scholarly journals RNA Interference: A Versatile Tool for Functional Genomics and Unraveling the Genes Required for Viral Disease Resistance in Plants

2019 ◽  
pp. 1-20 ◽  
Author(s):  
Tushar Ranjan ◽  
Namaste Kumari ◽  
Sangita Sahni ◽  
Bishun Deo Prasad
Keyword(s):  
Disease Resistance ◽  
Gene Silencing ◽  
Defense Mechanisms ◽  
Reverse Genetics ◽  
Viral Disease ◽  
Virus Induced Gene Silencing ◽  
Advantages And Disadvantages ◽  
Natural Defense ◽  
Versatile Tool ◽  
Plant Genes

Virus-induced gene silencing (VIGS) is a powerful reverse genetics technology used to unravel the functions of genes. It uses viruses as vectors to carry targeted plant genes. The virus vector is used to induce RNA-mediated silencing of a gene or genes in the host plant. The process of silencing is triggered by dsRNA molecules, the mechanism is explained in this chapter. Over the years a large number of viruses have been modified for use as VIGS vectors and a list of these vectors is also included. As the name suggests, virus-induced gene silencing uses the host plant’s natural defense mechanisms against viral infection to silence plant genes. VIGS is methodologically simple and is widely used to determine gene functions, including disease resistance, abiotic stress, biosynthesis of secondary metabolites and signal transduction pathways. Here, we made an attempt to describe the basic underlying molecular mechanism of VIGS, the methodology and various experimental requirements, as well as its advantages and disadvantages. Finally, we discuss the future prospects of VIGS in relation to CRISPR/Cas9 technology. Besides using it to overexpress or silence genes, VIGS has emerged as the preferred delivery system for the cutting edge CRISPR/Cas9 genome editing technology.

scholarly journals Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kunxin Wu ◽  
Yadan Wu ◽  
Chunwei Zhang ◽  
Yan Fu ◽  
Zhixin Liu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Double Mutant ◽  
Target Genes ◽  
Rna Binding ◽  
Protein Product ◽  
Antiviral Defense ◽  
Virus Induced Gene Silencing ◽  
Argonaute 2 ◽  
Plant Genes ◽  
Silencing Pathways

Abstract Background Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. Results In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. Conclusions The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

Evaluation of virus-induced gene silencing methods for forage legumes including alfalfa, sainfoin, and fenugreek

2019 ◽  
Vol 99 (6) ◽  
pp. 917-926 ◽  
Author(s):  
Champa Wijekoon ◽  
Stacy D. Singer ◽  
Randall J. Weselake ◽  
Udaya Subedi ◽  
Surya N. Acharya
Keyword(s):  
Gene Silencing ◽  
Plant Species ◽  
Reverse Genetics ◽  
Functional Characterization ◽  
Homologous Gene ◽  
Virus Induced Gene Silencing ◽  
Forage Legumes ◽  
Crop Species ◽  
Barrel Medic ◽  
Trigonella Foenum Graecum L

Virus-induced gene silencing (VIGS) is a rapid reverse genetics tool that has been developed in a wide variety of plant species for assessing gene functions. However, while VIGS has been utilized successfully in the diploid model leguminous species Medicago truncatula (Gaertn.) (barrel medic), such a platform has yet to be established in forage legume crop species. Therefore, we evaluated the effectiveness of this method in forage legumes using a previously developed PEBV (pea early browning virus) system whereby a fragment of the pea (Pisum sativum L.) PHYTOENE DESATURASE (PDS) gene was transferred into a range of alfalfa (Medicago sativa L.), sainfoin (Onobrychis viciifolia Scop.), and fenugreek (Trigonella foenum-graecum L.) cultivars using leaf infiltration and apical meristem injection. Barrel medic was used as a positive control. Gene silencing was observed after 10–15 d through the presence of a leaf bleaching phenotype, and was confirmed using quantitative real-time RT-PCR. Silencing of PDS was achieved in a selection of cultivars in all species assessed, with the highest silencing efficiency apparent in fenugreek. The introduction of a highly homologous gene fragment from a heterologous plant species to target endogenous genes for transient VIGS-based silencing in a range of species of interest represents a potentially useful strategy for the rapid functional characterization of candidate genes in forages.

scholarly journals Virus-induced gene silencing for Asteraceae-a reverse genetics approach for functional genomics in Gerbera hybrida

2012 ◽  
Vol 10 (8) ◽  
pp. 970-978 ◽  
Author(s):  
Xianbao Deng ◽  
Paula Elomaa ◽  
Cuong X. Nguyen ◽  
Timo Hytönen ◽  
Jari P. T. Valkonen ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Functional Genomics ◽  
Reverse Genetics ◽  
Virus Induced Gene Silencing ◽  
Gerbera Hybrida

scholarly journals Simultaneous Silencing of Two Different Arabidopsis Genes With A Novel Virus-Induced Gene Silencing Vector

2020 ◽  
Author(s):  
Xiuchun Zhang ◽  
Yadan Wu ◽  
Chunwei Zhang ◽  
Kunxin Wu ◽  
Zhixin Liu
Keyword(s):  
Gene Silencing ◽  
Rna Silencing ◽  
Target Genes ◽  
Rna Binding ◽  
Protein Product ◽  
Turnip Crinkle Virus ◽  
Virus Induced Gene Silencing ◽  
Argonaute 2 ◽  
Plant Genes ◽  
Silencing Pathways

Abstract Background: Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. Results: In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to evaluate the relative importance of several known RNA silencing pathway genes in PDS VIGS. This approach allowed us to validate the involvement of DICER-LIKE 4 (DCL4) and ARGONAUTE 2 (AGO2) in PDS silencing. Notably, double-stranded RNA-binding protein 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B. Conclusions: The CPB1B-based vector developed in this work is a valuable tool with tracable and visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

scholarly journals TaBln1 negatively regulates wheat resistance to stripe rust by reducing Ca2+ influx

2021 ◽  
Author(s):  
Shuangyuan Guo ◽  
Yanqin Zhang ◽  
Peng Zeng ◽  
Min Li ◽  
Qiong Zhang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Defense Mechanisms ◽  
Molecular Mechanisms ◽  
Puccinia Striiformis ◽  
Negative Regulator ◽  
Virus Induced Gene Silencing ◽  
Test Technique ◽  
Cereal Grain ◽  
Host Defense Response

Blufensin1 (Bln1) has been identified as a negative regulator of basal defense mechanisms that is unique to the cereal grain crops barley, wheat, and rice. However, the molecular mechanisms through which Blufensin1 regulates the wheat immune response are poorly understood. In this study, we found that TaBln1 is significantly induced by Puccinia striiformis f. sp. tritici (Pst) virulent race CYR31 infection. Knockdown the expression of TaBln1 by virus-induced gene silencing reduced Pst growth and development, and enhanced the host defense response. In addition, TaBln1 was found to physically interact with TaCaM3 on the plasma membrane. Silencing TaCaM3 with virus-induced gene silencing increased fungal infection areas and sporulation and reduced wheat resistance to the Pst CYR23 and CYR31. Moreover, we found that the TaCaM3 transcription level could be induced by treatment with chitin but not flg22. Silencing TaCaM3 decreased the Ca2+ influx induced by chitin, but silencing TaBln1 increased the Ca2+ influx in vivo using a non-invasive micro-test technique. Taken together, we identified the wheat negative regulator TaBln1, which interacts with TaCaM3 to impair Ca2+ influx and inhibits plant defenses.

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