Masamichi Isogai

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.

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.

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.

Characterization of virus-induced gene silencing in tobacco plants infected with apple latent spherical virus

SummaryApple latent spherical virus (ALSV) expressing green fluorescent protein (GFP-ALSV) was used for analysis of virus-induced gene silencing (VIGS) in tobacco plants expressing GFP (GFP-tobacco). In GFP-tobacco inoculated with GFP-ALSV, small dark spots appeared on inoculated leaves at 5 days post-inoculation (dpi), then expanded, and finally covered the whole area of the leaves after 12 dpi. Most of the fluorescence of upper leaves above the 12th true leaf disappeared at 21 dpi. Thus, GFP-ALSV infection efficiently triggered VIGS of a transgene (GFP gene) in tobacco plants. Analysis of GFP-silenced leaves showed that viral RNAs and proteins accumulated in all leaves where most GFP mRNA had been degraded. The siRNAs derived from ALSV-RNAs were not detected in samples from which siRNA of GFP mRNA could be easily detected. Direct tissue blot analysis showed that the spread of GFP-ALSV always preceded the induction of VIGS in infected leaves of GFP-tobacco. GFP leaf patch tests using Nicotiana benthamiana line 16c showed that Vp20, one of the three capsid proteins, is a silencing suppressor which interferes with systemic silencing.

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.

Molecular detection of five cherry viruses from sweet cherry trees in Japan

To investigate the occurrence of viruses in sweet cherry in Japan, leaf samples were collected from sweet cherry trees in Aomori, Iwate, and Yamagata prefectures. Analysis of dsRNAs from sweet cherry leaves showed that 73% of 49 samples contained dsRNAs more than 6 kbp in size. Reverse transcription-polymerase chain reaction analysis of cherry viruses indicated that 14% of the samples had Little cherry virus-1, 65% had Little cherry virus-2, 14% had Cherry necrotic rusty mottle virus, 49% had Cherry virus A, and 92% had Cherry green ring mottle virus. This is the first molecular detection of these five cherry viruses in Japan.

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.

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.

Complete nucleotide sequence of the rice tungro spherical virus genome of the highly virulent strain Vt6.

The complete nucleotide sequence of rice tungro spherical virus (RTSV) strain Vt6, originally from Mindanao, the Philippines, with higher virulence to resistant rice cultivars, was determined and compared with the published sequence for the Philippine-type strain A (RTSV-A-Shen). It was reported that RTSV-A was not able to infect a rice resistant cultivar TKM 6 (10). RTSV-Vt6 and RTSV-A-Shen share 90% and 95% homology at nucleotide and amino-acid levels, respectively. The N-terminal leader sequence of RTSV-Vt6 contained a 39-amino acids-region (positions 65 to 103) which was totally different from that of RTSV-A-Shen; the difference resulted from frame shifting by nucleotide insertions and deletions. To confirm the amino-acid sequence differences of the leader polypeptide, the same region was cloned and sequenced using a newly obtained variant of RTSV-type 6, which had been collected in the field of IRRI, and seven field isolates from Mindanao, the Philippines. Since all the sequences of the target region are identical to that of the Vt6 leader polypeptide, the sequence difference in the leader region seems not to correlate with the virulence of Vt6.

First report of blueberry red ringspot disease caused by Blueberry red ringspot virus in Japan

Virus-like symptoms—red ringspots on stems and leaves, circular blotches or pale spots on fruit—were found on commercial highbush blueberry (Vacciniumcorymbosum) cultivars Blueray, Weymouth, Duke and Sierra in Japan. In PCR testing, single DNA fragments were amplified from total nucleic acid samples of the diseased blueberry bushes using primers specific to Blueberry red ringspot virus (BRRV). Sequencing analysis of the amplified products revealed 95.7–97.7% nucleotide sequence identity with the BRRV genome. This paper is the first report of blueberry red ringspot disease caused by BRRV in Japan.