An Inexpensive System for Rapid and Accurate On-site Detection of Garlic-Infected Viruses by Agarose Gel Electrophoresis Followed by Array Assay.

Garlic can be infected by a variety of viruses, but mixed infections with leek yellow stripe virus, onion yellow dwarf virus, and allexiviruses are the most damaging, so an easy, inexpensive on-site method to simultaneously detect at least these three viruses with a certain degree of accuracy is needed to produce virus-free plants. The most common laboratory method for diagnosis is multiplex reverse transcription polymerase chain reaction (RT-PCR). However, allexiviruses are highly diverse even within the same species, making it difficult to design universal PCR primers for all garlic-growing regions in the world. To solve this problem, we developed an inexpensive on-site detection system for the three garlic viruses that uses a commercial mobile PCR device and a compact electrophoresis system with a blue light. In this system, virus-specific bands generated by electrophoresis can be identified by eye in real time because the PCR products are labeled with a fluorescent dye, FITC. Because the electrophoresis step might eventually be replaced with a lateral flow assay (LFA), we also demonstrated that a uniplex LFA can be used for virus detection; however, multiplexing and a significant cost reduction are needed before it can be used for on-site detection.

Metagenomic Analyses of Viruses in the Orchid Mycorrhizal Interaction Using Improved Assemble Tools.

In nature, mycorrhizal association with soil-borne fungi is indispensable for orchid species. Compatible mycorrhizal fungi form endo-mycorrhizal structures in orchid cells, and the fungal structures are digested in orchid cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until nonphotosynthetic young seedlings develop leaves and become photoautotrophic. Seeds of all orchids germinate with the help of their own fungal partners, and therefore, specific partnership has been acquired in a long evolutionary history between orchids and fungi. Assuming that horizontal transmission of viruses may occur in such a close relationship, we are focusing on viruses that infect orchids and their mycorrhizal fungi. We prepared aseptically germinated orchid plants (i.e., fungi-free plants) together with pure-cultured fungal isolates and conducted transcriptome analyses (RNA-seq) by next-generation sequencing (NGS) approach. To reconstruct virus-related sequences that would have been present in the RNA sample of interest, de novo assembly process is required using short read sequences obtained from RNA-seq. In the previous version of our protocol (see Viral Metagenomics, first edition 2018), virus searches were conducted using contig sets constructed by a single assembler, but this time we devised a method to construct more reliable contigs using multiple assemblers and again reinvestigated that viruses could be detected. Because the virus detection efficiency and number of detected virus species clearly differed depending on the assembly pipeline and the number of the input data, multiple methods should be used to identify viral infection, if possible.

Generation and Retention of Defective RNA3 from Cucumber Mosaic Virus and Relevance of the 2b Protein to Generation of the Subviral RNA.

A defective RNA3 (D3Yα) of strain Y of cucumber mosaic virus (CMV-Y) was examined on host-specific maintenance, experimental conditions, and a viral factor required for its generation in plants. D3Yα was stably maintained in cucumber but not in tomato plants for 28 days post inoculation (dpi). D3Yα was generated in Nicotiana tabacum or N. benthamiana after prolonged infection in the second and the third passages, but not in plants of N. benthamiana grown at low temperature at 28 dpi or infected with CMV-Y mutant that had the 2b gene deleted. Collectively, we suggest that generation and retention of D3Yα depends on potential host plants and experimental conditions, and that the 2b protein has a role for facilitation of generation of D3Yα.

Host-specific adaptation drove the coevolution of leek yellow stripe virus and Allium plants.

Host adaptation plays a crucial role in virus evolution and is a consequence of long-term interactions between virus and host in a complex arms race between host RNA silencing and viral RNA silencing suppressor (RSS) as counterdefense. Leek yellow stripe virus (LYSV), a potyvirus causing yield loss of garlic, infects several species of Allium plants. The unexpected discovery of an interspecific hybrid of garlic, leek, and great-headed (GH) garlic motivated us to explore the host-adaptive evolution of LYSV. Here, using Bayesian phylogenetic comparative methods and a functional assay of viral RSS activity, we show that the evolutionary context of LYSV has been shaped by the host adaptation of the virus during its coevolution with Allium plants. Our phylogenetic analysis revealed that LYSV isolates from leek and their taxonomic relatives (Allium ampeloprasum complex; AAC) formed a distinct monophyletic clade separate from garlic isolates and are likely to be uniquely adapted to AAC. Our comparative studies on viral accumulation indicated that LYSV accumulated at a low level in leek, whereas LYSVs were abundant in other Allium species such as garlic and its relatives. When RSS activity of the viral P1 and HC-Pro of leek LYSV isolate was analyzed, significant synergism in RSS activity between the two proteins was observed in leek but not in other species, suggesting that viral RSS activity may be important for the viral host-specific adaptation. We thus consider that LYSV may have undergone host-specific evolution at least in leek, which must be driven by speciation of its Allium hosts. IMPORTANCE Potyviruses are the most abundant plant RNA viruses and are extremely diversified in terms of their wide host range. Due to frequent host switching during their evolution, host-specific adaptation of potyviruses may have been shaped by numerous host factors. However, any critical determinants for viral host range remain largely unknown, possibly because of the repeated gain and loss of virus infectivity of plants. Leek yellow stripe virus (LYSV) is a species of the genus Potyvirus, which has a relatively narrow host range, generally limited to hosts in the genus Allium. Our investigations on leek and leek relatives (Allium ampeloprasum complex), which must have been generated through interspecies hybridization, revealed that LYSV accumulation remained low in leek as a result of viral host adaptation in competition with host resistance such as RNA silencing. This study presents LYSV as an ideal model to study the process of host-adaptive evolution and virus-host coevolution.

VIGS as a strategy to reverse aphid wing induction by Y-satellite RNA of cucumber mosaic virus.

Y-satellite RNA (Y-sat) of cucumber mosaic virus upregulates the expression of the aphid ABCG4 gene, which promotes aphid wing formation. We used ABCG4 virus-induced gene silencing (VIGS) to prevent the wing-induction mechanism of Y-sat and thus inhibited aphid wing formation. Of the aphids on plants with VIGS of ABCG4, only about 30% had wings, and 60-70% of the winged aphids were small and likely impaired in flying ability. In addition, we showed that double-stranded RNAs (dsRNAs) and small RNAs were transferred from the plant to the aphid to adequately silence aphid genes. Supplying ABCG4 dsRNA by VIGS to aphids is thus a potential strategy to inhibit aphid wing formation.

Two mutually exclusive evolutionary scenarios for allexiviruses that overcome host RNA silencing and autophagy by regulating viral CRP expression.

The genus Allexivirus currently includes eight virus species that infect allium plants. Previously, we showed that there are two distinct groups of allexiviruses (deletion [D]-type and insertion [I]-type) based on the presence or absence of a 10- to 20-base insert (IS) between the coat protein (CP) and cysteine rich protein (CRP) genes. In the present study of CRPs to analyze their functions, we postulated that evolution of allexiviruses may have been largely directed by CRPs and thus proposed two evolutionary scenarios for allexiviruses based mainly on the presence or absence of IS and determined by how the allexiviruses challenge host resistance mechanisms (RNA silencing and autophagy). We found that both CP and CRP are RNA silencing suppressors (RSS), that they can inhibit each other's RSS activity in the cytoplasm, and that CRP becomes a target of host autophagy in the cytoplasm but not CP. To mitigate CRP interference with CP, and to increase the CP's RSS activity, allexiviruses developed two strategies: confinement of D-type CRP in the nucleus and degradation of I-type CRP by autophagy in the cytoplasm. Here, we demonstrate that viruses of the same genus achieve two completely different evolutionary scenarios by controlling expression and subcellular localization of CRP.

Battle for control of anthocyanin biosynthesis in two Brassicaceae species infected with turnip mosaic virus.

It has previously been found that turnip mosaic virus (TuMV) greatly suppresses anthocyanin accumulation (AA) in Brassica rapa leaves, and that such leaves become infected whilst anthocyanin-enriched leaves on the same plants are rarely infected. To clarify whether AA is a defense against TuMV, in this study we examined tissue-level patterns of spontaneous AA in relation to the cellular localization of a TuMV strain that expresses a yellow fluorescent protein. We found that TuMV infection was significantly blocked by AA, suggesting that it functions as a chemical barrier against TuMV. We next analysed changes in expression of genes related to anthocyanin biosynthesis in TuMV-infected leaves of Arabidopsis. TuMV also suppressed AA that is induced by high light in Arabidopsis, and this this suppression was mainly due to inhibited expression of anthocyanin late-biosynthesis genes (LBGs). Most positive transcription factors of LBGs were also down-regulated, while the negative regulator SPL15 was highly up-regulated. Cucumber mosaic virus (CMV) also moderately suppressed AA in Arabidopsis, but in a different manner. Since it appeared that anthocyanin-enriched leaves of Arabidopsis were resistant to TuMV but not CMV, our results suggested that the anthocyanin-associated resistance that we observed was specific to TuMV.

Diversity of viral RNA silencing suppressors and their involvement in virus-specific symptoms.

RNA silencing is an evolutionarily conserved and homology-dependent gene inactivation system that regulates most biological processes at either the transcriptional or post-transcriptional level. In plants, insects and certain mammalian systems, RNA silencing constitutes the basis of the antiviral defense mechanism. To counteract RNA silencing-based antiviral responses viruses adopt strategies of replication and host invasion that include mechanisms of RNA silencing suppression. Indeed, viruses can express proteins known as RNA silencing suppressors (RSSs). Over the last two decades, silencing studies in plant virology have been largely devoted to the discovery and description of RSSs. The result has been exciting and these studies have revealed (i) an incredible diversity of proteins and mechanisms of RSSs belonging to various viral taxonomic groups, (ii) the multifunctionality of RSSs: they can fulfill several functions during viral infection and target one or more key points in the RNA silencing machinery. Some RSSs of model viral systems have been the subject of exceptional in-depth studies; they have proven to be real molecular tools for studying plant physiology, plant biology and virus-plant interactions, even in some cases extending the knowledge of the response of plants to other biotic and abiotic stressors. RSS diversity in phylogenesis, in mechanism of action and the frequent presence of more than one RSS in a single viral genome all suggest that they are extremely plastic in evolving to overcome host defenses. In this chapter, we present and discuss the most recent findings related to the well-studied RSSs of four viral taxonomic groups: geminiviruses, potyviruses, tombusviruses and cucumoviruses.

Leek Yellow Stripe Virus Can Adjust for Host Adaptation by Trimming the N-Terminal Domain to Allow the P1 Protein to Function as an RNA Silencing Suppressor.

In Japan, the P1 protein (S-type) encoded by leek yellow stripe virus (LYSV) isolates detected in Honshu and southward is shorter than the P1 (N-type) of LYSV isolates from garlic grown in Hokkaido due to a large deletion in the N-terminal half. In garlic fields in Hokkaido, two types of LYSV isolate with N- and S-type P1s are sometimes found in mixed infections. In this study, we confirmed that N- and S-type P1 sequences were present in the same plant and that they belong to different evolutionary phylogenetic groups. To investigate how LYSV with S-type P1 (LYSV-S) could have invaded LYSV with N-type P1 (LYSV-N)-infected garlic, we examined wild Allium spp. plants in Hokkaido and found that LYSV was almost undetectable. On the other hand, in Honshu, LYSV-S was detected at a high frequency in Allium spp. other than garlic, suggesting that the LYSV-S can infect a wider host range of Allium spp. compared to LYSV-N. Because P1 proteins of potyviruses have been reported to promote RNA silencing suppressor (RSS) activity of HC-Pro proteins, we analyzed whether the same was true for P1 of LYSV. In onion, contrary to expectation, the P1 protein itself had RSS activity. Moreover, the RSS activity of S-type P1 was considerably stronger than that of N-type P1, suggesting that LYSV P1 may be able to enhance its RSS activity when the deletion is in the N-terminal half and that acquiring S-type P1 may have enabled LYSV to expand its host range.

Coat protein of partitiviruses isolated from mycorrhizal fungi functions as an RNA silencing suppressor in plants and fungi.

Orchid seeds depend on colonization by orchid mycorrhizal (OM) fungi for their germination; therefore, the orchids and OM fungi have long maintained a close relationship (e.g., formation of the hyphal mass structure, peloton) during their evolution. In the present study, we isolated new partitiviruses from OM fungi; partitivirus were separately found in different subcultures from the same fungi. Partitiviruses have been believed to lack an RNA silencing suppressor (RSS), which is generally associated with viral pathogenicity, because most partitiviruses isolated so far are latent in both plants and fungi. However, we found that the coat protein (CP) of our partitiviruses indeed had RSS activity, which differed among the virus isolates from OM fungi; one CP showed RSS activity in both plants and fungi, while another CP showed no activity. The family Partitiviridae include viruses isolated from plants and fungi, and it has been suggested that these viruses may occasionally be transmitted between plant and fungal hosts. Given that there are several reports showing that viruses can adapt to nonhost using strong RSS, we here discussed the idea that partitiviruses may be better able to migrate between the orchid and fungus probably through the pelotons formed in the orchid cells, if host RNA silencing is suppressed by partitivirus RSS.

Progressive DNA demethylation in epigenetic hybrids between parental plants with and without methylation of the transgene promoter.

KEY MESSAGE: Crosses of parents that differ in their DNA methylation states leads to progressive demethylation in the F1 hybrids. In plant breeding research, hybrid vigor in F1 hybrids is known to be a very important phenomenon. Hybrid vigor, or heterosis, refers to the fact that F1 hybrids from crosses with a certain combination of parents have traits that are superior to those of the parents. In addition, DNA methylation is an important factor that affects gene expression in plant genomes and contributes to hybrid vigor. We introduced the 35S promoter sequence into the cucumber mosaic virus (CMV)-based vector and inoculated the GFP-expressing transgenic Nicotiana benthamiana line 16c with the recombinant virus specifically to induce DNA methylation on the 35S promoter. For plants that had transcriptional gene silencing (TGS) of GFP established by methylation of the 35S promoter (35S-TGS), TGS was fully maintained in their later self-pollinated generations. When the 35S-TGS plants were crossed with 16c, which does not contain DNA methylation in the 35S promoter, the F1 hybrids unexpectedly became progressively DNA demethylated as the plants grew. We hypothesis that in F1 hybrids that are produced by a cross between parents with extremely different gene methylation states, the methylation state of the genes in question may shift more and more to hypomethylation as the plants grow. This progressive demethylation phenomenon observed in this study may be important in plant breeding to reactivate the genes which were silenced by DNA methylation.

Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b's efficient suppression of RNA silencing.

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b's nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b's C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b's nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b's efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.

A plant virus satellite RNA directly accelerates wing formation in its insect vector for spread.

Cucumber mosaic virus (CMV) often accompanies a short RNA molecule called a satellite RNA (satRNA). When infected with CMV in the presence of Y-satellite RNA (Y-sat), tobacco leaves develop a green mosaic, then turn yellow. Y-sat has been identified in the fields in Japan. Here, we show that the yellow leaf colour preferentially attracts aphids, and that the aphids fed on yellow plants, which harbour Y-sat-derived small RNAs (sRNAs), turn red and subsequently develop wings. In addition, we found that leaf yellowing did not necessarily reduce photosynthesis, and that viral transmission was not greatly affected despite the low viral titer in the Y-sat-infected plants. Y-sat-infected plants can therefore support a sufficient number of aphids to allow for efficient virus transmission. Our results demonstrate that Y-sat directly alters aphid physiology via Y-sat sRNAs to promote wing formation, an unprecedented survival strategy that enables outward spread via the winged insect vector.

Resistance induction based on the understanding of molecular interactions between plant viruses and host plants.

BACKGROUND: Viral diseases cause significant damage to crop yield and quality. While fungi- and bacteria-induced diseases can be controlled by pesticides, no effective approaches are available to control viruses with chemicals as they use the cellular functions of their host for their infection cycle. The conventional method of viral disease control is to use the inherent resistance of plants through breeding. However, the genetic sources of viral resistance are often limited. Recently, genome editing technology enabled the publication of multiple attempts to artificially induce new resistance types by manipulating host factors necessary for viral infection. MAIN BODY: In this review, we first outline the two major (R gene-mediated and RNA silencing) viral resistance mechanisms in plants. We also explain the phenomenon of mutations of host factors to function as recessive resistance genes, taking the eIF4E genes as examples. We then focus on a new type of virus resistance that has been repeatedly reported recently due to the widespread use of genome editing technology in plants, facilitating the specific knockdown of host factors. Here, we show that (1) an in-frame mutation of host factors necessary to confer viral resistance, sometimes resulting in resistance to different viruses and that (2) certain host factors exhibit antiviral resistance and viral-supporting (proviral) properties. CONCLUSION: A detailed understanding of the host factor functions would enable the development of strategies for the induction of a new type of viral resistance, taking into account the provision of a broad resistance spectrum and the suppression of the appearance of resistance-breaking strains.

Artificially Edited Alleles of the Eukaryotic Translation Initiation Factor 4E1 Gene Differentially Reduce Susceptibility to Cucumber Mosaic Virus and Potato Virus Y in Tomato.

Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.

RNA silencing-related genes contribute to tolerance of infection with potato virus X and Y in a susceptible tomato plant.

BACKGROUND: In plants, the RNA silencing system functions as an antiviral defense mechanism following its induction with virus-derived double-stranded RNAs. This occurs through the action of RNA silencing components, including Dicer-like (DCL) nucleases, Argonaute (AGO) proteins, and RNA-dependent RNA polymerases (RDR). Plants encode multiple AGOs, DCLs, and RDRs. The functions of these components have been mainly examined in Arabidopsis thaliana and Nicotiana benthamiana. In this study, we investigated the roles of DCL2, DCL4, AGO2, AGO3 and RDR6 in tomato responses to viral infection. For this purpose, we used transgenic tomato plants (Solanum lycopersicum cv. Moneymaker), in which the expression of these genes were suppressed by double-stranded RNA-mediated RNA silencing. METHODS: We previously created multiple DCL (i.e., DCL2 and DCL4) (hpDCL2.4) and RDR6 (hpRDR6) knockdown transgenic tomato plants and here additionally did multiple AGO (i.e., AGO2 and AGO3) knockdown plants (hpAGO2.3), in which double-stranded RNAs cognate to these genes were expressed to induce RNA silencing to them. Potato virus X (PVX) and Y (PVY) were inoculated onto these transgenic tomato plants, and the reactions of these plants to the viruses were investigated. In addition to observation of symptoms, viral coat protein and genomic RNA were detected by western and northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). Host mRNA levels were investigated by quantitative RT-PCR. RESULTS: Following inoculation with PVX, hpDCL2.4 plants developed a more severe systemic mosaic with leaf curling compared with the other inoculated plants. Systemic necrosis was also observed in hpAGO2.3 plants. Despite the difference in the severity of symptoms, the accumulation of PVX coat protein (CP) and genomic RNA in the uninoculated upper leaves was not obviously different among hpDCL2.4, hpRDR6, and hpAGO2.3 plants and the empty vector-transformed plants. Moneymaker tomato plants were asymptomatic after infection with PVY. However, hpDCL2.4 plants inoculated with PVY developed symptoms, including leaf curling. Consistently, PVY CP was detected in the uninoculated symptomatic upper leaves of hpDCL2.4 plants through western blotting. Of note, PVY CP was rarely detected in other asymptomatic transgenic or wild-type plants. However, PVY was detected in the uninoculated upper leaves of all the inoculated plants using reverse transcription-polymerase chain reactions. These findings indicated that PVY systemically infected asymptomatic Moneymaker tomato plants at a low level (i.e., no detection of CP via western blotting). CONCLUSION: Our results indicate that the tomato cultivar Moneymaker is susceptible to PVX and shows mild mosaic symptoms, whereas it is tolerant and asymptomatic to systemic PVY infection with a low virus titer. In contrast, in hpDCL2.4 plants, PVX-induced symptoms became more severe and PVY infection caused symptoms. These results indicate that DCL2, DCL4, or both contribute to tolerance to infection with PVX and PVY. PVY CP and genomic RNA accumulated to a greater extent in DCL2.4-knockdown plants. Hence, the contribution of these DCLs to tolerance to infection with PVY is at least partly attributed to their roles in anti-viral RNA silencing, which controls the multiplication of PVY in tomato plants. The necrotic symptoms observed in the PVX-infected hpAGO2.3 plants suggest that AGO2, AGO3 or both are also distinctly involved in tolerance to infection with PVX.

Apple latent spherical virus (ALSV)-induced gene silencing in a medicinal plant, Lithospermum erythrorhizon.

Lithospermum erythrorhizon is a medicinal plant that produces shikonin, a red lipophilic naphthoquinone derivative that accumulates exclusively in roots. The biosynthetic steps required to complete the naphthalene ring of shikonin and its mechanism of secretion remain unclear. Multiple omics studies identified several candidate genes involved in shikonin production. The functions of these genes can be evaluated using virus-induced gene silencing (VIGS) systems, which have been shown advantageous in introducing iRNA genes into non-model plants. This study describes the development of a VIGS system using an apple latent spherical virus (ALSV) vector and a target gene, phytoene desaturase (LePDS1). Virus particles packaged in Nicotiana benthamiana were inoculated into L. erythrorhizon seedlings, yielding new leaves with albino phenotype but without disease symptoms. The levels of LePDS1 mRNAs were significantly lower in the albino plants than in mock control or escape plants. Virus-derived mRNA was detected in infected plants but not in escape and mock plants. Quantitative PCR and deep sequencing analysis indicated that transcription of another hypothetical PDS gene (LePDS2) also decreased in the defective leaves. Virus infection, however, had no effect on shikonin production. These results suggest that virus-based genetic transformation and the VIGS system silence target genes in soil-grown L. erythrorhizon.

Virus-Induced Gene Silencing in Lilies Using Cucumber Mosaic Virus Vectors.

Virus-induced gene silencing (VIGS) systems are effective for rapid analysis of gene functions in plants that require a long period of growth such as Lilium. We successfully developed a VIGS system using the cucumber mosaic virus (HL strain, CMV-HL) vector to induce RNA silencing of the L. leichtlinii phytoene desaturase gene (LlPDS), where at 30 days postinoculation (dpi), photo-bleaching was observed in the upper leaves of L. leichtlinii, and at 57 dpi, white regions appeared on flower tepals that accumulate orange carotenoids. This vector spreads in bulbs, and it could induce silencing on emerged shoots in the following year. The CMV-HL vector can be easily constructed by insertion of a 30-60 nt fragment into the cloning site of the RNA3 genome. In this chapter, we describe how to use the CMV-HL vector system in the context of Lilium plants.

Application of Virus-Induced Gene Silencing to Arbuscular Mycorrhizal Fungi.

Downregulation of AM fungal genes using a plant viral vector is feasible. A partial sequence of a target fungal gene is cloned into the multicloning site of CMV2-A1 vector developed from RNA2 of Cucumber mosaic virus Y strain, and the RNA2, together with RNA1 and RNA3 of the virus, are in vitro-transcribed. Inoculation of Nicotiana benthamiana with these viral RNAs results in reconstitution of the virus in the plant, which triggers silencing of the fungal gene. Here, we describe not only the methods but also several tips for successful application of virus-induced gene silencing to AM fungi.

Application of cucumber mosaic virus to efficient induction and long-term maintenance of virus-induced gene silencing in spinach.

Virus-induced gene silencing (VIGS) is a useful tool for functional genomics in plants. In this study, we tried to apply cucumber mosaic virus (CMV) to efficient induction of VIGS in spinach. Although VIGS for spinach had been previously developed based on two viruses (beet curly top virus and tobacco rattle virus), they still have some problems with systemic movement and long-term maintenance of VIGS in spinach. Although ordinary CMV strains infect spinach inducing distinct mosaic symptoms, using a CMV pseudorecombinant, we can modify the viral pathogenicity to attenuate viral symptoms that may mask the silencing phenotype. We here successfully demonstrated the viral ability to silence the phytoene desaturase (PDS) and the dihydroflavonol 4-reductase (DFR) genes in spinach. Because CMV could quickly induce VIGS even at 7-10 days postinoculation and the virus did not disappear even at the flowering stage, this CMV-based VIGS system would contribute to functional genomics in spinach and especially to the elucidation of molecular mechanisms for some properties unique to spinach such as plasticity of sex expression; the CMV-induced VIGS can last until the flowering stage after the virus was inoculated onto the seedling.