scholarly journals Applications of virus-induced gene silencing for identification of gene function in fruit

2021 ◽  
Vol 5 ◽  
Author(s):  
Gangshuai Liu ◽  
Hongli Li ◽  
Daqi Fu
Keyword(s):  
Gene Silencing ◽  
Fruit Quality ◽  
Gene Function ◽  
Biological Process ◽  
Virus Induced Gene Silencing ◽  
Functional Identification ◽  
Advantages And Disadvantages ◽  
Research Fields ◽  
Complex Biological Process

Abstract With the development of bioinformatics, it is easy to obtain information and data about thousands of genes, but the determination of the functions of these genes depends on methods for rapid and effective functional identification. Virus-induced gene silencing (VIGS) is a mature method of gene functional identification developed over the last 20 years, which has been widely used in many research fields involving many species. Fruit quality formation is a complex biological process, which is closely related to ripening. Here, we review the progress and contribution of VIGS to our understanding of fruit biology and its advantages and disadvantages in determining gene function.

scholarly journals Optimization of a virus-induced gene silencing system and functional elucidation of MaBAM9b in postharvest banana fruits

2020 ◽  
Author(s):  
juhua liu ◽  
Meng Li ◽  
Jing Zhang ◽  
Hongxia Miao ◽  
Jingyi Wang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Fruit Quality ◽  
Gene Function ◽  
Fruit Ripening ◽  
Starch Content ◽  
Soluble Sugar ◽  
Starch Degradation ◽  
Banana Fruit ◽  
Virus Induced Gene Silencing ◽  
Starch Conversion

Abstract Background: Banana is a typical starch conversion fruit, and fruit ripening involves a process of fruit quality formation. To improve the nutritional value of banana, it is necessary to understand the genetic basis of the metabolic pathways that operate during fruit ripening processes. MaBAM9b is a key enzyme gene that might play an important role in starch degradation during the banana fruit ripening process. The identification of gene function by stable genetic transformation is time- and energy-consuming. Thus, developing an efficient and rapid method for functional identification is imperative. Virus-Induced Gene Silencing (VIGS) is a reverse-genetics method based on RNA-mediated antiviral plant defense that has been used to rapidly identify gene function in plants. Results: In this report, 0.5% iodine-potassium-iodide (I2-KI) staining for 150 s determined that a 1:3 ratio of TRV1: TRV2-MaBAM9b cultivated at an optical density of 600 nm (OD600) 0.8 at 30 mmHg for 30 sec and kept on Murashige & Skoog (MS) media for 5 d produced the best silencing results. Under these conditions, the total starch content was greatly increased, while the β-amylase activity, soluble sugar contents, and the expression of endogenous MaBAM9b were greatly decreased. Conclusions: The developed system is particularly useful for studying genes and networks for starch conversion in fruit, which alone would not produce a visual phenotype. This system will provide a platform for banana functional genomics and for banana fruit quality improvement.

scholarly journals Rapid Determination of Gene Function by Virus-induced Gene Silencing in Wheat and Barley

Crop Science ◽  
2010 ◽  
Vol 50 ◽  
pp. S-77-S-84 ◽  
Author(s):  
Cahid Cakir ◽  
Megan E. Gillespie ◽  
Steven R. Scofield
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Rapid Determination ◽  
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.

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 Rose Phytoene Desaturase Gene Silencing by Apple Latent Spherical Virus Vectors

HortScience ◽  
2012 ◽  
Vol 47 (9) ◽  
pp. 1278-1282 ◽  
Author(s):  
Hiroaki Ito ◽  
Masaki Ochiai ◽  
Hiroaki Kato ◽  
Katsuhiro Shiratake ◽  
Daigo Takemoto ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Molecular Breeding ◽  
Phytoene Desaturase ◽  
Virus Induced Gene Silencing ◽  
Desaturase Gene ◽  
Apple Latent Spherical Virus ◽  
The Rose ◽  
Highly Uniform ◽  
Secondary Shoot

We have succeeded in establishing a virus-induced gene silencing (VIGS) of rose using Apple latent spherical virus (ALSV) vectors. An ALSV infection on rose did not cause any symptoms like those observed on other plant species and grew healthy. We have cloned and sequenced the phytoene desaturase (PDS) gene in wild rose, then used its fragment for silencing the rose internal PDS gene. The silencing phenotypes such as the highly uniform photo-bleached phenotype with PDS inhibitions were observed on the upper leaves of primary shoots and on a secondary shoot of R. rugosa for more than 5 months. ALSV vectors seemed useful for analyzing gene function and for the molecular breeding of rose.

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.

Induction of virus-induced gene silencing and in planta validation in cucurbits using the CFMMV-Cm vector

2021 ◽  
Author(s):  
Gung Pyo Lee ◽  
Sun-Ju Rhee ◽  
Yoon Jeong Jang ◽  
Jun-Young Park
Keyword(s):  
Gene Silencing ◽  
Male Sterility ◽  
Differentially Expressed Genes ◽  
High Throughput ◽  
Gene Function ◽  
De Novo ◽  
The Novel ◽  
Virus Induced Gene Silencing ◽  
Male Sterile ◽  
High Throughput Analysis

Virus-induced gene silencing (VIGS) has been employed for the high-throughput analysis of endogenous gene function. We developed a CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) for the efficient generation of viral transcripts in plants. Using the novel pCF93 vector, we identified genes related to male sterility in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. We previously reported reference-based and de novo transcriptomic profiling for the detection of differentially expressed genes between a male fertile line (DAH3615) and its near isogenic male sterile line (DAH3615-MS). Based on the RNA-seq results, we identified 38 de novo-exclusive differentially expressed genes (DEDEGs) that are potentially responsible for male sterility. Partial genes of 200~300bp were cloned into pCF93 which was then inoculated into DAH, a small type of watermelon that enables high-throughput screening with a small cultivation area. In this manner, we simultaneously characterized phenotypes associated with the 38 candidate genes in a common-sized greenhouse. Eight out of the 38 gene-silenced plants produced male sterile flowers with abnormal stamens and no pollens. Gene expression levels in flowers were validated via RT-qPCR. Stamen histological sections from male sterile floral buds and mature flowers showed developmental disruption and shrunken pollen sacs. Based on the current findings, we believe that the novel pCF93 vector and our VIGS system facilitate high-throughput analysis for the study of gene function in watermelons.

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.

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