Tomohide Natsuaki

Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani

Abstract Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66 – 100% for isolates of different subgroups within an AG, and 55 – 96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

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.

The simultaneous differentiation of Potato virus Y strains including the newly described strain PVYNTN-NW by multiplex PCR assay

New recombinant strain and genotype of PVY, designated as PVY(NTN-NW) and SYR-III, respectively, shared properties with PVY(NTN) and PVY(N)W has been reported recently. PVY(NTN-NW) predominated in potato fields in Syria and was able to induce potato tuber necrotic ringspot disease (PTNRD). Due to the rapid spread of the recombinant strains of PVY which might be the case of PVY(NTN-NW), a specific and reliable detection method is an essential step to control this strain and minimize its spread. The shared properties of PVY(NTN-NW) and SYR-III with PVY(NTN) and PVY(N)W, however, complicate their identification involving multiple detection methods. Therefore, a multiplex polymerase chain reaction (PCR), that relies on a combination of previously published and newly designed primers was developed for the detection and identification of PVY(NTN-NW) and SYR-III in single or mixed infections with the main PVY strains, PVY(O), PVY(N), PVY(NTN) and PVY(N)W. In addition, the present PCR assay was able to detect the recombination points in the P1 region enabling the differentiation of the variable genotypes of the recombinant strains PVY(NTN-NW), PVY(NTN) and PVY(N)W. The reliability of this PCR assay was confirmed using a significant number of well characterized PVY isolates collected from Syria and Japan including those of PVY(NTN-NW), SYR-III, PVY(O), NA-PVY(N), PVY(N)W and PVY(NTN).

Nucleotide Sequence of Citrus Tristeza Virus Seedling Yellows Isolate

The complete nucleotide sequence of a seedling-yellows-inducing isolate NUagA of Citrus tristeza virus (CTV) was determined. It consisted of 19302 nucleotides and contained 12 open reading frames (ORF) organized identically to those of previously sequenced isolates. This genome is the largest among the CTV genome sequenced so far ; it is 6 nucleotides (nt), 76 nt, 43 nt, and 53 nt longer than that of T36 (quick decline, Florida), VT (seedling yellows, Israel), T385 (mild, Spain), and SY568 (stem pitting, California), respectively. Sequence comparison of NUagA and the other isolates revealed approximately 90% identities throughout the 3′ half of the genome. The 5′ half of the genome was only about 70% identical to that of T36 but still high at about 90% to those of VT, SY568, and T385. Comparison of amino acid sequences on ORF1a encoding polyproteins, the most variable region, reflects the CTV isolate relationship ; NUagA is closely related to VT, SY568, and T385, but distantly related to T36.

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.

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.

Fine genetic mapping of the TuNI locus causing systemic veinal necrosis by turnip mosaic virus infection in Arabidopsis thaliana

In the pathosystem of turnip mosaic virus (TuMV) and Arabidopsis thaliana, two distinct symptoms (mosaic symptom and veinal necrosis) were observed that were dependent upon the combination of the TuMV isolate and the Arabidopsis ecotype. The Col-0 ecotype developed mosaic symptoms after infection with the TuMV isolate Azu while the Ler ecotype developed veinal necrosis after infection with the same TuMV isolate. The Ler phenotype is controlled by a single dominant gene TuNI (TuMV necrosis inducer) which is located on chromosome 1. The TuNI gene was precisely mapped to the ~105 kb interval between the two markers of mXF41 and mRF28 by using several types of DNA polymorphism markers. Within this region, which included largely duplicated sequences, a total of 19 putative genes were predicted and 15 of these were classified into five gene families. The genes belonging to the gene families At1g58480 and At1g58602 may function in response to infection by pathogens. The gene family At1g58480 encodes lipase-like proteins, which might be involved in the induction of defence responses that are mediated by salicylic acid. The gene family At1g58602 encodes the CC-NBS-LRR (CNL) proteins, which are known to function as one of the plant resistance (R) proteins against pathogens. In the present study, the possibility that TuNI might function as an R gene was discussed.

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.

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.

Complete nucleotide sequence of the genome organization of RNA2 of patchouli mild mosaic virus, a new favavirus

SummaryThe complete nucleotide sequence and the genome organization of the RNA 2 of a patchouli mild mosaic virus (PaMMV) was determined. The sequence consists of 3591 nucleotides and contains a single long open reading frame sufficient to code for 118K protein. Three proteins of 52 K, 44 K and 22 K could be encoded by the PaMMV RNA 2 genome. Our analysis of the N-terminal sequences of two species of coat protein (CP) allowed precise location of the CP cistrons within the polyprotein. 44 K and 22 K proteins are the coat proteins. The positions of the cleavage sites are Gln/Ala between 44 K and 22 K coat proteins and Gln/Gly between 52 K and 44 K proteins. Comparison of PaMMV RNA 2 with comoviral and nepoviral RNA 2 showed no sequence similarity. These results as well as previous serological studies strongly suggest that PaMMV is a member in the genus Fabavirus.