scholarly journals Secondary siRNAs in Plants: Biosynthesis, Various Functions, and Applications in Virology

2021 ◽  
Vol 12 ◽  
Author(s):  
Neeti Sanan-Mishra ◽  
A. Abdul Kader Jailani ◽  
Bikash Mandal ◽  
Sunil K. Mukherjee
Keyword(s):  
Gene Silencing ◽  
Small Rnas ◽  
Rna Silencing ◽  
Viral Infections ◽  
Natural Resistance ◽  
Virus Induced Gene Silencing ◽  
Systemic Spread ◽  
Secondary Sirnas ◽  
Silencing Pathways ◽  
Virus Interactions

The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, and trans-species transfer have also been detailed. The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant–virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.

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.

A Virus-Induced Gene-Silencing Vector Based on a Mild Isolate of Watermelon Mosaic Virus for Reverse Genetic Analyses in Cucurbits

2021 ◽  
Author(s):  
Fakhreddine Houhou ◽  
Verónica Aragonés ◽  
Anamarija Butković ◽  
Cristina Sáez ◽  
Belén Picó ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Rna Silencing ◽  
Viral Infections ◽  
Watermelon Mosaic Virus ◽  
Reverse Genetic ◽  
Virus Induced Gene Silencing ◽  
Genetic Analyses ◽  
Endogenous Gene ◽  
Mild Isolate

Abstract As a response to viral infections, host plants trigger an RNA-mediated gene silencing defense, to which viruses respond with the expression of viral-encoded RNA silencing suppressors. If virus clones are manipulated to include sequences homologous to host endogenous genes, these are also targeted by the plant RNA silencing machinery. This so-called virus-induced gene silencing (VIGS) has become a powerful technique for reverse genetic analyses in plants, as an alternative to labor-intensive genome transformation. We show that a mild isolate of Watermelon mosaic virus (WMV, genus Potyvirus) can be used as a VIGS vector for reverse genetic analyses in melon. Recombinant WMV clones —in which fragments of the melon Phytoene desaturase (PDS) mRNA were inserted in sense, antisense, and hairpin modalities— induced a distinctive phenotype and significant silencing of the endogenous gene. While the foreign fragments in sense and antisense orientations were stable in the viral progeny, the hairpin was quickly lost. Nevertheless, the hairpin construct triggered a maintained silencing effect comparable to those of the sense and antisense constructs. The suitability of WMV as a VIGS vector was further confirmed targeting melon Magnesium chelatase subunit I (CHLI). These results also support that, although potyviruses express a strong silencing suppressor that usually precludes VIGS, mild isolates of this kind of viruses can be used as VIGS vectors. Finally, to facilitate the use of this new tool by cucurbit geneticists, we describe plasmid pGWMV-VIGS that allows easy cloning fragments of the genes of interest in a single Gibson assembly reaction.

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.

scholarly journals Development of a Viral RdRp-Assisted Gene Silencing System and Its Application in the Identification of Host Factors of Plant (+)RNA Virus

2021 ◽  
Vol 12 ◽  
Author(s):  
Wang Zhang ◽  
Yanglin Qiu ◽  
Lingyun Zhou ◽  
Jinlong Yin ◽  
Liqun Wang ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Secretory Pathway ◽  
Rna Virus ◽  
Expression System ◽  
Mrna Levels ◽  
Virus Induced Gene Silencing ◽  
Hairpin Rna ◽  
Transgenic Expression ◽  
Early Secretory Pathway ◽  
Virus Interactions

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions.

scholarly journals Virus and Viroid-Derived Small RNAs as Modulators of Host Gene Expression: Molecular Insights Into Pathogenesis

2021 ◽  
Vol 11 ◽  
Author(s):  
S. V. Ramesh ◽  
Sneha Yogindran ◽  
Prabu Gnanasekaran ◽  
Supriya Chakraborty ◽  
Stephan Winter ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Small Rnas ◽  
Rna Silencing ◽  
Transcriptional Gene Silencing ◽  
Genomic Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Viroid Infection ◽  
Wide Range ◽  
Associated Symptoms ◽  
Sequence Complementarity

Virus-derived siRNAs (vsiRNAs) generated by the host RNA silencing mechanism are effectors of plant’s defense response and act by targeting the viral RNA and DNA in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) pathways, respectively. Contrarily, viral suppressors of RNA silencing (VSRs) compromise the host RNA silencing pathways and also cause disease-associated symptoms. In this backdrop, reports describing the modulation of plant gene(s) expression by vsiRNAs via sequence complementarity between viral small RNAs (sRNAs) and host mRNAs have emerged. In some cases, silencing of host mRNAs by vsiRNAs has been implicated to cause characteristic symptoms of the viral diseases. Similarly, viroid infection results in generation of sRNAs, originating from viroid genomic RNAs, that potentially target host mRNAs causing typical disease-associated symptoms. Pathogen-derived sRNAs have been demonstrated to have the propensity to target wide range of genes including host defense-related genes, genes involved in flowering and reproductive pathways. Recent evidence indicates that vsiRNAs inhibit host RNA silencing to promote viral infection by acting as decoy sRNAs. Nevertheless, it remains unclear if the silencing of host transcripts by viral genome-derived sRNAs are inadvertent effects due to fortuitous pairing between vsiRNA and host mRNA or the result of genuine counter-defense strategy employed by viruses to enhance its survival inside the plant cell. In this review, we analyze the instances of such cross reaction between pathogen-derived vsiRNAs and host mRNAs and discuss the molecular insights regarding the process of pathogenesis.

Silence is green

2004 ◽  
Vol 32 (6) ◽  
pp. 946-951 ◽  
Author(s):  
A.J. Herr
Keyword(s):  
Nucleic Acids ◽  
Small Rnas ◽  
Rna Silencing ◽  
Recent Progress ◽  
Gene Families ◽  
Regulatory Mechanisms ◽  
Endogenous Genes ◽  
Silencing Pathways

Small RNAs serve as the specificity determinant for a collection of regulatory mechanisms known as RNA silencing. Plants use these mechanisms to control the expression of endogenous genes and to suppress unwanted foreign nucleic acids. Several gene families implicated in silencing have undergone expansion and evidence exists for multiple RNA silencing pathways. Recent progress in defining the components of a number of these pathways is examined here.

scholarly journals Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain

2021 ◽  
Author(s):  
Shirui Chen ◽  
Wei Liu ◽  
Masahiro Naganuma ◽  
Yukihide Tomari ◽  
Hiro-oki Iwakawa
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Functional Differentiation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Secondary Sirnas ◽  
Fixed End ◽  
Paz Domain ◽  
Reproductive Processes

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt heterochromatic small interfering RNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs). The former small RNAs are linked to plant heterochromatin, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient "eudicot-type" DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 3′ overhangs and 5′ phosphates, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5 which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

scholarly journals Expression dynamics of ARGONAUTE proteins during meiosis in Arabidopsis

2021 ◽  
Author(s):  
Cecilia Oliver ◽  
German Martinez
Keyword(s):  
Genetic Diversity ◽  
Gene Silencing ◽  
Rna Silencing ◽  
Potential Role ◽  
Specific Activity ◽  
Male Meiosis ◽  
Transcriptional Gene Silencing ◽  
Posttranscriptional Gene Silencing ◽  
Argonaute Proteins ◽  
Silencing Pathways

Meiosis is a specialized cell division that is key for reproduction and genetic diversity in sexually reproducing plants. Recently, different RNA silencing pathways have been proposed to carry a specific activity during meiosis, but the pathways involved during this process remain unclear. Here, we explored the subcellular localization of different ARGONAUTE (AGO) proteins, the main effectors of RNA silencing, during male meiosis in Arabidopsis thaliana using immunolocalizations with commercially available antibodies. We detected the presence of AGO proteins associated with posttranscriptional gene silencing (AGO1, 2 and 5) in the cytoplasm or the nucleus, while AGOs associated with transcriptional gene silencing (AGO4 and 9) localized exclusively in the nucleus. These results indicate that the localization of different AGOs correlates with their predicted roles at the transcriptional and posttranscriptional levels and provide an overview of their timing and potential role during meiosis.

scholarly journals Efficient Infection of Nicotiana benthamiana by Tomato bushy stunt virus Is Facilitated by the Coat Protein and Maintained by p19 Through Suppression of Gene Silencing

2002 ◽  
Vol 15 (3) ◽  
pp. 193-202 ◽  
Author(s):  
Feng Qu ◽  
T. Jack Morris
Keyword(s):  
Coat Protein ◽  
Gene Silencing ◽  
Vascular System ◽  
Systemic Infection ◽  
Red Clover ◽  
Plant Viruses ◽  
Tomato Bushy Stunt Virus ◽  
Careful Examination ◽  
Virus Induced Gene Silencing ◽  
Systemic Spread

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana ben-thamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. ben-thamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.

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