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Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

ABSTRACT: Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia.The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms.UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed.We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

SUBMITTER: Fang P

PROVIDER: S-EPMC4619528 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

Fang Peng P Lu Rongfei R Sun Feng F Lan Ying Y Shen Wenbiao W Du Linlin L Zhou Yijun Y Zhou Tong T

Virology journal 20151024

<h4>Background</h4>Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia.<h4>Methods</h4>The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normali ...[more]

PMID: 26497487

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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.

Project description:BackgroundSouthern rice black-streaked dwarf virus (SRBSDV) has spread from the south of China to the north of Vietnam in the past few years, and has severely influenced rice production. However, previous study of traditional SRBSDV transmission method by the natural virus vector, the white-backed planthopper (WBPH, Sogatella furcifera), in the laboratory, researchers are frequently confronted with lack of enough viral samples due to the limited life span of infected vectors and rice plants and low virus acquisition and inoculation efficiency by the vector. Meanwhile, traditional mechanical inoculation of virus only apply to dicotyledon because of the higher content of lignin in the leaves of the monocot. Therefore, establishing an efficient and persistent-transmitting model, with a shorter virus transmission time and a higher virus transmission efficiency, for screening novel anti-SRBSDV drugs is an urgent need.MethodsIn this study, we firstly reported a novel method for transmitting SRBSDV in rice using the bud-cutting method. The transmission efficiency of SRBSDV in rice was investigated via the polymerase chain reaction (PCR) method and the replication of SRBSDV in rice was also investigated via the proteomics analysis.ResultsRice infected with SRBSDV using the bud-cutting method exhibited similar symptoms to those infected by the WBPH, and the transmission efficiency (>80.00%), which was determined using the PCR method, and the virus transmission time (30 min) were superior to those achieved that transmitted by the WBPH. Proteomics analysis confirmed that SRBSDV P1, P2, P3, P4, P5-1, P5-2, P6, P8, P9-1, P9-2, and P10 proteins were present in infected rice seedlings infected via the bud-cutting method.ConclusionThe results showed that SRBSDV could be successfully transmitted via the bud-cutting method and plants infected SRBSDV exhibited the symptoms were similar to those transmitted by the WBPH. Therefore, the use of the bud-cutting method to generate a cheap, efficient, reliable supply of SRBSDV-infected rice seedlings should aid the development of disease control strategies. Meanwhile, this method also could provide a new idea for the other virus transmission in monocot.

Dataset Information

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

Publications

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

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