup, rather than the AG/FAR subgroup. In expression analyses, GsAG1 transcripts were detected in the second to fourth floral whorls, while GsAG2 transcripts were detected in only the inner two whorls. Transgenic Arabidopsis expressing GsAG1 lacked petals and formed carpeloid organs instead of sepals. Compared with a single-flowered gentian cultivar, a double-flowered gentian mutant showed decreased expression of GsAG1 but unchanged expression of GsAG2. An analysis of the genomic structure of GsAG1 revealed that the gene had nine exons and eight introns, and that a 5,150-bp additional sequence was inserted into the sixth intron of GsAG1 in the double-flowered mutant. This insert had typical features of a Ty3/gypsy-type LTR-retrotransposon, and was designated as Tgs1. Virus-induced gene silencing of GsAG1 by the Apple latent spherical virus vector resulted in the conversion of the stamen to petaloid organs in early flowering transgenic gentian plants expressing an Arabidopsis FT gene.ConclusionsThese results revealed that GsAG1 plays a key role as a C-functional gene in stamen organ identity. The identification of the gene responsible for the double-flowered phenotype will be useful in further research on the floral morphogenesis of Japanese gentian.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0569-3) contains supplementary material, which is available to authorized users.

An effective and convenient method for the delivery of apple latent spherical virus (ALSV)-based vectors into plant cells by agroinoculation.

Virus infection leads to the synthesis of double-stranded RNA during virus replication, and then this infection produces small RNA molecules in the antiviral RNA silencing pathway. Here, we develop an Agrobacterium-mediated inoculation system for ALSV-based vectors. This system is more effective and convenient for inoculation of the ALSV vectors into plants compared to direct inoculation of ALSV-RNA2-based vectors in pUC plasmids reported previously. In addition, cointroduction of various plant viral RNA-silencing suppressors increased the efficiency of agroinoculation of the ALSV-based vector. An ALSV vector could be successfully used to silence an endogenous gene in plants. Therefore, the ALSV-based VIGS/agroinoculation system provides a valuable tool for functional genomics among a broad range of plant species.

Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing.

Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification.

Apple latent spherical virus vector as vaccine for the prevention and treatment of mosaic diseases in pea, broad bean, and eustoma plants by bean yellow mosaic virus.

We investigated the protective effects of a viral vector based on an Apple latent spherical virus (ALSV) harboring a segment of the Bean yellow mosaic virus (BYMV) genome against mosaic diseases in pea, broad bean, and eustoma plants caused by BYMV infection. In pea plants pre-inoculated with the ALSV vaccine and challenge inoculated with BYMV expressing green fluorescence protein, BYMV multiplication occurred in inoculated leaves, but was markedly inhibited in the upper leaves. No mosaic symptoms due to BYMV infection were observed in the challenged plants pre-inoculated with the ALSV vaccine. Simultaneous inoculation with the ALSV vaccine and BYMV also prevented mosaic symptoms in broad bean and eustoma plants, and BYMV accumulation was strongly inhibited in the upper leaves of plants treated with the ALSV vaccine. Pea and eustoma plants were pre-inoculated with BYMV followed by inoculation with the ALSV vaccine to investigate the curative effects of the ALSV vaccine. In both plant species, recovery from mosaic symptoms was observed in upper leaves and BYMV accumulation was inhibited in leaves developing post-ALSV vaccination. These results show that ALSV vaccination not only prevents mosaic diseases in pea, broad bean, and eustoma, but that it is also effective in curing these diseases.

Virus-induced gene silencing of N gene in tobacco by apple latent spherical virus vectors.

Virus infections induce an RNA-mediated defense that targets viral RNAs in a nucleotide sequence-specific manner in plants, commonly referred to as virus-induced gene silencing (VIGS). When the virus carries sequences of plant genes, it triggers virus-induced gene silencing (VIGS) and results in the degradation of mRNA of endogenous homologous gene. VIGS has been shown to have great potential as a reverse-genetics tool for studying of gene functions in plants, and it has several advantages over other functional genomics approaches. Here, we describe VIGS of N gene in tobacco cv. Xanthi nc by ALSV vectors containing fragments of N gene from Nicotiana glutinosa.

Presentation of epitope sequences from foreign viruses on the surface of apple latent spherical virus particles

Apple latent spherical virus (ALSV) has small isometric particles that are comprised of two single-stranded RNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp20, and Vp24). We constructed ALSV vectors for presenting foreign peptides on the surface of virus particles. In these vectors, peptides can be fused to either of two C-terminal regions of Vp20 (amino acid positions between G171 and P172 or between P172 and L173) or the C-terminus (T192) of Vp24. An ALSV vector presenting the epitope sequences of the coat protein (CP) of zucchini yellow mosaic virus (ZYMV) could systemically infect host plants and was specifically recognized by antiserum against ZYMV by ELISA, immunoelectron microscopy, and immunoblotting. RT-PCR showed that the epitope sequences up to 20 amino acids were stably maintained in the chimeric ALSV for more than 10 serial passages and at least six months. Purified chimeric ALSV particles induced an immune response and the production of antibodies against ZYMV-CP in rabbits. The ALSV vector was also used for expression of an epitope from VP1 of foot-and-mouth disease virus.

Detection of apple latent spherical virus in seeds and seedlings from infected apple trees by reverse transcription quantitative PCR and deep sequencing: evidence for lack of transmission of the virus to most progeny seedlings

We used reverse transcription quantitative PCR (RT-qPCR) to test for apple latent spherical virus (ALSV) in cotyledons of seeds and true leaves of seedlings from apple trees infected with ALSV to accurately determine the status of infection in seedlings from ALSV-infected trees. The amplification curves of ALSV-RNAs from cotyledons were classified into three patterns: fast (F), slow (S), and no amplification (N/A). Viral RNA was detected in high concentrations (F pattern) in cotyledons from approximately 1 % of the seeds, a proportion broadly consistent with previously reported ALSV seed transmission rates (from zero to a few percent). In the majority of seeds (94.4 %); however, minute amounts of RNA related to the virus were detected (S pattern). Minute amounts of viral-related RNA were also detected by siRNA analysis using a next-generation sequencer. After the seeds germinated, however, no viral RNA was detected in the true leaves, so we concluded that the viral RNA in the seeds lacked the capacity to replicate. No ALSV-RNA at all was detected (N/A pattern) in the remaining 4.6 % of seeds. We concluded that almost all apple seedlings from ALSV-infected trees (approx. 99 %) can be considered as virus-free. Therefore, we think that apple seedlings from the generation obtained after the use of ALSV vector technology can be exempted from restrictions on genetically modified plants after they have been confirmed to be virus-free by virus testing.

Preventive and curative effects of Apple latent spherical virus vectors harboring part of the target virus genome against potyvirus and cucumovirus infections

Apple latent spherical virus (ALSV)-based vectors experimentally infect a broad range of plant species without causing symptoms and can effectively induce stable virus-induced gene silencing in plants. Here, we show that pre-infection of ALSV vectors harboring part of a target viral genome (we called ALSV vector vaccines here) inhibits the multiplication and spread of the corresponding challenge viruses [Bean yellow mosaic virus, Zucchini yellow mosaic virus (ZYMV), and Cucumber mosaic virus (CMV)] by a homology-dependent resistance. Further, the plants pre-infected with an ALSV vector having genome sequences of both ZYMV and CMV were protected against double inoculation of ZYMV and CMV. More interestingly, a curative effect of an ALSV vector vaccine could also be expected in ZYMV-infected cucumber plants, because the symptoms subsided on subsequent inoculation with an ALSV vector vaccine. This may be due to the invasion of ALSV, but not ZYMV, in the shoot apical meristem of cucumber.

Development of apple latent spherical virus-based vaccines against three tospoviruses

Apple latent spherical virus (ALSV) is characterized by its relatively broad host range, latency in most host plants, and ability to induce gene silencing in host plants. Herein, we focus on the above characteristic of ALSV and describe our development of ALSV vector vaccines against three tospoviruses, namely, Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), and Tomato spotted wilt virus (TSWV). DNA fragments of 201nt of three tospovirus S-RNAs (silencing suppressor (NSS) and nucleocapsid protein (N) coding regions for each tospovirus) were inserted into an ALSV-RNA2 vector to obtain six types of ALSV vector vaccines. Nicotiana benthamiana plants at the five-leaf stage were inoculated with each ALSV vector vaccine and challenged with the corresponding tospovirus species. Tospovirus-induced symptoms and tospovirus replication after challenge were significantly suppressed in plants preinoculated with all ALSV vector vaccines having the N region fragment, indicating that strong resistance was acquired after infection with ALSV vector vaccines. On the other hand, cross protection was not significant in plants preinoculated with ALSV vectors having the NSs region fragment. Similarly, inoculation with an ALSV-RNA1 vector having the N region fragment in the 3′-noncoding region, but not the NSs region fragment, induced cross protection, indicating that cross protection is via RNA silencing, not via the function of the protein derived from the N region fragment. Our approach, wherein ALSV vectors and selected target inserts are used, enables rapid establishment of ALSV vector vaccines against many pathogenic RNA viruses with known sequences.