Seed transmission of raspberry bushy dwarf virus is blocked in Nicotiana benthamiana plants by preventing virus entry into the embryo from the infected embryo sac and endosperm.

Raspberry bushy dwarf virus (RBDV) is transmitted through seed in infected red raspberry plants after pollination with pollen grains from healthy red raspberry plants. Here, we show that RBDV is not transmitted through seeds in infected Nicotiana benthamiana (Nb) plants after pollination with virus-free Nb pollen grains. Chromogenic in situ hybridization revealed that the virus invades the shoot apical meristem and the ovule, including the embryo sac, of RBDV-infected Nb plants; however, in seeds that developed from infected embryo sacs after fertilization by virus-free sperm cells, RBDV was absent in the embryos and present in the endosperms. When we analyzed seed transmission of RBDV in Nb mutants with mutations in dicer-like enzyme 2 and 4 (NbDCL2&4) or RNA-dependent RNA polymerase 6 (NbRDR6), RBDV was not present in the offspring from seeds with embryos and endosperms that did not express NbDCL2&4 or NbRDR6. These results suggest that seed transmission of RBDV is prevented by evasion of infection by the embryo and that RNA silencing is not essential for preventing seed transmission of RBDV in Nb plants.

Characterization of horizontal transmission of blueberry latent spherical virus by pollen.

Nicotiana benthamiana plants became infected with blueberry latent spherical virus (BLSV) after pollination with pollen grains produced by BLSV-infected N. benthamiana plants. Interestingly, pollen grains produced by BLSV-infected Vaccinium corymbosum (blueberry), Nicotiana alata, and Petunia × hybrida (petunia) plants also transmitted the virus to healthy N. benthamiana plants after pollination. As seen using aniline blue staining and fluorescence microscopy, pollen grains from BLSV-infected blueberry, N. alata, and petunia plants germinated on stigmas of N. benthamiana, and the pollen tubes penetrated the stigmas in a manner similar to that of N. benthamiana pollen grains on N. benthamiana stigmas. Whole-mount in situ hybridization and chromogenic in situ hybridization analysis showed that infected blueberry and N. benthamiana pollen grains germinated on N. benthamiana stigmas, and virus-containing pollen tubes penetrated the stigmas. Tissue blot hybridization analysis revealed that the initial infection sites were the N. benthamiana stigmas pollinated with infected pollen grains from blueberry and N. benthamiana. In addition, the virus spread from the initial infection sites to the phloem in the stigma and style. Taken together, we suggest that penetrating pollen tubes that harbored the virus results in infection foci in the stigma, and the virus then moves to the vascular tissues in the stigma and style and eventually establishes systemic infection.

Apple latent spherical virus structure with stable capsid frame supports quasi-stable protrusions expediting genome release.

Picorna-like plant viruses are non-enveloped RNA spherical viruses of ~30 nm. Part of the survival of these viruses depends on their capsid being stable enough to harbour the viral genome and yet malleable enough to allow its release. However, molecular mechanisms remain obscure. Here, we report a structure of a picorna-like plant virus, apple latent spherical virus, at 2.87 Å resolution by single-particle cryo-electron microscopy (cryo-EM) with a cold-field emission beam. The cryo-EM map reveals a unique structure composed of three capsid proteins Vp25, Vp20, and Vp24. Strikingly Vp25 has a long N-terminal extension, which substantially stabilises the capsid frame of Vp25 and Vp20 subunits. Cryo-EM images also resolve RNA genome leaking from a pentameric protrusion of Vp24 subunits. The structures and observations suggest that genome release occurs through occasional opening of the Vp24 subunits, possibly suppressed to a low frequency by the rigid frame of the other subunits.

The raspberry bushy dwarf virus 1b gene enables pollen grains to function efficiently in horizontal pollen transmission.

Horizontal pollen transmission by the raspberry bushy dwarf virus 1b deletion mutant (RBΔ1bstop), which is defective in virus virulence, was significantly decreased compared to wild-type raspberry bushy dwarf virus (wtRBDV). We assessed accumulation of viral genomic (g) RNAs in pollen grains from RBΔ1bstop-infected plants and found that the pollen grains had less viral gRNA than those from wtRBDV-infected plants. In addition, pollen grains from 1b-expressing transgenic plants (1b-plants) infected with RBΔ1bstop were more efficient in horizontal virus transmission to healthy plants after pollination than pollen from RBΔ1bstop-infected wild type plants. Moreover, viral gRNA accumulation in pollen grains from RBΔ1bstop-infected 1b-plants was higher than in pollen from RBΔ1bstop-infected wild type plants. We suggest that 1b increases the amount of viral gRNAs released from elongating pollen grains.

The 1b gene of raspberry bushy dwarf virus is a virulence component that facilitates systemic virus infection in plants.

A product translated from the 1b gene of raspberry bushy dwarf virus (RBDV) was specifically detected in RBDV-infected Nicotiana benthamiana plants by immunoblot analysis. To analyze the effects of the 1b gene on virus infection in host plants, an RBDV deletion mutant virus (RB∆1bstop), which is unable to express the 1b gene, was constructed and inoculated to N. benthamiana plants. The results showed that accumulation of the virus genomic (g) RNAs 1 and 2 decreased in inoculated leaves, and that systemic virus spread was delayed compared with wild-type RBDV. In contrast, accumulation of the viral gRNAs 1 and 2 was elevated in RB∆1bstop-infected leaf tissues during ectopic expression of the 1b gene. Furthermore, we found that the 1b has weak RNA silencing suppressor activity.

Horizontal pollen transmission of Gentian ovary ring-spot virus is initiated during penetration of the stigma and style by infected pollen tubes.

Gentian ovary ring-spot virus (GORV) infected gentian plants by pollination with GORV-infected gentian pollen grains, but the virus was not horizontally transmitted to gentian plants by transfer of pollen from GORV-infected Nicotiana benthamiana plants. However, N. benthamiana plants were infected with the virus by pollination with infected gentian pollen as well as by pollination with infected N. benthamiana pollen. When infected gentian pollen grains were placed on N. benthamiana stigmas, germinating pollen tubes penetrated into the stigmas and the styles (stigma-style). Virus infection occurred during penetration of the stigma-style, and the virus subsequently spread systemically to the mother plant. On the other hand, most infected N. benthamiana pollen grains failed to germinate on gentian stigmas, and virus infections were not detected in the stigma-style.

Pollen tubes introduce Raspberry bushy dwarf virus into embryo sacs during fertilization processes.

We developed a fertilization method in which pollen tubes entered into embryo sacs without any need to contact surrounding female sporophytic cells by using Torenia fournieri (Torenia) plants under the condition of hindering movement of the virus from a stigma, which is the first infection site leading to systemic infection. When RBDV-infected Torenia pollen grains were used for the developed fertilization method, the virus was transmitted to the seeds by pollen tubes germinating from them. On the other hand, no seeds were infected with the virus when Torenia plants were pollinated with healthy Torenia pollen grains in combination with RBDV-infected raspberry pollen grains, which caused the virus infection in the stigma by penetration of their pollen tubes arrested in its style. Our results indicate that vertical transmission of RBDV by pollen occurs in the transport of the virus into embryo sacs by pollen tubes reaching the embryo sacs.

Penetration of pollen tubes with accumulated Raspberry bushy dwarf virus into stigmas is involved in initial infection of maternal tissue and horizontal transmission.

Torenia fournieri (Torenia) plants were infected with Raspberry bushy dwarf virus (RBDV) by pollination with RBDV-infected raspberry pollen grains. The infected raspberry pollen grains germinated on Torenia stigmas, and then the pollen tubes penetrated into the stigma, even though the pollen tubes were arrested in the styles. In whole-mount in situ hybridization of germinating infected raspberry pollen grains, RBDV accumulated in the tips of the pollen tubes. Tissue blot hybridization of Torenia plants pollinated with infected raspberry pollen grains revealed that the first virus infection site leading to systemic infection is the stigma. When infected raspberry pollen grains that had lost germination capacity were pollinated on Torenia stigmas, RBDV could not infect the stigmas, and no horizontal transmission occurred. These results indicate that penetration of pollen tubes with accumulated RBDV into stigmas is essential in causing the first viral infection in the stigma to lead to systemic infection.

Characterization of plant virus-encoded gene silencing suppressors.

Agroinfiltration assay using green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c is a powerful method for screening of putative plant virus-encoded gene silencing suppressors. This method allows the investigator to know whether the putative viral suppressor inhibits silencing in a cell (local silencing) and/or spreading of silencing throughout a plant (systemic silencing). Additionally, grafting experiments using transgenic plants expressing the suppressor and the GFP will indicate whether the suppressor blocks systemic silencing steps, which include the production of a silencing signal in a silenced cell, and the cell-to-cell and long-distance movement of a silencing signal throughout a plant. Here, we describe methods and techniques of an agroinfiltration assay and grafting experiments, which were used for the characterization of Apple chlorotic leaf spot virus 50 kDa movement protein as a gene silencing suppressor. This protocol should allow the investigator to characterize putative plant virus-encoded gene silencing suppressors.

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 30 nm in diameter, which were composed of a single coat protein (CP) of 55 kDa. 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 1,379 nt and 1,392 nt 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.

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.

Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement protein.

Apple chlorotic leaf spot virus 50 kDa movement protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.

Combinations of two amino acids (Ala40 and Phe75 or Ser40 and Tyr75) in the coat protein of apple chlorotic leaf spot virus are crucial for infectivity.

Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala(40)-Val(59)-Phe(75)-Ser(130)-Met(184) or Ser(40)-Leu(59)-Tyr(75)-Thr(130)-Leu(184)) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala(40) and Phe(75) or Ser(40) and Tyr(75)) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala(40) to Ser(40) or Phe(75) to Tyr(75)) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.

Apple chlorotic leaf spot virus 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana.

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single- and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals.

Analysis of the spatial distribution of identical and two distinct virus populations differently labeled with cyan and yellow fluorescent proteins in coinfected plants.

ABSTRACT Apple latent spherical virus (ALSV) expressing yellow and cyan fluorescent proteins (ALSV-YFP and ALSV-CFP) was used to investigate the distribution of identical virus populations in coinfected plants. In Chenopodium quinoa plants inoculated with a mixture of ALSV-YFP and ALSV-CFP, fluorescence from YFP and CFP was always distributed separately in both inoculated and upper uninoculated leaves. Inoculation of each ALSV-YFP and ALSV-CFP to different leaves of a C. quinoa plant resulted in the separate distribution of each virus population among different upper leaves. When C. quinoa leaves were first inoculated with ALSV-CFP and then ALSV-YFP was reinoculated into the same leaves at various times after the first inoculation, ALSV-YFP infected only tissues where ALSV-CFP infection had not been established. The spatial separation was also found in Nicotiana benthamiana leaves coinoculated with Bean yellow mosaic virus (BYMV)-YFP and BYMV-CFP. In contrast, both YFP and CFP fluorescence signals were observed in the same tissues of N. benthamiana leaves mixed infected with ALSV-YFP and BYMV-CFP. YFP fluorescence from ALSV-YFP in mixed-infected leaves was brighter and longer than in leaves infected with ALSV-YFP singly.

Protein-protein- and protein-RNA-binding properties of the movement protein and VP25 coat protein of Apple latent spherical virus.

To elucidate the mechanism of Apple latent spherical virus (ALSV) movement, various properties of its cell-to-cell movement protein (MP) were analyzed. ELISA and blot overlay assays demonstrated that the MP bound specifically to ALSV virions and in particular to one of the three coat proteins (VP25) but not to the other two coat proteins (VP20 and VP24). Mutational analyses have revealed that the MP contains two domains with independent VP25-binding activity (amino acid residues 1-188 and 189-281). Furthermore, nucleotide-binding experiments showed that the MP and VP25 bound to single-stranded RNA (ssRNA) and ssDNA without any sequence specificity, but these two proteins did not bind to double-stranded RNA (dsRNA) and dsDNA. The MP contains three potentially independent single-stranded nucleic acid-binding domains between amino acid residues 95-188, 189-281 and 277-376. The MP demonstrated cooperative and VP25 demonstrated non-cooperative binding to ssRNA in gel-retardation analyses. The cooperative RNA binding of the MP became non-cooperative when MP and VP25 were tested together in competition binding experiments, even though a sufficient amount of the MP for fully cooperative RNA binding the MP was supplied. The roles of the MP and VP25 interactions and nucleic acid binding activities in ALSV movement are discussed.

Mapping the RNA-binding domain on the Apple chlorotic leaf spot virus movement protein.

The RNA-binding properties of the cell-to-cell movement protein (MP) of Apple chlorotic leaf spot virus were analysed. MP was expressed in Escherichia coli and was used in UV-crosslinking analysis, using a digoxigenin-UTP-labelled RNA probe and gel-retardation analysis. The analyses demonstrated that MP bound cooperatively to single-stranded RNA (ssRNA). When analysed for NaCl dependence of the RNA-binding activity, the majority of the MP could bind ssRNA even in binding buffer with 1 M NaCl. Furthermore, competition binding experiments showed that the MP bound preferentially to ssRNA and single-stranded DNA without sequence specificity. MP deletion mutants were used to identify the RNA-binding domain by UV-crosslinking analysis. Amino acid residues 82-126 and 127-287 potentially contain two independently active, single-stranded nucleic acid-binding domains.

Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein.

The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP-GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287-457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.