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Development of proteomic technology of shotgun and label free combined with multiple reaction monitoring to simultaneously detect southern rice black-streaked dwarf virus and rice ragged stunt virus.

ABSTRACT: The co-infection of rice caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) was widely found at many regions, such as Yunnan Province, China, and North and Central Vietnam. These rice viral diseases lead to seriously yield loss of rice. In this study, the proteomics technology of shotgun and label free combined with multiple reaction monitoring (MRM) was developed to detect rice sample of a single or/and co-infection. The shotgun assay indicated that some proteins coded by SRBSDV and RRSV were detected via the mode of in-gel digestion, except for P5-2, P7-2 and P9-2 of SRBSDV and P4b, P5, P6, P8a and P8b of RRSV. The technology of label free combined with MRM indicated that P2, P5-1, P4, P8, P7-1, P6, P9-1 and P10 of SRBSDV and P1, P3 and P9 of RRSV were higher abundance in rice plant, and P5-2, P7-2 and P9-2 of SRBSDV and P4b and P5 of RRSV were lower abundance in viruliferous-rice plant. So SRBSDV P9-1 and RRSV P3 was selected as marker molecule to be used in detection technology, and the label free combined with MRM technology was established to detect two kinds of rice virus.

SUBMITTER: Chen Z

PROVIDER: S-EPMC4188197 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Development of proteomic technology of shotgun and label free combined with multiple reaction monitoring to simultaneously detect southern rice black-streaked dwarf virus and rice ragged stunt virus.

Chen Zhuo Z Guo Qin Q Chen Bing-Hua BH Li Xiang-Yang XY Wang Zhen-Chao ZC He Peng P Yan Fei F Hu De-Yu DY Yang Song S

Virusdisease 20140227 3

The co-infection of rice caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) was widely found at many regions, such as Yunnan Province, China, and North and Central Vietnam. These rice viral diseases lead to seriously yield loss of rice. In this study, the proteomics technology of shotgun and label free combined with multiple reaction monitoring (MRM) was developed to detect rice sample of a single or/and co-infection. The shotgun assay indicated that s ...[more]

PMID: 25674599

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Project description:Rice black-streaked dwarf disease, caused by rice black-streaked dwarf virus (RBSDV), is a serious constraint in Chinese rice production. Breeding disease-resistant varieties through multigene aggregation is considered an effective way to control diseases, but few disease-resistant resources have been characterized thus far. To develop novel resources for resistance to RBSDV through CRISPR/Cas9-mediated genome editing, a guide RNA sequence targeting exon 1 of eIF4G was designed and cloned into a binary vector, pHUE401. This recombinant vector was used to generate mutations in the rice cultivar Nipponbare via Agrobacterium-mediated transformation. This approach produced heritable homozygous mutations in the transgene-free T1 generation. Sequence analysis of the eIF4G target region from T1 transgenic plants identified 3 bp deletion mutants, and analysis of the predicted amino acid sequence identified one amino acid deletion in mutants that possess near full-length eIF4G. Furthermore, our data suggest that eIF4G may plays an important role in rice normal development, as there were no eIF4G knock-out homozygous mutants in T1 generation plants. When homozygous mutant lines were inoculated with RBSDV, they exhibited enhanced tolerance to virus infection, without visibly affecting plant growth and development. However, the eif4g mutant plants showed the same sensitivity to rice stripe virus (RSV) infection as wild-type plants. Notably, the wild-type and mutant N-termini of eIF4G interacted directly with RBSDV P8 in yeast and in planta. Additionally, compared to wild-type plants, the eIF4G transcript level was reduced twofold in the mutant plants. These results indicate that site-specific mutation of rice eIF4G successfully conferred partial resistance specific to RBSDV associated with less transcription of eIF4G in mutants. Therefore, this study demonstrates that the novel eIF4G alleles generated by CRISPR/Cas9 represent valuable disease-resistant resources that can be used to develop RBSDV-resistant varieties.

Project description:Rice black-streaked dwarf virus (RBSDV) (species Rice black-streaked dwarf virus, genus Fijivirus, family Reoviridae) is the causal agent of rice black-streaked dwarf and maize rough dwarf diseases, which occur in intermittent epidemics in East Asian countries and are responsible for considerable yield losses. Intermittency of epidemics make accurate forecasting and designing of effective management strategies difficult. However, recent insights into host-virus-vector insect interactions are now informing forecasting and disease control measures. Resistance genes are also being identified and mapped.Symptomatology and host rangeRBSDV induces extreme stunting, darkened, and stiff leaves of crops and weeds only in the family Poaceae, including Oryza sativa, Zea mays, and Triticum aestivum. Infected plants produce totally or partially deformed panicles and remain alive through harvest.Genome and gene functionThe nonenveloped virus particles comprise a double-layered capsid, 50-nm core with genomic double-stranded RNA (dsRNA), and six proteins. The genome of RBSDV contains 10 segments of dsRNA, named S1 to S10 in decreasing order of molecular weight. Segments 1, 2, 3, 4, 6, 8, and 10 encode the RNA-dependent RNA polymerase (RdRp), the major core structural protein, a protein with guanylyltransferase activity, an outer-shell B-spike protein, viral RNA-silencing suppressor, the major capsid protein, and the outer capsid protein, respectively. Each of the segments 5, 7, and 9 encodes two proteins: P5-1, a component of viroplasms; P5-2 of unknown function; nonstructural protein P7-1, involved in forming the structural matrix of tubular structures in infected tissues; P7-2 of unknown function; P9-1, the main component of viroplasms in infected cells and involved in viral replication; and P9-2 of unknown function.Transmission and epidemiologyRBSDV is transmitted by Laodelphax striatellus in a persistent propagative manner. The vector insect is the only means of virus spread in nature, so its migration and transmission efficiency are obligatory for disease epidemics to develop. Susceptible varieties are widely planted, but efficient transmission by vectors is the primary reason for the epidemics. Cultivation system, pesticide overuse, and climatic conditions also contribute to epidemics by affecting the development of the vector insects and their population dynamics.Disease managementIn the absence of resistant varieties, integrated disease management aims at disrupting the cycle of virus transmission by the insect vector. Inheritance studies have indicated that resistance is mostly governed by quantitative trait loci or multiple genes. Genetic engineering through RNA-interference and gene-editing strategies are potential approaches for disease control.

Dataset Information

Development of proteomic technology of shotgun and label free combined with multiple reaction monitoring to simultaneously detect southern rice black-streaked dwarf virus and rice ragged stunt virus.

Publications

Development of proteomic technology of shotgun and label free combined with multiple reaction monitoring to simultaneously detect southern rice black-streaked dwarf virus and rice ragged stunt virus.

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