Yoichi Takanami

Inoculation with RNAs 1 and 2 of Cucumber Mosaic Virus Induces Viral RNA Replicase Activity in Tobacco Mesophyll Protoplasts

Summary The activity of membrane-bound RNA-dependent RNA polymerase in tobacco mesophyll protoplasts was measured and in vitro products were characterized after inoculation with cucumber mosaic virus (CMV) or with different combinations of the four virus RNAs and satellite RNA. Activity increased rapidly 4 to 6 h after inoculation with virus particles. When the inoculum contained RNA 1, RNA 2 and RNA 3, most of the in vitro products of the enzyme were full-length positive-sense RNAs (RNAs 1, 2, 3 and 4) that migrated as dsRNA; inoculation of RNA 1 or RNA 2 alone did not increase RNA polymerase activity and no virus RNA was produced. After inoculation with a mixture of RNA 1 and RNA 2, however, the enzyme activity increased and full-length positive-sense RNA 1 and RNA 2 were synthesized, and we speculate that the induced enzyme is CMV RNA replicase as a complex comprising the translation products of RNA 1 and RNA 2, membrane components of the host cell and CMV RNA templates. Protoplasts inoculated with RNA 1, RNA 2 and RNA 4 did not synthesize RNA 4 which suggests that positive-sense RNA 4 does not serve as a template for the replicase complex. Satellite RNA was synthesized in protoplasts inoculated with RNA 1, RNA 2 and satellite RNA which suggests that satellite RNA is replicated by the replicase complex.

Point mutations in the coat protein of cucumber mosaic virus affect symptom expression and virion accumulation in tobacco

We examined the correlation of the amino acid at position 129 in the coat protein (CP) of cucumber mosaic virus (CMV) with the phenotype of the viral pathology in tobacco by using CP mutants in which several amino acid substitutions had been introduced. An exchange between Ser129 in CMV-Y, a chlorosis-inducing strain, and Pro129 of CMV-O, a green-mosaic-inducing strain, reciprocally altered the phenotypes of those virus strains on tobacco. Replacement of either Ser129 in CMV-Y or Pro129 in CMV-O with a Leu, as is found in a chlorosis-inducing strain, CMV-M, resulted in veinal necrosis. Furthermore, we created mutants that have a Phe or a Gly at position 129. Two Phe129 mutants induced necrotic lesions on the inoculated leaves, and a Gly129 mutant induced green mosaic symptoms. In inoculated protoplasts, the mutant viruses and the wild-type virus all replicated RNA well, and accumulated CP; however, infection with the Leu129 and Phe129 mutants yielded few virions. The Phe129 mutants lacked the capacity to move systemically in tobacco; by 2 weeks post-inoculation, the Phe129 mutants occasionally gave rise to revertants that elicited chlorosis, green mosaic or veinal necrosis. Sequence analysis revealed that one had reverted to the parental Y strain, and the others had additional single amino acid changes (positions 138, 144 or 147). We suggest that amino acids at specific sites affect the whole structure of the CP and affect virus assembly, virus transport and symptom expression.

Combination of amino acids in the 3a protein and the coat protein of Cucumber mosaic virus determines symptom expression and viral spread in bottle gourd

Summary. Bottle gourd plants infected with an isolate of cucumber mosaic virus (CMV-KM) developed severe chronic mosaic symptoms (SCMS) with stunting, but two other isolates (CMV-Y and CMV-D8) did not. CMV-KM and CMV-D8 induced enlarged chlorotic spots and rapidly spread over the inoculated cotyledons, whereas CMV-Y elicited a hypersensitive response (HR) producing pin-point necrotic lesions. Reassortment analysis among the three isolates revealed that the local and systemic symptoms on the plants were regulated by RNA3. Reciprocal recombination and site-directed point mutation analyses of the three RNA3s demonstrated that a combination of genetic information encoded by the movement protein (MP) gene and the coat protein (CP) gene determines the induction of SCMS in bottle gourd. SCMS occurred when Ser51 in the MP of CMV-D8 was changed to Asn51, whereas substitution of Ser51 for Asn51 in the MP of CMV-KM eliminated its ability to induce SCMS. Furthermore, Ser129 in the CPs was shown to be responsible for induction of HR and blocking of efficient cell-to-cell and long-distance movement.

RESISTANCE AGAINST CUCUMBER MOSAIC VIRUS IN PLANTS EXPRESSING THE VIRAL REPLICON

CMV RNAs 1 and 2 are considered to constitute the viral replicon. Tobacco plants were transformed with either RNA1 or RNA2 to produce plant lines V1 and V2, respectively. Plants homozygous for each of the RNAs were generated and crossed to produce V1V2 (V2V1) lines that expressed both RNA1 and RNA2. An RNase protection assay indicated that RNA1 and RNA2 multiplied in V1V2 (V2V1) plants. Surprisingly, V1V2 (V2V1) plants, unlike their parent lines, showed a remarkably high level of resistance to CMV; this resistance was more effective against RNA inoculation than against virion inoculation. Experiments using protoplasts showed that the resistance was expressed at the single cell level. All the data together suggested that the observed resistance does not fit the criteria for either ‘RNA‐mediated’ or ‘replicase‐mediated’ resistance.

Unique grouping of the Far East Asian begomovirus complex based on sequence analyses of the DNA-A genome and associated DNAβ satellite molecules isolated from tomato, honeysuckle and Eupatorium plants in Japan

Nucleotide (nt) sequencing has contributed to the identification of virus species and has also proved diagnostically useful in the control of tomato-infecting begomoviruses disease. We determined the complete nt sequences of the DNA-A genome and its cognate DNAβ satellite molecules in isolates of Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus, Eupatorium yellow vein virus in Japan. Pairwise comparison analyses based on the nt sequences of DNA-A from the genetic group of these viruses tentatively named as TbLCJV, HYVMV and EpYVV (TbJV/HYV/EpV) revealed that this group had a significance threshold of 84 % identity. Phylogenetic relationship analyses of the nt sequences of DNA-A and DNAβ revealed that their isolates were separated into a discrete Far East Asian clade, distinct from all other begomoviruses. This clade was divided into two distinct clusters comprising the subgroups TbJV/HYV and EpV. Furthermore, recombination analysis revealed that members of the TbJV/HYV/EpV group had the genetic variation indicative of many recombination events. Our study demonstrates that this group forms a unique species complex, but that members have discrete lineages depending on their natural perennial host plants.

Involvement of cucumber mosaic cucumovirus RNA2 and RNA3 in viral systemic spread in radish plant

SummaryThe genetics of cucumber mosaic cucumovirus (CMV) and the pathogenicity of the virus for Raphanus sativus were analyzed using pseudorecombinants constructed from the infectious transcripts of two naturally occurring strains of cucumber mosaic cucumovirus (CMV-D8 and CMV-Y). CMV-D8, but not CMV-Y, could cause systemic infection of the plant. Viral accumulation and systemic movement in the plants was examined using immuno-tissue blot analysis, dot blot and Northern blot hybridization. Virus was equally distributed and CMV RNAs accumulated to similar levels in the inoculated cotyledons of radish irrespective of the pseudorecombinant, suggesting that there are no apparent differences in the ability of infection and viral accumulation between CMV-D8 and CMV-Y. We found, however, that both RNAs 2 and 3 of CMV-D8 are involved in determining the efficiency for the systemic infection of R. sativus. Co-operated interactions between genetic information of RNAs 2 and 3 would control the efficient translocation of virus from the inoculated leaves to the uninoculated upper leaves of radish plant.

Characteristics of Potato virus Y Isolated from Paprika in Korea

A virus isolate collected from infected paprika (Capsicum annuum var. grossum) was characterized as Potato virus Y (PVY) based on biological, serological, cytopathological, and molecular properties. In host range studies, the paprika isolate produced the mosaic symptom on some tobacco, tomato and pepper (Capsicum annuum). A new paprika isolate also infected potato cultivars which is different biological characteristic compared to the other popular potyvirus infecting paprika, Pepper mottle virus (PepMoV). Previously reported PVY strains, and did not infect pepper and typical PepMoV isolates did not infect potato. Distinctive inclusion patterns of the scroll, pinwheel, long laminated inclusions, and helper components in the cytoplasm of infected cells were also different to those observed by the typical PVY isolate infections. However, the paprika isolate reacted to the monoclonal antibody of strain with high absorbance readings. RT-PCR amplification, cloning, and sequencing of the 3` untranslated region and a part of coat protein gene also added additional evidence of the paprika isolate as the -related isolate. Multiple alignments as well as cluster dendrograms of PVY-paprika isolate revealed close phylogenetic relationship to the subgroup. Altogether, these results suggest that a new PVY isolate infecting paprika contained distinct characteristics compared to the other previously described PVY strains with closer relationship to the strain.

Specific Oligonucleotide Primers Based on Sequences of the 16S-23S rDNA Spacer Region for the Detection of Burkholderia gladioli by PCR

Specific primers were designed based on the sequences of the spacer region between the 16S and 23S ribosomal DNA (rDNA) for direct, rapid and specific detection of Burkholderia gladioli. These primers were named GLA-f and GLA-r. PCR performed on boiled bacterial suspensions yielded an amplification product of approximately 300 bp. No products from other bacterial species, including B. glumae were amplified, even after complete DNA extraction by the cetyltrimethyl-ammonium bromide (CTAB) method. Using the specific primers designed in this study, the PCR method can detect B. gladioli in plant samples within 6 hr. These data demonstrate the potential of specific PCR for the detection of B. gladioli.

RFLP analysis of the PCR-amplified 28S rDNA in Rhizoctonia solani

RFLP analyses of a portion of the 28S rDNA gene region were conducted by using four restriction endonucleases for 57 isolates of 13 intraspecific groups (ISGs) representing 7 anastomosis groups (AGs) ofRhizoctonia solani. Variations in the PCR-amplified rDNA products and the polymorphisms on digestion with restriction enzymes (BamHI,HaeIII,HhaI andHpaII) were observed among three AGs, AG 1, 2 and 4. These differences were also conserved among some ISGs of AG 1 and AG 2. Among ISGs of AG 1, the pattern of rDNA fragments of AG 1-IA obtained by digestion withHpaII was significantly different from those of AG 1-IB and IC. Such difference in the fragment pattern was also observed among AG 2-1, 2-2 IIIB and 2-2 IV by the digestion withHhaI andHpaII. A dendrogram derived from the restriction enzyme data showed that ISGs from AG 1 and AG 2 can each be subdivided into distinct groups, those are distantly related to the majority isolates of the other AGs.

Competition between wild-type virus and a reassortant from subgroups I and II of CMV and activation of antiviral responses in cowpea

Summary.To investigate the interactions between RNA3 and RNA4 from subgroups I and II in mixed infections, accumulation of CMV RNA were analyzed. In the mixed inoculation assays with CMV-LE (LE, subgroup I) and a reassortant LLm consisting of RNA1 and RNA2 from LE, and RNA3 from CMV-m2 (m2, subgroup II), LE RNA3 and RNA4 could systemically spread in the plants, whereas those of m2 could not. Furthermore, accumulation of virus short RNA and a cowpea-encoded RNA-directed RNA polymerase gene (VuRdRP1) mRNA were found in the plants, suggesting that VIGS and/or distinct antiviral responses (was) were activated by infection with CMV.