scholarly journals Development and Use of an Efficient DNA-Based Viral Gene Silencing Vector for Soybean

2009 ◽  
Vol 22 (2) ◽  
pp. 123-131 ◽  
Author(s):  
Chunquan Zhang ◽  
Chunling Yang ◽  
Steven A. Whitham ◽  
John H. Hill
Keyword(s):  
Virus Infection ◽  
Gene Silencing ◽  
Functional Genomics ◽  
Mosaic Virus ◽  
Soybean Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Viral Gene ◽  
35S Promoter ◽  
Virus Induced Gene Silencing ◽  
Wide Range

Virus-induced gene silencing (VIGS) is increasingly being used as a reverse genetics tool to study functions of specific plant genes. It is especially useful for plants, such as soybean, that are recalcitrant to transformation. Previously, Bean pod mottle virus (BPMV) was shown to be an effective VIGS vector for soybean. However, the reported BPMV vector requires in vitro RNA transcription and inoculation, which is not reliable or amenable to high-throughput applications. To increase the efficiency of the BPMV vector for soybean functional genomics, a DNA-based version was developed. Reported here is the construction of a Cauliflower mosaic virus 35S promoter-driven BPMV vector that is efficient for the study of soybean gene function. The selection of a mild rather than a severe BPMV strain greatly reduced the symptom interference caused by virus infection. The DNA-based BPMV vector was used to silence soybean homologues of genes involved in plant defense, translation, and the cytoskeleton in shoots and in roots. VIGS of the Actin gene resulted in reduced numbers of Soybean mosaic virus infection foci. The results demonstrate the utility of this new vector as an efficient tool for a wide range of applications for soybean functional genomics.

scholarly journals Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing

2004 ◽  
Vol 78 (17) ◽  
pp. 9487-9498 ◽  
Author(s):  
Ramachandran Vanitharani ◽  
Padmanabhan Chellappan ◽  
Justin S. Pita ◽  
Claude M. Fauquet
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
African Cassava Mosaic Virus ◽  
35S Promoter ◽  
Viral Dna ◽  
Posttranscriptional Gene Silencing ◽  
Cassava Mosaic Virus

ABSTRACT Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.

scholarly journals Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

2011 ◽  
Vol 101 (11) ◽  
pp. 1264-1269 ◽  
Author(s):  
Xiuchun Zhang ◽  
Shirley Sato ◽  
Xiaohong Ye ◽  
Anne E. Dorrance ◽  
T. Jack Morris ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Virus Resistance ◽  
Soybean Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Systemic Resistance ◽  
35S Promoter ◽  
Virus Infections ◽  
Bean Pod Mottle Virus ◽  
Soybean Plants

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

scholarly journals Engineering Resistance to Multiple Prunus Fruit Viruses Through Expression of Chimeric Hairpins

2007 ◽  
Vol 132 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Zongrang Liu ◽  
Ralph Scorza ◽  
Jean-Michel Hily ◽  
Simon W. Scott ◽  
Delano James
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Fruit Production ◽  
Cauliflower Mosaic Virus ◽  
Ringspot Virus ◽  
35S Promoter ◽  
Plum Pox Virus ◽  
In Planta ◽  
Posttranscriptional Gene Silencing ◽  
Transgenic Lines

Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

scholarly journals (285) Characterization of a Composite Promoter from Genomic Sequences of Grapevine

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1053C-1053
Author(s):  
Manjul Dutt ◽  
Zhijian T. Li ◽  
Sadanand Dhekney ◽  
Dennis J. Gray
Keyword(s):  
Mosaic Virus ◽  
Genomic Sequence ◽  
Fusion Gene ◽  
Core Promoter ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
2S Albumin ◽  
Wide Range ◽  
Constitutive Gene Expression

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

scholarly journals Cucumber mosaic virus-induced gene silencing in banana

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yuh Tzean ◽  
Ming-Chi Lee ◽  
Hsiao-Hsuan Jan ◽  
Yi-Shu Chiu ◽  
Tsui-Chin Tu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Virus Induced Gene Silencing

scholarly journals A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Decai Tuo ◽  
Peng Zhou ◽  
Pu Yan ◽  
Hongguang Cui ◽  
Yang Liu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Viral Vector ◽  
Function Analysis ◽  
Systemic Infection ◽  
Cdna Clones ◽  
Virus Induced Gene Silencing ◽  
Efficient Gene ◽  
Gene Function Analysis

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

2012 ◽  
Vol 39 (9) ◽  
pp. 764 ◽  
Author(s):  
Gi-Ho Lee ◽  
Seong-Han Sohn ◽  
Eun-Young Park ◽  
Young-Doo Park
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Camv 35S ◽  
Histone Deacetylase 1 ◽  
Transgenic Lines ◽  
Green Fluorescent

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

Discovery and Characterization of Differentially Expressed Soybean MiRNAs and Their Targets During Soybean Mosaic Virus Infection Unveils Novel Insight Into Soybean-SMV Interaction

2021 ◽  
Author(s):  
Bowen Li ◽  
Adhimoolam Karthikeyan ◽  
Liqun Wang ◽  
Jinlong Yin ◽  
Tongtong Jin ◽  
...  
Keyword(s):  
Virus Infection ◽  
Mosaic Virus ◽  
Target Genes ◽  
Soybean Mosaic Virus ◽  
Expression Patterns ◽  
Defense Responses ◽  
Pcr Analysis ◽  
Functional Annotations ◽  
Additional Information ◽  
Insight Into

Abstract Background: Soybean mosaic virus (SMV) is the most devastating pathogen of soybean. MicroRNAs (miRNAs) are a class of non-coding RNAs (21-24 nucleotides) and play important roles in regulating defense responses against pathogens. However, miRNA's response to SMV in soybean is not as well documented. Result: In this study, we analyzed 18 miRNA libraries, including three biological replicates from two soybean lines (Resistant and susceptible lines to SMV strain SC3 selected from the near-isogenic lines of Qihuang No. 1× Nannong1138-2) after virus infection at three different time intervals (0 dpi, 7 dpi, and 14 dpi). A total of 1,092 miRNAs, including 608 known miRNAs and 484 novel miRNAs were detected. Differential expression analyses identified the miRNAs responded during soybean-SMV interaction. Then, miRNAs potential target genes were predicted via data mining, and functional annotation was done by Gene Ontology (GO) analysis. Eventually, the expression patterns of several miRNAs validated by quantitative real-time PCR analysis are consistent with sequencing results. Conclusion: We have identified a large number of miRNAs and their target genes and also functional annotations. Our study provides additional information on soybean miRNAs and an insight into the role of miRNAs during SMV-infection in soybean.

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