Characterization of a ToMV isolate overcoming Tm-22 resistance gene in tomato.

Tomato mosaic virus (ToMV) is easily transmitted in soil and by contact. By these reasons, it is relatively difficult to control ToMV disease in tomato. Incorporation of the Tm-22 gene has been widely used as a control method for ToMV, but ToMV isolates that overcome this resistance gene have been reported worldwide in recent years. In this study, we determined the entire nucleotide sequences of ToMV isolate [named ToMV-KMT (LC650928)], which was isolated from tomato plants showing symptoms of systemic necrosis in Kumamoto prefecture, Japan. We also analyzed the viral gene of ToMV-KMT that overcome the Tm-22 gene by constructing its infectious cDNA clone and by generating chimeric viruses with a non-breaking strain. According to previous research, Tm-22 recognizes the viral movement protein (MP) and exerts resistance by inducing hypersensitive reaction or hypersensitive cell death. We discovered that a mutation in the 240th amino acid (aspartic acid to tyrosine) of the MP of ToMV-KMT, which may stabilize the protein's structure, is responsible for the ability of this isolate to overcome the resistance of Tm-22.

Characterization of tulip streak virus, a novel virus associated with the family Phenuiviridae.

In Japan, tulip-growing areas have been plagued by viral diseases for decades, but the viruses causing the damage remain undescribed. In this study, Nicotiana benthamiana and Chenopodium quinoa plants mechanically inoculated with crude sap from a symptomatic tulip flower exhibited necrosis symptoms. Additionally, flexuous and filamentous virus particles were detected by electron microscopy analysis. Moreover, we determined the complete sequences of two genomic segments of the tulip streak virus (TuSV), which is a new virus associated with streaking symptoms, on the basis of a next-generation sequencing analysis. Homology analyses of the amino acid sequence of RNA-dependent RNA polymerase and the terminal sequence of the genomic RNA indicated that TuSV is associated with viruses in the family Phenuiviridae, but differs substantially from other reported viruses.

Construction of an infectious dahlia common mosaic virus clone.

Dahlia is a major ornamental plant that is cultivated worldwide. However, dahlia plants, which are mainly propagated through vegetative reproduction, are susceptible to widespread damage by viruses, and viral control requires that the nature of the infecting virus(es) be known. In this study, dahlia common mosaic virus (DCMV) was detected for the first time in Japan and sequenced. This is the first report of an infectious DCMV clone being constructed, and it will aid in the characterization of DCMV.

Utility of a GFP-expressing Barley yellow mosaic virus for analyzing disease resistance genes.

The soil-borne plasmodiophorid Polymyxa graminis is a vector for Barley yellow mosaic virus (BaYMV), which can severely damage barley plants. Although 22 disease resistance genes have been identified, only a few have been used for breeding virus-resistant cultivars. Recently, BaYMV strains capable of overcoming the effects of some of these genes have been detected. In this study, green fluorescent protein (GFP)-expressing BaYMV was constructed and used to examine viral dynamics in inoculated barley plants. Leaf inoculations resulted in higher infection rates than root or crown inoculations. Additionally, inoculations of some resistant cultivars produced infections that were similar to those observed in a field test. The results of this study indicate that the GFP-expressing virus is a useful tool for visualizing virus replication and dynamics, and for understanding resistance mechanisms.

Detection of Fusarium oxysporum f. sp. fragariae by Using Loop-Mediated Isothermal Amplification.

We developed a loop-mediated isothermal amplification (LAMP) assay for detecting Fusarium oxysporum f. sp. fragariae, the causal agent of wilt in strawberry plants. This assay was based on genomic regions between the portions of transposable elements Han and Skippy of the fungus. The LAMP assay allowed the efficient detection of F. oxysporum f. sp. fragariae DNA by visual inspection, without requiring gel electrophoresis. The detection limit was 100 pg of genomic DNA, which is comparable to that of PCR. The LAMP primers successfully discriminated F. oxysporum f. sp. fragariae strains from nonpathogenic F. oxysporum strains and other fungi. The LAMP assay at 63°C, which was found to be the optimal treatment temperature, for 1.5 h successfully detected F. oxysporum f. sp. fragariae California strains GL1270 and GL1385. When the assay was performed using a Genelyzer FIII portable fluorometer, these California strains were successfully detected in 1 h. The assay facilitated the detection of conidia in soil samples after they were precultured on a selective medium for F. oxysporum (FoG2) as well as latent infection in strawberry plants after preculturing. The LAMP assay for visual inspection of DNA required only a heating block and an incubator, reducing the cost of this assay. Thus, it could be suitable for the detection of F. oxysporum f. sp. fragariae strains in centers that store prefoundation and foundation stocks of strawberry, including plant nurseries.

ICTV Virus Taxonomy Profile: Ophioviridae.

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.

Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato.

Yellowing symptoms caused by tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), both assigned to the genus Crinivirus, resemble nutrient deficiencies. Therefore, early diagnosis of infections will prevent crop damage and the spread of the viruses. In this study, we established a rapid detection method for ToCV and TICV by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). We first designed primer sets for RT-LAMP specific for ToCV and TICV. Next, by selecting the optimum primer set and determining the optimum conditions for the RT-LAMP reaction, each virus was detected within 50 min by piercing the diseased area of a tomato leaf with a toothpick, immersing the toothpick in the reaction solution, and conducting the RT-LAMP reaction. To verify the accuracy of the procedure, 61 tomato leaf samples showing disease symptoms were collected from five regions of Indonesia, and the RT-LAMP results for the samples were identical to those obtained with the commonly used reverse transcription-polymerase chain reaction.

Infection dynamics in viral spread and interference under the synergism between Cucumber mosaic virus and Turnip mosaic virus.

Mixed infection of Cucumber mosaic virus (CMV) and Turnip mosaic virus (TuMV) induced more severe symptoms on Nicotiana benthamiana than single infection. To dissect the relationships between spatial infection patterns and the 2b protein (2b) of CMV in single or mixed infections, the CMV vectors expressing enhanced green fluorescent or Discosoma sp. red fluorescent proteins (EGFP [EG] or DsRed2 [Ds], respectively were constructed from the same wild-type CMV-Y and used for inoculation onto N. benthamiana. CMV2-A1 vector (C2-A1 [A1]) has a functional 2b while CMV-H1 vector (C2-H1 [H1]) is 2b deficient. As we expected from the 2b function as an RNA silencing suppressor (RSS), in a single infection, A1Ds retained a high level of accumulation at initial infection sites and showed extensive fluorescence in upper, noninoculated leaves, whereas H1Ds disappeared rapidly at initial infection sites and could not spread efficiently in upper, noninoculated leaf tissues. In various mixed infections, we found two phenomena providing novel insights into the relationships among RSS, viral synergism, and interference. First, H1Ds could not spread efficiently from vasculature into nonvascular tissues with or without TuMV, suggesting that RNA silencing was not involved in CMV unloading from vasculature. These results indicated that 2b could promote CMV to unload from vasculature into nonvascular tissues, and that this 2b function might be independent of its RSS activity. Second, we detected spatial interference (local interference) between A1Ds and A1EG in mixed infection with TuMV, between A1Ds (or H1Ds) and TuMV, and between H1Ds and H1EG. This observation suggested that local interference between two viruses was established even in the synergism between CMV and TuMV and, again, RNA silencing did not seem to contribute greatly to this phenomenon.

Quick adaptation of Ralstonia Solanacearum to copper stress to recover culturability and growth in water and soil.

Cells of Ralstonia solanacearum were exposed to Cu in distilled water, and the resulting Cu-stressed non-culturable cells were inoculated to natural (non-pasteurized) and pasteurized soils in order to examine their culturability and recovery. Exposing the cells to 20 µM CuSO4 produced transitory non-culturable cells, which exhibited a remarkable recovery in culturability after incubation in the solution for 36 h, reaching a density near the initial level by 108 h. To determine whether such non-culturable cells actually "resuscitated" or multiplied after adapting to Cu toxicity, growth curves were constructed in order to contrast the rates of increase in culturable cell numbers between Cu-stressed or non-stressed inocula. Additionally, fresh non-stressed cells were exposed to CuSO4 in the presence of nalidixic acid by adding the antibiotic at different times after the onset of Cu stress to verify any cell multiplication during the population increase. The results revealed that the non-culturable cells surviving Cu toxicity adapted very quickly to Cu and began multiplying within 12 h, because only the Cu-stressed cells that were increasing in the exponential growth phase, but not those in the stationary phase, were killed by the antibiotic. Such cells exhibited an apparent tolerance to this metal when inoculated to a freshly prepared solution of CuSO4, and also detoxified the ion in the solution in which they grew. The presence of nutrients greatly counteracted the effect of Cu in water microcosms, since culturable cells were detected and increased in number even when exposed to 40 µM CuSO4. In contrast, when inoculated to non-pasteurized soil, Cu-stressed cells showed no such recoveries. However, when the soil was pasteurized before inoculation or added with nutrients, culturable cells were recovered and increased in number. This indicates that increased nutrient availability in soil allows Cu-stressed cells to quickly overcome the stress and increase in culturable populations.

The P3 protein of turnip mosaic virus can alone induce hypersensitive response-like cell death in Arabidopsis thaliana carrying TuNI.

Strains TuR1 and TuC of Turnip mosaic virus (TuMV) induce different symptoms on Arabidopsis thaliana ecotype Landsberg erecta (Ler); plants infected with TuR1 develop systemic necrosis, while TuC causes mosaics. We previously found that the Ler systemic necrosis was controlled by a single dominant gene, TuNI (TuMV necrosis inducer), and that it was actually a form of host defense response leading to a hypersensitive reaction (HR)-like cell death. To identify the viral factor interacting with TuNI, the domain swapping between the genomic clones of TuR1 and TuC was carried out, and we identified the TuMV symptom determinant interacting with TuNI as the P3 gene. Moreover, it was found that the central 0.5-kb domain of P3, including three different amino acids between the two isolates, was responsible for the systemic HR. To verify that the P3 gene can alone induce necrosis, we analyzed the constitutive P3 expression in Ler transgenic plants and the transient P3 expression in Ler protoplasts. These results indicated that P3 alone caused HR-like cell death. In this study, we successfully demonstrated that the systemic necrosis by TuMV in Arabidopsis was determined by the gene-for-gene interaction between TuNI and P3 using the protoplast system for direct verification.

Complete Genome Sequence of a Pepper Yellow Leaf Curl Indonesia Virus Isolated from Tomato in Bali, Indonesia.

We report a complete genome sequence of a pepper yellow leaf curl Indonesia virus (PepYLCIV) isolated in Bali, Indonesia. This virus shares around 90% identity with other PepYLCIV DNA-As and 86% identity with DNA-Bs, suggesting that it is a novel isolate of PepYLCIV.

Cross-protection against bean yellow mosaic virus (BYMV) and clover yellow vein virus by Attenuated BYMV isolate M11.

Attenuated isolate M11 of Bean yellow mosaic virus (BYMV), obtained after exposing BYMV-infected plants to low temperature, and its efficacy in cross-protecting against infection by BYMV isolates from gladiolus, broad bean (Vicia faba) and white clover (Trifolium repens) was assessed with western blotting and reverse transcription-polymerase chain reaction. The level of cross-protection varied depending on the challenge virus isolates. Cross-protection was complete against BYMV isolates from gladiolus, but incomplete against BYMV isolates from other hosts. M11 also partially cross-protected against an isolate of Clover yellow vein virus. A comparison of the nucleotide sequence of M11 and those of BYMV isolates from gladiolus and from other hosts showed higher homology among gladiolus isolates than the homology between gladiolus isolates and nongladiolus isolates. In the phylogenetic trees, constructed using the nucleotide sequences of an overall polyprotein of the genomes, five gladiolus isolates clustered together, completely separated from the three BYMV isolates from other hosts. A comparison of the amino acid sequences between M11 and its parental isolate IbG, and analysis of recombinant infectious clones between M11 and IbG revealed that an amino acid at position 314 was involved in the attenuation of BYMV.

Complete Genome Sequence of Tomato Leaf Curl New Delhi Virus from Luffa in Indonesia.

This is the first report of a begomovirus infecting luffa in Indonesia. The genome of this virus shares a close identity with that of Tomato leaf curl New Delhi virus (ToLCNDV). There is a 36-nucleotide duplicated sequence in the DNA-B component, suggesting the occurrence of an intraviral recombination.

Development of a LAMP assay with a portable device for real-time detection of begomoviruses under field conditions.

The emergence of begomovirus infection is one of the most important problems affecting production of a variety of vegetable crops worldwide. Infection by begomoviruses has been detected and spread rapidly on Cucurbitaceae and Solanaceae plants in Indonesia. A rapid and simple detection assay for begomoviruses under field conditions for routine sampling of plants is needed. Primers for a loop-mediated isothermal amplification (LAMP) assay were designed based on the sequences of three Indonesian begomoviruses, namely Tomato leaf curl New Delhi virus (ToLCNDV), Pepper yellow leaf curl Indonesia virus (PepYLCIV), and Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), infecting Cucurbitaceae and Solanaceae plants. LAMP assays using a Genelyzer™ III portable fluorometer with a toothpick method successfully detected these begomoviruses in infected melon, pepper, and eggplant samples. LAMP assays conducted during a field survey for detection of the three begomoviruses on 104 fresh leaves indicated that most of the samples were positive; the findings were confirmed by PCR using universal primers of begomovirus as a common detection method. These results demonstrate that this simple and rapid LAMP assay using a fluorometer portable device may be used to achieve real-time detection of begomoviruses under field conditions.

Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF(iso)4E and inhibits in vitro translation.

The interaction between turnip mosaic virus (TuMV) viral protein linked to the genome (VPg) and Arabidopsis thaliana eukaryotic initiation factor (iso)4E (eIF(iso)4E) was investigated to address the influence of potyviral VPg on host cellular translational initiation. Affinity chromatographic analysis showed that the region comprising amino acids 62-70 of VPg is important for the interaction with eIF(iso)4E. In vitro translation analysis showed that the addition of VPg significantly inhibited translation of capped RNA in eIF(iso)4E-reconstituted wheat germ extract. This result indicates that VPg inhibits cap-dependent translational initiation via binding to eIF(iso)4E. The inhibition by VPg of in vitro translation of RNA with wheat germ extract did not depend on RNase activity. Our present results may indicate that excess VPg produced at the encapsidation stage shuts off cap-dependent translational initiation in host cells by inhibiting complex formation between eIF(iso)4E and cellular mRNAs.

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.

Binding analyses for the interaction between plant virus genome-linked protein (VPg) and plant translational initiation factors.

The turnip mosaic virus (TuMV) genome-linked protein (VPg) and Arabidopsis thaliana translation initiation factors were expressed and purified in order to investigate their binding properties and kinetics. Affinity chromatography on m(7)GTP-sepharose showed that bound A. thaliana eIF(iso)4E was eluted with crude TuMV VPg. Further column studies with purified VPg and other A. thaliana eIF4E isoforms showed that VPg preferentially bound eIF(iso)4E. Structural data implicate Trp-46 and Trp-92 in eIF(iso)4E in cap recognition. When Trp-46 or Trp-92 were changed to Leu, eIF(iso)4E lost the ability to form a complex with both VPg and m(7)GTP-sepharose. This suggests that the VPg-binding site is located in or near the cap-recognition pocket on eIF(iso)4E. Affinity constants for the interactions with eIF(iso)4E of VPg and capped RNA oligomer were determined using surface plasmon resonance (SPR). The K(D) values showed that the binging affinity of VPg for eIF(iso)4E is stronger than that of capped RNA. This suggests that viral VPg can interfere with formation of a translational initiation complex on host plant cellular mRNA by sequestering eIF(iso)4E. Further experiments with affinity chromatography showed that VPg forms a ternary complex with eIF(iso)4E and eIF(iso)4G. Thus, VPg may participate in viral translational initiation by functioning as an alternative cap-like structure.

A simplified method for obtaining plant viral RNA for RT-PCR.

An easy and fast procedure (named the simple-direct-tube (SDT) method) was developed for preparing plant virus RNA for cDNA synthesis. The SDT method can be completed in approximately 15min and does not require the use of antiserum, filtering or centrifugation. The procedure to grind plant tissues in phosphate-buffered saline containing Tween-20 (PBST) and to place the extract in a microfuge tube for a few minutes allow adsorption of the virus particles to the tube wall. The sap is then removed and the tube is washed with PBST before the addition of RNase-free water. This manipulation can be performed at room temperature. Using this method followed by reverse transcription-polymerase chain reaction (RT-PCR), infections by turnip mosaic virus, cucumber mosaic virus, and cucumber green mottle mosaic virus (CGMMV) were readily detected, indicating that the SDT method can be used in assays to detect different viruses. For the detection of CGMMV, it was necessary to heat the tubes before cDNA synthesis, suggesting that the immobilized CGMMV particles required disruption by heat treatment to release RNA.

A New Technique to Select Mild Strains of Cucumber mosaic virus.

An efficient technique to select a good attenuated virus to control Cucumber mosaic virus (CMV) disease was developed. Preliminary screenings were conducted to assess the virulence of virus recovered from dark-green islands and yellow tissues of mosaic leaves of Nicotiana rustica after co-inoculation with an attenuated mutant P2bR46C of CMV and its original severe isolate Pepo. All single-lesion isolates (SLIs) obtained from dark-green islands had the attenuated P2bR46C phenotype, but the SLIs from yellow tissue had either the virulent Pepo or the P2bR46C phenotype. When Pepo-infected N. rustica and tomato plants were grown at 15 or 36°C for 30 days, 17 of 288 SLIs obtained from the treated leaves elicited mosaic and dark-green spots without malformation. Dark-green tissue from each plant infected with 1 of these 17 SLIs then was used to inoculate one plant of N. rustica. All 17 plants had either very mild mosaic or no visible symptoms. One of these potential mild strains, 36R37, had an amino acid substitution on the 2b gene encoding the 2b protein. Isolate 36R37 also was highly cross-protective, and its symptom attenuation was stable for three serial host passages. After cold or heat treatment, the dark-green tissue proved to be a good source for isolating mild strains of the virus.