scholarly journals An Efficient Brome mosaic virus-Based Gene Silencing Protocol for Hexaploid Wheat (Triticum aestivum L.)

2021 ◽  
Vol 12 ◽  
Author(s):  
Yongqin Wang ◽  
Chenglin Chai ◽  
Behnam Khatabi ◽  
Wolf-Rüdiger Scheible ◽  
Michael K. Udvardi ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Hexaploid Wheat ◽  
Time Course ◽  
Fluorescent Protein ◽  
Brome Mosaic Virus ◽  
Post Inoculation ◽  
Wheat Cultivars ◽  
Wheat Roots

Virus-induced gene silencing (VIGS) is a rapid and powerful method to evaluate gene function, especially for species like hexaploid wheat that have large, redundant genomes and are difficult and time-consuming to transform. The Brome mosaic virus (BMV)-based VIGS vector is widely used in monocotyledonous species but not wheat. Here we report the establishment of a simple and effective VIGS procedure in bread wheat using BMVCP5, the most recently improved BMV silencing vector, and wheat genes PHYTOENE DESATURASE (TaPDS) and PHOSPHATE2 (TaPHO2) as targets. Time-course experiments revealed that smaller inserts (~100 nucleotides, nt) were more stable in BMVCP5 and conferred higher silencing efficiency and longer silencing duration, compared with larger inserts. When using a 100-nt insert and a novel coleoptile inoculation method, BMVCP5 induced extensive silencing of TaPDS transcript and a visible bleaching phenotype in the 2nd to 5th systemically-infected leaves from nine to at least 28 days post inoculation (dpi). For TaPHO2, the ability of BMVCP5 to simultaneously silence all three homoeologs was demonstrated. To investigate the feasibility of BMV VIGS in wheat roots, ectopically expressed enhanced GREEN FLUORESCENT PROTEIN (eGFP) in a transgenic wheat line was targeted for silencing. Silencing of eGFP fluorescence was observed in both the maturation and elongation zones of roots. BMVCP5 mediated significant silencing of eGFP and TaPHO2 mRNA expression in roots at 14 and 21 dpi, and TaPHO2 silencing led to the doubling of inorganic phosphate concentration in the 2nd through 4th systemic leaves. All 54 wheat cultivars screened were susceptible to BMV infection. BMVCP5-mediated TaPDS silencing resulted in the expected bleaching phenotype in all eight cultivars examined, and decreased TaPDS transcript was detected in all three cultivars examined. This BMVCP5 VIGS technology may serve as a rapid and effective functional genomics tool for high-throughput gene function studies in aerial and root tissues and in many wheat cultivars.

scholarly journals Characterization of a Brome mosaic virus Strain and Its Use as a Vector for Gene Silencing in Monocotyledonous Hosts

2006 ◽  
Vol 19 (11) ◽  
pp. 1229-1239 ◽  
Author(s):  
Xin Shun Ding ◽  
William L. Schneider ◽  
Srinivasa Rao Chaluvadi ◽  
M. A. Rouf Mian ◽  
Richard S. Nelson
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Festuca Arundinacea ◽  
Systemic Infection ◽  
Brome Mosaic Virus ◽  
Expression Vectors ◽  
Virus Induced Gene Silencing ◽  
Dicotyledonous Plants ◽  
Virus Expression

Virus-induced gene silencing (VIGS) is used to analyze gene function in dicotyledonous plants but less so in monocotyledonous plants (particularly rice and corn), partially due to the limited number of virus expression vectors available. Here, we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb. (tall fescue) that caused systemic mosaic symptoms on barley, rice, and a specific cultivar of maize (Va35) under greenhouse conditions. Through sequencing, the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca), and genetic determinants that controlled the systemic infection of rice were mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Coinoculation of RNAs 1 and 2 from F-BMV and RNA 3 from R-BMV expressing a portion of a plant gene to leaves of barley, rice, and maize plants resulted in visual silencing-like phenotypes. The visual phenotypes were correlated with decreased target host transcript levels in the corresponding leaves. The VIGS visual phenotype varied from maintained during silencing of actin 1 transcript expression to transient with incomplete penetration through affected tissue during silencing of phytoene desaturase expression. F-BMV RNA 3 was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector C-BMVA/G (C for chimeric) was shown to be useful for VIGS. These BMV vectors will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

scholarly journals Tobacco mosaic virus 126-kDa Protein Increases the Susceptibility of Nicotiana tabacum to Other Viruses and Its Dosage Affects Virus-Induced Gene Silencing

2008 ◽  
Vol 21 (12) ◽  
pp. 1539-1548 ◽  
Author(s):  
Phillip A. Harries ◽  
Karuppaiah Palanichelvam ◽  
Sumana Bhat ◽  
Richard S. Nelson
Keyword(s):  
Nicotiana Tabacum ◽  
Gene Silencing ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Brome Mosaic Virus ◽  
Virus Induced Gene Silencing ◽  
Suppressor Activity

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein–green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

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.

scholarly journals Temperature-Dependent Wsm1 and Wsm2 Gene-Specific Blockage of Viral Long-Distance Transport Provides Resistance to Wheat streak mosaic virus and Triticum mosaic virus in Wheat

2016 ◽  
Vol 29 (9) ◽  
pp. 724-738 ◽  
Author(s):  
Satyanarayana Tatineni ◽  
Everlyne N. Wosula ◽  
Melissa Bartels ◽  
Gary L. Hein ◽  
Robert A. Graybosch
Keyword(s):  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Wheat Streak Mosaic Virus ◽  
Viral Resistance ◽  
Wheat Cultivars ◽  
Temperature Dependent ◽  
Long Distance ◽  
Long Distance Transport ◽  
Long Distance Movement ◽  
Distance Transport

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are economically important viral pathogens of wheat. Wheat cvs. Mace, carrying the Wsm1 gene, is resistant to WSMV and TriMV, and Snowmass, with Wsm2, is resistant to WSMV. Viral resistance in both cultivars is temperature sensitive and is effective at 18°C or below but not at higher temperatures. The underlying mechanisms of viral resistance of Wsm1 and Wsm2, nonallelic single dominant genes, are not known. In this study, we found that fluorescent protein–tagged WSMV and TriMV elicited foci that were approximately similar in number and size at 18 and 24°C, on inoculated leaves of resistant and susceptible wheat cultivars. These data suggest that resistant wheat cultivars at 18°C facilitated efficient cell-to-cell movement. Additionally, WSMV and TriMV efficiently replicated in inoculated leaves of resistant wheat cultivars at 18°C but failed to establish systemic infection, suggesting that Wsm1- and Wsm2-mediated resistance debilitated viral long-distance transport. Furthermore, we found that neither virus was able to enter the leaf sheaths of inoculated leaves or crowns of resistant wheat cultivars at 18°C but both were able to do so at 24°C. Thus, wheat cvs. Mace and Snowmass provide resistance at the long-distance movement stage by specifically blocking virus entry into the vasculature. Taken together, these data suggest that both Wsm1 and Wsm2 genes similarly confer virus resistance by temperature-dependent impairment of viral long-distance movement.

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 A High-Throughput Gene-Silencing System for the Functional Assessment of Defense-Related Genes in Barley Epidermal Cells

2005 ◽  
Vol 18 (8) ◽  
pp. 755-761 ◽  
Author(s):  
D. Douchkov ◽  
D. Nowara ◽  
U. Zierold ◽  
P. Schweizer
Keyword(s):  
Gene Silencing ◽  
High Throughput ◽  
Gene Function ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Epidermal Cells ◽  
Powdery Mildew Fungus ◽  
Nonspecific Resistance ◽  
Knock Down ◽  
Highly Efficient

Large-scale gene silencing by RNA interference (RNAi) offers the possibility to address gene function in eukaryotic organisms at a depth unprecedented until recently. Although genome-wide RNAi approaches are being carried out in organisms like Caenorhabditis elegans, Drosophila spp. or human after the corresponding tools had been developed, knock-down of only single or a few genes by RNAi has been reported in plants thus far. Here, we present a method for high-throughput, transient-induced gene silencing (TIGS) by RNAi in barley epidermal cells that is based on biolistic transgene delivery. This method will be useful to address gene function of shoot epidermis resulting in cell-autonomous phenotypes such as resistance or susceptibility to the powdery-mildew fungus Blumeria graminis f. sp. hordei. Gene function in epidermal cell elongation, stomata regulation, or UV resistance might be addressed as well. Libraries of RNAi constructs can be built up by a new, cost-efficient method that combines highly efficient ligation and recombination by the Gateway cloning system. This method allows cloning of any blunt-ended DNA fragment without the need of adaptor sequences. The final RNAi destination vector was found to direct highly efficient RNAi, as reflected by complete knock-down of a cotransformed green fluorescent protein reporter gene as well as by complete phenolcopy of the recessive loss-of-function mlo resistance gene. By using this method, a role of the t-SNARE proteinin three types of durable, race-nonspecific resistance was observed.

scholarly journals An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits

2021 ◽  
Vol 12 ◽  
Author(s):  
Yifan Wang ◽  
Ning Huang ◽  
Niu Ye ◽  
Lingyu Qiu ◽  
Yadong Li ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Function Analysis ◽  
Juglans Regia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tobacco Rattle Virus ◽  
Post Inoculation ◽  
Virus Induced Gene Silencing ◽  
Gene Function Analysis

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

scholarly journals A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants

2019 ◽  
Author(s):  
Mei Liu ◽  
Zhiling Liang ◽  
Miguel A. Aranda ◽  
Ni Hong ◽  
Liming Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Nicotiana Benthamiana ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Economic Significance ◽  
Potential Alternative ◽  
Low Efficiency

AbstractCucurbits produce fruits or vegetables that have great dietary importance and economic significance worldwide. The published genomes of at least 11 cucurbit species are boosting gene mining and novel breeding strategies, however genetic transformation in cucurbits is impractical as a tool for gene function validation due to low transformation efficiencies. Virus-induced gene silencing (VIGS) is a potential alternative tool. So far, very few ideal VIGS vectors are available for cucurbits. Here, we describe a new VIGS vector derived from cucumber green mottle mosaic virus (CGMMV), a monopartite virus that infects cucurbits naturally. We show that the CGMMV vector is competent to induce efficient silencing of the phytoene desaturase (PDS) gene in the model plant Nicotiana benthamiana and in cucurbits, including watermelon, melon, cucumber and bottle gourd. Infection with the CGMMV vector harboring PDS sequences of 69-300 bp in length in the form of sense-oriented or hairpin cDNAs resulted in photobleaching phenotypes in N. benthamiana and cucurbits by PDS silencing. Additional results reflect that silencing of the PDS gene could persist for over two months and the silencing effect of CGMMV-based vectors could be passaged. These results demonstrate that CGMMV vector could serve as a powerful and easy-to-use tool for characterizing gene function in cucurbits.One sentence summaryA CGMMV-based vector enables gene function studies in cucurbits, an extremely low efficiency species for genetic transformation.

Efficient and high-throughput pseudorecombinant-chimeric Cucumber mosaic virus-based VIGS in maize

2021 ◽  
Author(s):  
Huangai Li ◽  
Danfeng Zhang ◽  
Ke Xie ◽  
Yan Wang ◽  
Qiansheng Liao ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
High Throughput ◽  
Large Scale ◽  
Growth Period ◽  
Post Inoculation ◽  
Virus Induced Gene Silencing ◽  
Combination Strategy ◽  
Gene Characterization

Abstract Virus-induced gene silencing (VIGS) is a versatile and attractive approach for functional gene characterization in plants. Although several VIGS vectors for maize (Zea mays) have been previously developed, their utilities are limited due to low viral infection efficiency, insert instability, short maintenance of silencing, inadequate inoculation method, or abnormal requirement of growth temperature. Here, we established a Cucumber mosaic virus (CMV)-based VIGS system for efficient maize gene silencing that overcomes many limitations of VIGS currently available for maize. Using two distinct strains, CMV-ZMBJ and CMV-Fny, we generated a pseudorecombinant-chimeric (Pr) CMV. Pr CMV showed high infection efficacy but mild viral symptoms in maize. We then constructed Pr CMV-based vectors for VIGS, dubbed Pr CMV VIGS. Pr CMV VIGS is simply performed by mechanical inoculation of young maize leaves with saps of Pr CMV-infected Nicotiana benthamiana under normal growth conditions. Indeed, suppression of isopentenyl/dimethylallyl diphosphate synthase (ZmIspH) expression by Pr CMV VIGS resulted in non-inoculated leaf bleaching as early as 5 d post-inoculation (dpi) and exhibited constant and efficient systemic silencing over the whole maize growth period up to 105 dpi. Furthermore, utilizing a ligation-independent cloning (LIC) strategy, we developed a modified Pr CMV-LIC VIGS vector, allowing easy gene cloning for high-throughput silencing in maize. Thus, our Pr CMV VIGS system provides a much-improved toolbox to facilitate efficient and long-duration gene silencing for large-scale functional genomics in maize, and our pseudorecombination-chimera combination strategy provides an approach to construct efficient VIGS systems in plants.

scholarly journals Brome mosaic virus defective RNAs generated during infection of barley plants

1999 ◽  
Vol 80 (9) ◽  
pp. 2511-2518 ◽  
Author(s):  
Tri Asmira Damayanti ◽  
Hideaki Nagano ◽  
Kazuyuki Mise ◽  
Iwao Furusawa ◽  
Tetsuro Okuno
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Helper Virus ◽  
Brome Mosaic Virus ◽  
Cdna Clones ◽  
Post Inoculation ◽  
Dependent Manner ◽  
Genomic Rnas ◽  
Defective Rnas

Brome mosaic virus (BMV) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective RNAs (D-RNAs). The D-RNAs were detected in total and virion RNAs extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking D-RNA. The D-RNAs were derived from genomic RNA3 by double or mainly single deletions in the 3a protein ORF, and formed a heterogeneous population. By using in vitro transcripts of D-RNA synthesized from full-length cDNA clones, the D-RNAs were shown to replicate in a helper virus-dependent manner and to be packaged into virions in barley protoplasts. Subgenomic RNA4 was produced from the D-RNA and the coat protein was also expressed. Existence of the D-RNAs together with BMV genomic RNAs in inoculated protoplasts decreased the accumulation of 3a protein but it had no apparent effect on the accumulation of BMV genomic RNA3 or the coat protein. This is the first report of naturally occurring D-RNAs generated during prolonged infection with BMV.

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