Wheat Yellow Mosaic Virus Research Articles

Resistance to wheat yellow mosaic virus in Madsen wheat is controlled by two major complementary QTLs.

Wheat yellow mosaic virus resistance of Madsen is governed by two complementary QTLs, Qym1 and Qym2 , located on chromosome arms 2DL and 3BS. Wheat yellow mosaic, caused by Wheat yellow mosaic virus (WYMV), is one of the most serious wheat diseases in East Asia. In this study, recombinant inbred lines (RILs, F9) from a cross between cultivars Madsen (resistant) and Hokushin (susceptible) grown in a WYMV-infected nursery field were tested for the presence of WYMV in leaves by enzyme-linked immunosorbent assay (ELISA) and genotyped by using genome-wide molecular markers. Two major QTLs were detected: Qym1 located between Xgwm539 and Xgwm349 on chromosome 2DL and Qym2 located between Xbarc147 and Xwmc623 on chromosome 3BS. The resistance alleles for both QTLs originated from Madsen. The third QTL Qym3 located near Xwmc457 on chromosome 4D, where the resistant allele for this QTL originated from Hokushin. Although the Qym3 was rather minor, it was essential to complement Qym1 and Qym2 for complete avoidance of WYMV infection. Near-isogenic lines carrying the resistance QTLs were developed by repeated backcrosses using Madsen as the donor parent and Hokushin as the recurrent parent. The lines that were resistant to WYMV (as tested by ELISA) were homozygous for the Madsen alleles at both Qym1 and Qym2. Qym1 dominance was partial, whereas that of Qym2 was nearly complete. Qym1 was closely linked to Xwmc41; Qym2 was closely linked to Xwmc754. These markers will be useful in marker-assisted selection in wheat breeding for WYMV resistance; this study will facilitate cloning the WYMV resistance genes.

DAS-ELISA of Wheat yellow mosaic virus of Wheat Leaves Homogenized on a Multi-specimen Cell Disruption Device

DAS-ELISA of <i>Wheat yellow mosaic virus </i>of Wheat Leaves Homogenized on a Multi-specimen Cell Disruption Device

Association of VPg and eIF4E in the host tropism at the cellular level of Barley yellow mosaic virus and Wheat yellow mosaic virus in the genus Bymovirus

Barley yellow mosaic virus (BaYMV) and Wheat yellow mosaic virus (WYMV) are separate species in the genus Bymovirus with bipartite plus-sense RNA genomes. In fields, BaYMV infects only barley and WYMV infects only wheat. Here, we studied the replicative capability of the two viruses in barley and wheat mesophyll protoplasts. BaYMV replicated in both barley and wheat protoplasts, but WYMV replicated only in wheat protoplasts. The expression of wheat translation initiation factor 4E (eIF4E), a common host factor for potyviruses, from the WYMV genome enabled WYMV replication in barley protoplasts. Replacing the BaYMV VPg gene with that of WYMV abolished BaYMV replication in barley protoplasts, whereas the additional expression of wheat eIF4E from BaYMV genome restored the replication of the BaYMV mutant in barley protoplasts. These results indicate that both VPg and the host eIF4E are involved in the host tropism of BaYMV and WYMV at the replication level.

コムギ縞萎縮ウイルス媒介者Polymyxa graminis の感染を抑制する根圏細菌の選抜

コムギ縞萎縮ウイルス媒介者Polymyxa graminis の感染を抑制する根圏細菌の選抜

Impact of transgenic wheat with wheat yellow mosaic virus resistance on microbial community diversity and enzyme activity in rhizosphere soil.

The transgenic wheat line N12-1 containing the WYMV-Nib8 gene was obtained previously through particle bombardment, and it can effectively control the wheat yellow mosaic virus (WYMV) disease transmitted by Polymyxa graminis at turngreen stage. Due to insertion of an exogenous gene, the transcriptome of wheat may be altered and affect root exudates. Thus, it is important to investigate the potential environmental risk of transgenic wheat before commercial release because of potential undesirable ecological side effects. Our 2-year study at two different experimental locations was performed to analyze the impact of transgenic wheat N12-1 on bacterial and fungal community diversity in rhizosphere soil using polymerase chain reaction-denaturing gel gradient electrophoresis (PCR-DGGE) at four growth stages (seeding stage, turngreen stage, grain-filling stage, and maturing stage). We also explored the activities of urease, sucrase and dehydrogenase in rhizosphere soil. The results showed that there was little difference in bacterial and fungal community diversity in rhizosphere soil between N12-1 and its recipient Y158 by comparing Shannon's, Simpson's diversity index and evenness (except at one or two growth stages). Regarding enzyme activity, only one significant difference was found during the maturing stage at Xinxiang in 2011 for dehydrogenase. Significant growth stage variation was observed during 2 years at two experimental locations for both soil microbial community diversity and enzyme activity. Analysis of bands from the gel for fungal community diversity showed that the majority of fungi were uncultured. The results of this study suggested that virus-resistant transgenic wheat had no adverse impact on microbial community diversity and enzyme activity in rhizosphere soil during 2 continuous years at two different experimental locations. This study provides a theoretical basis for environmental impact monitoring of transgenic wheat when the introduced gene is derived from a virus.

Biological and Genetic Diversity of Wheat yellow mosaic virus (Genus Bymovirus)

The biological and genetic diversity of Wheat yellow mosaic virus (WYMV) isolates in Japan was characterized. On the basis of wheat cultivar reactions, 14 WYMV isolates from various places were classified into pathotypes I, II, or III. These were distributed in central, northern, and southern areas of Japan, respectively. WYMV isolates comprised three genotypes (A, A' and B) based on amino acid differences in RNA1 and two genotypes (a and b) based on amino acid differences in RNA2. A correlation was found between the WYMV RNA1-based genotype and pathotype, suggesting that factors associated with pathogenicity map to RNA1. Genotype Aa and A'a were distributed mainly in the central to southern areas of Japan, and genotype Bb was found in northern areas of Japan, as shown by reverse-transcription polymerase chain reaction restriction fragment length polymorphism analysis. Chinese isolates YA and YZ were closely related to genotypes Bb and Aa, respectively. Wheat was introduced from China to Japan in the 4th and 5th centuries, and the two genotypes of WYMV might also have been introduced with the crop from China and later adapted to local wheat cultivars in Japan.

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV.

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat-growing areas in China. Because it is vectored by the fungus-like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co-transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12-1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12-1 showed broad-spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild-type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus-derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad-spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.

Nucleo-cytoplasmic shuttling of VPg encoded by Wheat yellow mosaic virus requires association with the coat protein

VPg (virus protein, genome-linked) is a multifunctional protein that plays important roles in viral multiplication in the cytoplasm. However, a number of VPgs encoded by plant viruses target the nucleus and this appears to be biologically significant. These VPgs may therefore be translocated between nuclear and cytoplasmic compartments during virus infection, but such nucleo-cytoplasmic transport has not been demonstrated. We report that VPg encoded by Wheat yellow mosaic virus (WYMV, genus Bymovirus, family Potyviridae) accumulated in both the nucleus and cytoplasm of infected cells, but localized exclusively in the nucleus when expressed alone in plants. Computational analyses predicted the presence of a nuclear localization signal (NLS) and a nuclear export signal (NES) in WYMV VPg. Mutational analyses showed that both the N-terminal and the NLS domains of VPg contribute to the efficiency of nuclear targeting. In vitro and in planta assays indicated that VPg interacts with WYMV coat protein (CP) and proteinase 1 (P1) proteins. Observation of VPg fused to a fluorescent protein and subcellular fractionation experiments showed that VPg was translocated to the cytoplasm when co-expressed with CP, but not with P1. Mutations in the NES domain or treatment with leptomycin B prevented VPg translocation to the cytoplasm when co-expressed with CP. Our results suggest that association with CP facilitates the nuclear export of VPg during WYMV infection.

Selection Pressure and Founder Effects Constrain Genetic Variation in Differentiated Populations of Soilborne Bymovirus Wheat yellow mosaic virus (Potyviridae) in China

To study the population genetic structure and forces driving the evolution of Wheat yellow mosaic virus (WYMV), the nucleotide sequences encoding the coat protein (CP) (297 sequences) or the genome-linked virion protein (VPg) (87 sequences) were determined from wheat plants growing at 11 different locations distributed in five provinces in China. There were close phylogenetic relationships between all sequences but clustering on the phylogenetic trees was congruent with their provenance, suggesting an origin-dependent population genetic structure. There were low levels of genetic diversity, ranging from 0.00035 ± 0.00019 to 0.01536 ± 0.00043 (CP), and 0.00086 ± 0.00039 to 0.00573 ± 0.00111 (VPg), indicating genetic stability or recent emergence of WYMV in China. The results may suggest that founder effects play a role in shaping the genetic structure of WYMV. Between-population diversity was consistently higher than within-population diversity, suggesting limited gene flow between subpopulations (average FST 0.6241 for the CP and 0.7981 for the VPg). Consistent amino acid substitutions correlated with the provenance of the sequences were observed at nine positions in the CP (but none in the VPg), indicating an advanced stage in population structuring. Strong negative (purifying) selection was implicated on both the CP and VPg but positive selection on a few codons in the CP, indicating an ongoing molecular adaptation.

Induction of 4VS chromosome recombinants using the CS ph1b mutant and mapping of the wheat yellow mosaic virus resistance gene from Haynaldia villosa

The wheat spindle streak mosaic virus (WSSMV) or wheat yellow mosaic virus (WYMV) resistance gene, Wss1, from Haynaldia villosa, was previously mapped to the chromosome arm 4VS by the development of 4V (4D) substitution and T4DL·4VS translocationlines. For better utilization and more accurate mapping of the Wss1, in this research, the CS ph1b mutant was used to induce new translocations with shortened 4VS chromosome fragments. Thirty-five homozygous translocations with different alien fragment sizes and breakpoints of 4VS were identified by GISH and molecular marker analysis. By field test, it was found that all the identified terminal translocations characterized as having smaller 4VS chromosome segments in the chromosome 4DS were highly resistant to WYMV, while all the interstitial translocations with 4VS inserted into the 4DS were WYMV susceptible. Marker analysis using 32 4VS-specific markers showed that both the terminal and interstitial translocations had different alien fragment sizes. Five specific markers could be detected in the WYMV-resistant terminal translocation line NAU421 with the shortest introduced 4VS fragment, indicating they can be used for marker-assisted selection in wheat breeding. Based on the resistance evaluation, GISH and molecular marker analysis of the available translocations, the gene(s) conferring the WYMV resistance on 4VS could be further cytologically mapped to the distal region of 4VS, immersed in the bin of FL 0.78-1.00. The newly developed small fragment translocations with WYMV resistance and 4VS specific markers have laid solid groundwork for the utilization in wheat breeding for WYMV resistance as well as further cloning of Wss1.

A one step real-time RT-PCR assay for the quantitation of Wheat yellow mosaic virus (WYMV).

BackgroundWheat yellow mosaic virus (WYMV) is an important pathogen in China and other countries. It is the member of the genus Bymovirus and transmitted primarily by Polymyxa graminis. The incidence of wheat infections in endemic areas has risen in recent years. Prompt and dependable identification of WYMV is a critical component of response to suspect cases.MethodsIn this study, a one step real-time RT-PCR, followed by standard curve analysis for the detection and identification of WYMV, was developed. Two reference genes, 18s RNA and β-actin were selected in order to adjust the veracity of the real-time RT-PCR assay.ResultsWe developed a one-step Taqman-based real-time quantitative RT-PCR (RT-qPCR) assay targeting the conserved region of the 879 bp long full-length WYMV coat protein gene. The accuracy of normalized data was analyzed along with appropriate internal control genes: β-actin and 18s rRNA which were included in detecting of WYMV-infected wheat leaf tissues. The detectable end point sensitivity in RT-qPCR assay was reaching the minimum limit of the quantitative assay and the measurable copy numbers were about 30 at106-fold dilution of total RNA. This value was close to 104-fold more sensitive than that of indirect enzyme-linked immunosorbent assay. More positive samples were detected by RT-qPCR assay than gel-based RT-PCR when detecting the suspected samples collected from 8 regions of China. Based on presented results, RT-qPCR will provide a valuable method for the quantitative detection of WYMV.ConclusionsThe Taqman-based RT-qPCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of WYMV than other currently used methods.

Differential detection of Wheat yellow mosaic virus, Japanese soil-borne wheat mosaic virus and Chinese wheat mosaic virus by reverse transcription loop-mediated isothermal amplification reaction

A differential detection method for three wheat viruses: Wheat yellow mosaic virus (WYMV), Japanese soil-borne mosaic virus (JSBWMV) and Chinese wheat mosaic virus (CWMV) using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction was developed. All three primer sets, which were designed from the genome sequences of WYMV, JSBWMV and CWMV respectively, worked most efficiently at 65°C and could detect each virus RNA within 10min by fluorescence monitoring using an isothermal DNA amplification and fluorescence detection device. Furthermore, these primer sets showed unique annealing curves. The peak denaturing temperatures of WYMV, JSBWMV and CWMV primer sets were 87.6°C, 84.8°C and 86.4°C, respectively and were clearly distinguished by the isothermal DNA amplification and fluorescence detection device. The RT-LAMP assay including all three primer sets was found to be 100 times more sensitive than RT-PCR for WYMV and JSBWMV and as sensitive as RT-PCR for CWMV. The RT-LAMP method was validated for the simultaneous detection of these viruses in wheat and barley leaves.

Development of a multiplex polymerase chain reaction for simultaneous detection of wheat viruses and a phytoplasma in China

Barley yellow dwarf viruses (BYDVs, mainly consisting of three strains, GAV, GPV and PAV, in China), barley stripe mosaic virus (BSMV), wheat yellow mosaic virus (WYMV), wheat dwarf virus (WDV) and wheat blue dwarf phytoplasma (WBD) constitute a group of major wheat pathogens that have caused huge yield losses but are scarcely distinguished by their phenotype alone. For the simultaneous detection and discrimination of these seven pathogens in wheat, a multiplex polymerase chain reaction (M-PCR) method was developed in this study through a series of parameter optimizations. Detection and sensitivity tests using samples collected from the field indicate that the M-PCR method can rapidly, simultaneously and relatively effectively detect BYDV-GAV, -GPV, -PAV, BSMV, WYMV, WDV and WBD.

Rapid detection of wheat yellow mosaic virus by reverse transcription loop-mediated isothermal amplification.

For the detection of wheat yellow mosaic virus (WYMV), we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Using Primer Explorer software, four sets of primers were designed and RT-LAMP assay reaction conditions were optimized. The RT-LAMP was performed at different times by four primer sets. Agarose gel analysis showed that WYMV could be detected after 30 min with the primer set III and after 45 min with the other three primer sets, both under the 80-min reaction time. RT-LAMP had the same results with the four primer sets, thus primer set III and 65°C for 80 min reaction were selected for virus detection. There was no significant different when avian myeloblastosis virus (AMV) and moloney murine leukemia virus (M-MLV) RT-LAMP with the four primer sets and M-MLV was chosen due to its relatively cheap price. The result on specificity showed that the assay could amplify WYMV specifically, and the sensitivity comparison showed that the RT-LAMP was 100 times more sensitive than conventional reverse-transcriptase-polymerase chain reaction (RT-PCR). Overall, RT-LAMP was found to be a simple, specific, sensitive, convenient and time-saving method for WYMV detection.

Polymyxa graminis . [Distribution map

Abstract A new distribution map is provided for Polymyxa graminis Ledingham. Cercozoa: Plasmodiophorida. Hosts: groundnut ( Arachis hypogaea ), oats ( Avena sativa ), barley ( Hordeum vulgare ), rice ( Oryza sativa ), rye ( Secale cereale ), wheat ( Triticum aestivum ), millet ( Panicum miliaceum ) and sorghum ( Sorghum bicolor ). Information is given on the geographical distribution in Europe (Belgium, France, Mainland France, Germany, Greece, Mainland Greece, Italy, Mainland Italy, Netherlands, Spain, Mainland Spain, UK, England and Wales), Asia (China, Anhui, Gansu, Henan, Hubei, Hunan, Jiangsu, Jiangxi, Shaanxi, Shandong, Shanxi, Sichuan, Zhejiang, India, Andhra Pradesh, Gujarat, Punjab, Rajasthan, Iran, Japan, Honshu, Lebanon, Pakistan, Syria, Turkey), Africa (Burkina Faso, Cote d'Ivoire, Mali, Niger, Senegal), North America (Canada, Manitoba, Ontario, Prince Edward Island, USA, Arkansas, Florida, Georgia, Illinois, Iowa, Kansas, Kentucky, Maryland, Missouri, Nebraska, New Mexico, New York, Oklahoma, Pennsylvania, South Carolina, Virginia, Washington), South America (Bolivia, Brazil, Rio Grande do Sul, Colombia), Oceania (New Zealand). Vector of numerous plant viruses, including Soil-borne wheat mosaic virus, Wheat yellow mosaic virus, Wheat spindle streak mosaic virus, Barley yellow mosaic virus, Oat mosaic virus, Rice stripe necrosis virus and Peanut clump virus. Several viruses transmitted by P. graminis cause major widespread diseases on barley, wheat and groundnut. P. graminis is presumed to be present in all fields infested with the viral diseases it transmits, but is also present in virus-free soils. The distribution of the viruses transmitted by P. graminis is greater than the confirmed distribution of P. graminis shown on the map.

反復戻し交配によって育成したコムギ縞萎縮病抵抗性系統「滝系麦」の収量性

反復戻し交配によって育成したコムギ縞萎縮病抵抗性系統「滝系麦」の収量性

Mapping a gene conferring resistance to Wheat yellow mosaic virus in European winter wheat cultivar ‘Ibis’ (Triticum aestivum L.)

Wheat yellow mosaic, caused by Wheat yellow mosaic virus (WYMV), is one of the most devastating soil-borne diseases of winter wheat (Triticum aestivum L.) in Japan. Yellow-striped leaves and stunted spring growth, symptomatic of WYMV infection, result in severe yield loss. A new putative WYMV resistance gene in the European wheat cultivar ‘Ibis’ was mapped in the cluster of microsatellite markers including Xcfd16, Xwmc41, Xcfd168 and Xwmc181 on the long arm of chromosome 2D at the distances of 2.0 cM, 4.0 cM, 7.1 cM and 12.4 cM, respectively. WYMV-resistant cultivars contained a common haplotype of the four markers, whereas moderately susceptible and susceptible cultivars did not. These results should be useful in marker-assisted selection for WYMV resistance in wheat.

コムギ縞萎縮病抵抗性系統の育成と「Madsen」由来の抵抗性遺伝子 &lt;i&gt;YmMD&lt;/i&gt;の座乗領域の推定

コムギ縞萎縮病は，コムギ縞萎縮ウイルス（Wheat yellow mosaic virus: WYMV）を病原とする土壌伝染性の病害である．本病は東北以南では古くから発生の報告があったが，北海道では1991年に初めて発生が確認され，本病害に対する抵抗性を持たない北海道の優良品種「ホクシン」の普及に伴い被害が拡大した．本研究では，1995年から5カ年にわたってWYMV発生圃場においてコムギ品種・系統の抵抗性検定を行い，本病害に対する抵抗性が“強”の遺伝資源としてアメリカの品種「Madsen」を見出した．また，Madsen/ホクシンのF2集団を用いた検定において，「Madsen」由来のコムギ縞萎縮病抵抗性の遺伝様式は優性の1遺伝子によるものと推定した．一方で「Madsen」は，成熟期が遅く，穂発芽しやすく，雪腐れ病の発病も多いなど育種素材として利用しづらい形質を有していた．そこで，2001年から2005年の5カ年にわたり圃場検定による選抜を利用した反復戻し交配を5回行い，「ホクシン」に「Madsen」の縞萎縮病抵抗性を導入することにより，縞萎縮病抵抗性以外の特性が「ホクシン」に近い育種素材を4系統作出した．さらに，得られた反復戻し交配系統と「ホクシン」を用いて，SSRマーカーによるDNAの多型を調べることによって，「Madsen」由来のコムギ縞萎縮病抵抗性遺伝子YmMDが座乗している染色体領域を2DL上のwmc041とgwm349の間と推定した．

コムギ縞萎縮病(WYMV)抵抗性遺伝子YmMDの座乗領域と準同質遺伝子系統の収量性(平成21年度年次講演会一般講演)

コムギ縞萎縮病(WYMV)抵抗性遺伝子YmMDの座乗領域と準同質遺伝子系統の収量性(平成21年度年次講演会一般講演)