Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex, C3PO, Determines Their Regulation by Phospholipase Cβ

A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate–glutamate–alanine–histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

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Evidence That RNA Silencing-Mediated Resistance to Beet necrotic yellow vein virus Is Less Effective in Roots Than in Leaves

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0194 ◽

2005 ◽

Vol 18 (3) ◽

pp. 194-204 ◽

Cited By ~ 71

Author(s):

Ida Bagus Andika ◽

Hideki Kondo ◽

Tetsuo Tamada

Keyword(s):

Nicotiana Benthamiana ◽

Rna Silencing ◽

Defense Mechanism ◽

Virus Titer ◽

Virus Multiplication ◽

Open Reading Frame ◽

Yellow Vein ◽

Polymyxa Betae ◽

Small Interfering Rnas ◽

Reading Frame

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

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Role of RNA Silencing Suppression in the Pathogenicity and Host Specificity of the Grapevine Virus A

10.32747/2010.7592114.bard ◽

2010 ◽

Author(s):

Munir Mawassi ◽

Valerian Dolja

Keyword(s):

Rna Silencing ◽

Defense Mechanism ◽

Plant Viruses ◽

Small Interfering Rnas ◽

Grapevine Virus ◽

Virus Transport ◽

Systematic Analysis ◽

Grapevine Virus A ◽

Silencing Suppression ◽

And Function

RNA silencing is a defense mechanism that functions against virus infection and involves sequence-specific degradation of viral RNA. Diverse RNA and DNA viruses of plants encode RNA silencing suppressors (RSSs), which, in addition to their role in viral counterdefense, were implicated in the efficient accumulation of viral RNAs, virus transport, pathogenesis, and determination of the virus host range. Despite rapidly growing understanding of the mechanisms of RNA silencing suppression, systematic analysis of the roles played by diverse RSSs in virus biology and pathology is yet to be completed. Our research was aimed at conducting such analysis for two grapevine viruses, Grapevine virus A (GVA) and Grapevine leafroll-associated virus-2 (GLRaV- 2). Our major achievements on the previous cycle of BARD funding are as follows. 1. GVA and GLRaV-2 were engineered into efficient gene expression and silencing vectors for grapevine. The efficient techniques for grapevine infection resulting in systemic expression or silencing of the recombinant genes were developed. Therefore, GVA and GLRaV-2 were rendered into powerful tools of grapevine virology and functional genomics. 2. The GVA and GLRaV-2 RSSs, p10 and p24, respectively, were identified, and their roles in viral pathogenesis were determined. In particular, we found that p10 functions in suppression and pathogenesis are genetically separable. 3. We revealed that p10 is a self-interactive protein that is targeted to the nucleus. In contrast, p24 mechanism involves binding small interfering RNAs in the cytoplasm. We have also demonstrated that p10 is relatively weak, whereas p24 is extremely strong enhancer of the viral agroinfection. 4. We found that, in addition to the dedicated RSSs, GVA and GLRaV-2 counterdefenses involve ORF1 product and leader proteases, respectively. 5. We have teamed up with Dr. Koonin and Dr. Falnes groups to study the evolution and function of the AlkB domain presents in GVA and many other plant viruses. It was demonstrated that viral AlkBs are RNA-specific demethylases thus providing critical support for the biological relevance of the novel process of AlkB-mediated RNA repair.

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A Single dicer Gene Is Required for Efficient Gene Silencing Associated with Two Classes of Small Antisense RNAs in Mucor circinelloides

Eukaryotic Cell ◽

10.1128/ec.00191-09 ◽

2009 ◽

Vol 8 (10) ◽

pp. 1486-1497 ◽

Cited By ~ 47

Author(s):

Juan P. de Haro ◽

Silvia Calo ◽

María Cervantes ◽

Francisco E. Nicolás ◽

Santiago Torres-Martínez ◽

...

Keyword(s):

Rna Silencing ◽

Critical Role ◽

Gene Replacement ◽

Mucor Circinelloides ◽

Null Mutant ◽

Small Interfering Rnas ◽

Vegetative Development ◽

Hairpin Rna ◽

Antisense Rnas ◽

Efficient Gene

ABSTRACT RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2 − transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores.

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84. RNA Silencing: Role of Interferon System Components in Non_Specific Effects Induced by Chemically Synthesized Small Interfering RNAs, Enzymatically Synthesized Small Interfering RNAs and Short Hairpin RNAs

Molecular Therapy ◽

10.1016/j.ymthe.2006.08.102 ◽

2006 ◽

Vol 13 ◽

pp. S35

Author(s):

Christopher S. McAllister ◽

Charles E. Samuel

Keyword(s):

Rna Silencing ◽

Small Interfering Rnas ◽

System Components ◽

Interferon System ◽

Short Hairpin ◽

Short Hairpin Rnas

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RNA Silencing against Geminivirus: Complementary Action of Posttranscriptional Gene Silencing and Transcriptional Gene Silencing in Host Recovery

Journal of Virology ◽

10.1128/jvi.01474-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1332-1340 ◽

Cited By ~ 105

Author(s):

Edgar A. Rodríguez-Negrete ◽

Jimena Carrillo-Tripp ◽

Rafael F. Rivera-Bustamante

Keyword(s):

Gene Silencing ◽

Intergenic Region ◽

Rna Silencing ◽

Methylation Status ◽

Inverse Correlation ◽

Plant Viruses ◽

Transcriptional Gene Silencing ◽

Small Interfering Rnas ◽

Coding Region ◽

Natural Defense

ABSTRACT RNA silencing in plants is a natural defense system mechanism against invading nucleic acids such as viruses. Geminiviruses, a family of plant viruses characterized by a circular, single-stranded DNA genome, are thought to be both inducers and targets of RNA silencing. Some natural geminivirus-host interactions lead to symptom remission or host recovery, a process commonly associated with RNA silencing-mediated defense. Pepper golden mosaic virus (PepGMV)-infected pepper plants show a recovery phenotype, which has been associated with the presence of virus-derived small RNAs. The results presented here suggest that PepGMV is targeted by both posttranscriptional and transcriptional gene silencing mechanisms. Two types of virus-related small interfering RNAs (siRNAs) were detected: siRNAs of 21 to 22 nucleotides (nt) in size that are related to the coding regions (Rep, TrAP, REn, and movement protein genes) and a 24-nt population primarily associated to the intergenic regions. Methylation levels of the PepGMV A intergenic and coat protein (CP) coding region were measured by a bisulfite sequencing approach. An inverse correlation was observed between the methylation status of the intergenic region and the concentration of viral DNA and symptom severity. The intergenic region also showed a methylation profile conserved in all times analyzed. The CP region, on the other hand, did not show a defined profile, and its methylation density was significantly lower than the one found on the intergenic region. The participation of both PTGS and TGS mechanisms in host recovery is discussed.

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Inhibition of RNA silencing by the coat protein of Pelargonium flower break virus: distinctions from closely related suppressors

Journal of General Virology ◽

10.1099/vir.0.006098-0 ◽

2009 ◽

Vol 90 (2) ◽

pp. 519-525 ◽

Cited By ~ 25

Author(s):

Sandra Martínez-Turiño ◽

Carmen Hernández

Keyword(s):

Coat Protein ◽

Potato Virus ◽

Rna Silencing ◽

Potato Virus X ◽

Plant Viruses ◽

Small Interfering Rnas ◽

Silencing Suppression ◽

Viral Suppressors

Viral-derived double-stranded RNAs (dsRNAs) activate RNA silencing, generating small interfering RNAs (siRNAs) which are incorporated into an RNA-induced silencing complex (RISC) that promotes homology-dependent degradation of cognate RNAs. To counteract this, plant viruses express RNA silencing suppressors. Here, we show that the coat protein (CP) of Pelargonium flower break virus (PFBV), a member of the genus Carmovirus, is able to efficiently inhibit RNA silencing. Interestingly, PFBV CP blocked both sense RNA- and dsRNA-triggered RNA silencing and did not preclude generation of siRNAs, which is in contrast with the abilities that have been reported for other carmoviral CPs. We have also found that PFBV CP can bind siRNAs and that this ability correlates with silencing suppression activity and enhancement of potato virus X pathogenicity. Collectively, the results indicate that PFBV CP inhibits RNA silencing by sequestering siRNAs and preventing their incorporation into a RISC, thus behaving similarly to unrelated viral suppressors but dissimilarly to orthologous ones.

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

10.1101/2021.08.02.454693 ◽

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.

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Reprogramming of RNA silencing triggered by cucumber mosaic virus infection in Arabidopsis

Genome Biology ◽

10.1186/s13059-021-02564-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Maria Luz Annacondia ◽

German Martinez

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Virus Resistance ◽

Rna Silencing ◽

High Throughput Sequencing ◽

Small Interfering Rnas ◽

Genome Wide ◽

Endogenous Genes ◽

Silencing Pathways ◽

Relationship Of

Abstract Background RNA silencing has an important role mediating sequence-specific virus resistance in plants. The complex interaction of viruses with RNA silencing involves the loading of viral small interfering RNAs (vsiRNAs) into its host ARGONAUTE (AGO) proteins. As a side effect of their antiviral activity, vsiRNAs loading into AGO proteins can also mediate the silencing of endogenous genes. Here, we analyze at the genome-wide level both aspects of the interference of cucumber mosaic virus (CMV) with the RNA silencing machinery of Arabidopsis thaliana. Results We observe CMV-derived vsiRNAs affect the levels of endogenous sRNA classes. Furthermore, we analyze the incorporation of vsiRNAs into AGO proteins with a described antiviral role and the viral suppressor of RNA silencing (VSR) 2b, by combining protein immunoprecipitation with sRNA high-throughput sequencing. Interestingly, vsiRNAs represent a substantial percentage of AGO-loaded sRNAs and displace other endogenous sRNAs. As a countermeasure, the VSR 2b loaded vsiRNAs and mRNA-derived siRNAs, which affect the expression of the genes they derive from. Additionally, we analyze how vsiRNAs incorporate into the endogenous RNA silencing pathways by exploring their target mRNAs using parallel analysis of RNA end (PARE) sequencing, which allow us to identify vsiRNA-targeted genes genome-wide. Conclusions This work exemplifies the complex relationship of RNA viruses with the endogenous RNA silencing machinery and the multiple aspects of virus resistance and virulence that this interaction induces.

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Viral suppressors from members of the family Closteroviridae combating antiviral RNA silencing: a tale of a sophisticated arms race in host-pathogen interactions

Phytopathology Research ◽

10.1186/s42483-021-00104-y ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Muhammad Dilshad Hussain ◽

Tahir Farooq ◽

Xi Chen ◽

Muhammad Tariqjaveed ◽

Tong Jiang ◽

...

Keyword(s):

Rna Silencing ◽

Protein S ◽

Specific Protein ◽

Small Interfering Rnas ◽

Virus Infections ◽

Antiviral Responses ◽

The Family ◽

Inactivation Mechanism ◽

Recent Developments ◽

Viral Suppressors

AbstractRNA silencing is an evolutionarily homology-based gene inactivation mechanism and plays critical roles in plant immune responses to acute or chronic virus infections, which often pose serious threats to agricultural productions. Plant antiviral immunity is triggered by virus-derived small interfering RNAs (vsiRNAs) and functions to suppress virus further replication via a sequence-specific degradation manner. Through plant-virus arms races, many viruses have evolved specific protein(s), known as viral suppressors of RNA silencing (VSRs), to combat plant antiviral responses. Numerous reports have shown that VSRs can efficiently curb plant antiviral defense response via interaction with specific component(s) involved in the plant RNA silencing machinery. Members in the family Closteroviridae (closterovirids) are also known to encode VSRs to ensure their infections in plants. In this review, we will focus on the plant antiviral RNA silencing strategies, and the most recent developments on the multifunctional VSRs encoded by closterovirids. Additionally, we will highlight the molecular characters of phylogenetically-associated closterovirids, the interactions of these viruses with their host plants and transmission vectors, and epidemiology.

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