Beet Curly Top Iran Virus Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

Beet curly top Iran virus (BCTIV) is a yield-limiting geminivirus belonging to the becurtovirus genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles Mastrevirus, whereas the virion strand organization is similar to the Curtovirus genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels. Multiple geminivirus genes have been identified as viral suppressors of RNA silencing (VSR) but VSR activity remains mostly elusive in becurtoviruses. We found that BCTIV-V2 and -Rep could suppress specific Sense-PTGS mechanisms with distinct efficiencies depending on the nature of the silencing inducer and the target gene. Local silencing induced by GFP inverted repeat (IR) could not be suppressed by V2 but was partially reduced by Rep. Accordingly, we documented that Rep but not V2 could suppress systemic silencing induced by GFP-IR. In addition, we showed that the VSR activity of Rep was partly regulated by RNA-dependent RNA Polymerase 6 (RDR6), whereas the VSR activity of V2 was independent of RDR6. Domain mapping for Rep showed that an intact Rep protein was required for the suppression of PTGS. In summary, we showed that BCTIV-Rep and -V2 function as silencing suppressors with distinct modes of action.

High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing.

In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double-stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)-expressing Nicotiana benthamiana 16C line, high-pressure spraying protocol, and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3' of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5' and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3'-end of the target RNA.

Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants.

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host's RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM's mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

Host-induced gene silencing - mechanisms and applications.

Host-induced gene silencing (HIGS) technology has emerged as a powerful alternative to chemical treatments for protecting plants from pathogens or pests. More than 170 HIGS studies have been published so far, and HIGS products have been launched. First, we discuss the strengths and limitations of this technology in a pathosystem-specific context. Next, we highlight the requirement for fundamental knowledge on the molecular mechanisms (i.e. uptake, processing and translocation of transgene-expressed double-stranded RNAs) that determine the efficacy and specificity of HIGS. Additionally, we speculate on the contribution of host and target RNA interference machineries, which may be incompatible depending on the lifestyle of the pathogen or pest. Finally, we predict that closing these gaps in knowledge will lead to the development of novel integrative concepts, precise risk assessment and tailor-made HIGS therapy for plant diseases.

Turnip mosaic virus in oilseed rape activates networks of sRNA-mediated interactions between viral and host genomes.

Virus-induced plant diseases in cultivated plants cause important damages in yield. Although the mechanisms of virus infection are intensely studied at the cell biology level, only little is known about the molecular dialog between the invading virus and the host genome. Here we describe a combinatorial genome-wide approach to identify networks of sRNAs-guided post-transcriptional regulation within local Turnip mosaic virus (TuMV) infection sites in Brassica napus leaves. We show that the induction of host-encoded, virus-activated small interfering RNAs (vasiRNAs) observed in virus-infected tissues is accompanied by site-specific cleavage events on both viral and host RNAs that recalls the activity of small RNA-induced silencing complexes (RISC). Cleavage events also involve virus-derived siRNA (vsiRNA)-directed cleavage of target host transcripts as well as cleavage of viral RNA by both host vasiRNAs and vsiRNAs. Furthermore, certain coding genes act as virus-activated regulatory hubs to produce vasiRNAs for the targeting of other host genes. The observations draw an advanced model of plant-virus interactions and provide insights into the complex regulatory networking at the plant-virus interface within cells undergoing early stages of infection.

Critical view on RNA silencing-mediated virus resistance using exogenously applied RNA.

In almost all eukaryotes, RNA interference (RNAi) is a natural defence mechanism against foreign nucleic acids, including transposons and viruses. It is generally triggered by long double stranded RNA molecules (dsRNA, >50bp) that are processed into small interfering RNAs (siRNAs). RNAi can be artificially activated by the expression of RNAi triggers through viruses (virus-induced gene silencing, VIGS) and transgenes. Moreover, for almost 10 years, exogenous RNA application methods are developed as tools to induce RNAi in plants. In this review, exogenous RNA application techniques having the potential to activate RNAi with a focus on RNAi-mediated virus resistance will be discussed. Limitations of exogenous RNA applications, targeting of virus vectors and open questions related to mechanistic details that still require further investigation will be pointed out.

High-Pressure-Sprayed Double Stranded RNA Does Not Induce RNA Interference of a Reporter Gene.

In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing of long double stranded RNAs (dsRNA) into siRNAs by DICER-LIKE endonucleases (DCLs). SiRNAs are loaded onto ARGONAUTE proteins to constitute the RNA-induced silencing complex (RISC). Natural dsRNAs derive from transcription of inverted repeats or of specific RNA molecules that are transcribed by RNA-directed RNA polymerase 6 (RDR6). Moreover, replication of infecting viruses/viroids results in the production of dsRNA intermediates that can serve as substrates for DCLs. The high effectiveness of RNAi both locally and systemically implicated that plants could become resistant to pathogens, including viruses, through artificial activation of RNAi by topical exogenous application of dsRNA. The most preferable procedure to exploit RNAi would be to simply spray naked dsRNAs onto mature plants that are specific for the attacking pathogens serving as a substitute for pesticides applications. However, the plant cell wall is a difficult barrier to overcome and only few reports claim that topical application of naked dsRNA triggers RNAi in plants. Using a transgenic Nicotiana benthamiana line, we found that high-pressure-sprayed naked dsRNA did not induce silencing of a green fluorescence protein (GFP) reporter gene. Small RNA sequencing (sRNA-seq) of the samples from dsRNA sprayed leaves revealed that the dsRNA was, if at all, not efficiently processed into siRNAs indicating that the dsRNA was insufficiently taken up by plant cells.

The Rep proteins encoded by alphasatellites restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Alphasatellites are non-essential satellite-like components associated with geminiviruses. The precise selective advantage to a geminivirus infection of an alphasatellite remains unclear. The ability of the cotton leaf curl Multan alphasatellite (CLCuMuA)-encoded replication-associated protein (Rep) to suppress TGS was investigated by using Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant Potato virus X (PVX) vector carrying CLCuMuA Rep resulted in restoration of GFP expression. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. Inoculation with PVX vectors harbouring a further six Rep proteins, encoded by genetically distinct alphasatellites, were similarly shown to result in 16-TGS plants with restored GFP expression. These results indicate that the alphasatellite-encoded Rep can restore the expression of a transcriptionally silenced GFP transgene in N. benthamiana, indicating that alphasatellites are involved in overcoming host defence.

Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs.

MAIN CONCLUSION: In this study, we show that aberrant pre-mRNAs from non-spliced and non-polyadenylated intron-containing transgenes are channelled to the RNA silencing pathway. In plants, improperly processed transcripts are called aberrant RNAs (ab-RNAs) and are eliminated by either RNA silencing or RNA decay mechanisms. Ab-RNAs transcribed from intronless genes are copied by RNA-directed RNA polymerases (RDRs) into double-stranded RNAs which are subsequently cleaved by DICER-LIKE endonucleases into small RNAs (sRNAs). In contrast, ab-RNAs from intron-containing genes are suggested to be channelled post-splicing to exonucleolytic degradation. Yet, it is not clear how non-spliced aberrant pre-mRNAs are eliminated. We reasoned that transient expression of agroinfiltrated intron-containing transgenes in Nicotiana benthamiana would allow us to study the steady-state levels of non-spliced pre-mRNAs. SRNA deep sequencing of the agroinfiltrated transgenes revealed the presence of sRNAs mapping to the entire non-spliced pre-mRNA suggesting that RDRs (most likely RDR6) processed aberrant non-spliced pre-mRNAs. Primary and secondary sRNAs with lengths of 18-25 nucleotides (nt) were detected, with the most prominent sRNA size class of 22 nt. SRNAs also mapped to the terminator sequence, indicating that RDR substrates also comprised read-through transcripts devoid of polyadenylation tail. Importantly, the occurring sRNAs efficiently targeted cognate mRNA for degradation but failed to cleave the non-spliced pre-mRNA, corroborating the notion that sRNAs are not triggering RNA cleavage in the nucleus.

Delivery of Hairpin RNAs and Small RNAs Into Woody and Herbaceous Plants by Trunk Injection and Petiole Absorption.

Since its discovery, RNA interference has been widely used in crop protection. Recently, transgene-free procedures that were based on exogenous application of RNA molecules having the capacity to trigger RNAi in planta have been reported. Yet, efficient delivery of such RNA molecules to plants and particularly to trees poses major technical challenges. Here, we describe simple methods for efficient delivery of hairpin RNAs (hpRNAs) and small interfering RNAs (siRNAs) to Malus domestica, Vitis vinifera, and Nicotiana benthamiana that are based on trunk injection and/or petiole absorption. The applied RNA molecules were efficiently taken up and systemically transported. In apical leaves, the RNA was already detectable 1 day post-application (dpa) and could be detected at least up to 10 dpa, depending on the method of application. Confocal microscopy revealed that the uptaken and systemically transported RNA molecules were strictly restricted to the xylem and apoplast which may illustrate why the applied hpRNAs were not processed into siRNAs by plant DICER-LIKE (DCL) endonucleases. These innovative methods may have great impact in pest management against chewing and/or xylem sap-feeding vectors and eukaryotic pathogens that reside in the xylem.

Induction of Silencing in Plants by High-Pressure Spraying of In vitro-Synthesized Small RNAs.

In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction. High-pressure spraying of siRNAs with sizes of 21, 22, and 24 nucleotides (nt) led to local GFP silencing. Small RNA deep sequencing revealed that no shearing of siRNAs was detectable by high-pressure spraying. Systemic silencing was basically detected upon spraying of 22 nt siRNAs. Local and systemic silencing developed faster and more extensively upon targeting the apical meristem than spraying of mature leaves.

RNA-directed DNA methylation efficiency depends on trigger and target sequence identity.

RNA-directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt-TASVd) and a non-infectious potato spindle tuber viroid (PSTVd) transgene (Nt-SB2). The two viroid sequences exhibit 81% sequence identity. Nt-TASVd and Nt-SB2 plants were genetically crossed. In the progeny plants (Nt-SB2/TASVd), deep sequencing of small RNAs (sRNAs) showed that TASVd infection was associated with the accumulation of abundant small interfering RNAs (siRNAs) that mapped along the entire TASVd but only partially matched the SB2 transgene. TASVd siRNAs efficiently targeted SB2 RNA for degradation, but no transitivity was detectable. Bisulfite sequencing in the Nt-SB2/TASVd plants revealed that the TASVd transgene was targeted for dense cis-RdDM along its entire sequence. In the same plants, the SB2 transgene was targeted for trans-RdDM. The SB2 methylation pattern, however, was weak and heterogeneous, pointing to a positive correlation between trigger-target sequence identity and RdDM efficiency. Importantly, trans-RdDM on SB2 was also detected at sites where no homologous siRNAs were detected. Our data indicate that RdDM efficiency depends on the trigger-target sequence identity, and is not restricted to siRNA occupancy. These findings support recent data suggesting that RNAs with sizes longer than 24 nt (>24-nt RNAs) trigger RdDM.

Functional Analysis of Cotton Leaf Curl Kokhran Virus/Cotton Leaf Curl Multan Betasatellite RNA Silencing Suppressors.

In South Asia, Cotton leaf curl disease (CLCuD) is caused by a complex of phylogenetically-related begomovirus species and a specific betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). The post-transcriptional gene silencing (PTGS) suppression activities of the transcriptional activator protein (TrAP), C4, V2 and ßC1 proteins encoded by Cotton leaf curl Kokhran virus (CLCuKoV)/CLCuMuB were assessed in Nicotiana benthamiana. A variable degree of local silencing suppression was observed for each viral protein tested, with V2 protein exhibiting the strongest suppression activity and only the C4 protein preventing the spread of systemic silencing. The CLCuKoV-encoded TrAP, C4, V2 and CLCuMuB-encoded ßC1 proteins were expressed in Escherichia coli and purified. TrAP was shown to bind various small and long nucleic acids including single-stranded (ss) and double-stranded (ds) RNA and DNA molecules. C4, V2, and ßC1 bound ssDNA and dsDNA with varying affinities. Transgenic expression of C4 under the constitutive 35S Cauliflower mosaic virus promoter and ßC1 under a dexamethasone inducible promoter induced severe developmental abnormalities in N. benthamiana. The results indicate that homologous proteins from even quite closely related begomoviruses may differ in their suppressor activity and mechanism of action. The significance of these findings is discussed.

Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA.

In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS. Transgenic tobacco plants carrying cytoplasmic and nuclear PTGS sensor constructs were PSTVd-infected resulting in the generation of abundant PSTVd-derived small interfering RNAs (vd-siRNAs). Northern blot analysis revealed that, in contrast to the cytoplasmic sensor, the nuclear sensor transcript was not targeted for RNA degradation. Bisulfite sequencing analysis showed that the nuclear PTGS sensor transgene was efficiently targeted for RdDM. Our data suggest that PSTVd fails to trigger nuclear PTGS, and that RdDM and nuclear PTGS are not necessarily coupled.

Engineering viroid resistance.

Viroids are non-encapsidated, non-coding, circular, single-stranded RNAs (ssRNAs). They are classified into the families Pospiviroidae and Avsunviroidae, whose members replicate in the nucleus and chloroplast of plant cells, respectively. Viroids have a wide host range, including crop and ornamental plants, and can cause devastating diseases with significant economic losses. Thus, several viroids are world-wide, classified as quarantine pathogens and, hence, there is an urgent need for the development of robust antiviroid strategies. RNA silencing-based technologies seem to be a promising tool in this direction. Here, we review the recent advances concerning the complex interaction of viroids with the host's RNA silencing machinery, evaluate past and present antiviroid approaches, and finally suggest alternative strategies that could potentially be employed in the future in order to achieve transgenic and non-transgenic viroid-free plants.

Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and ßC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or ßC1 resulted in re-expression of GFP. Northern blot analysis confirmed that the observed GFP fluorescence was associated with GFP mRNA accumulation. These results indicated that Rep, TrAP and ßC1 proteins of CLCuKoV-CLCuMuB can re-activate the expression of a transcriptionally silenced GFP transgene in N. benthamiana. Although Rep, TrAP, or ßC1 proteins have, for other begomoviruses or begomoviruses-betasatellites, been previously shown to have TGS suppressor activity, this is the first report demonstrating that a single begomovirus-betasatellite complex encodes three suppressors of TGS.

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing.

In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFP(endo)). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFP(endo) (Nb-GFP(endo)) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFP(endo) coding region. Importantly, hpF-agroinfiltrated Nb-GFP(endo) plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFP(cDNA)) carrying a GFP cDNA transgene (GFP(cDNA)) were included in the analysis. HpF-agroinfiltrated Nb-GFP(cDNA) plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFP(cDNA) plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3' of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFP(cDNA) coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.

In Nicotiana species, an artificial microRNA corresponding to the virulence modulating region of Potato spindle tuber viroid directs RNA silencing of a soluble inorganic pyrophosphatase gene and the development of abnormal phenotypes.

Potato spindle tuber viroid (PSTVd) is a small non-protein-coding RNA pathogen that can induce disease symptoms in a variety of plant species. How PSTVd induces disease symptoms is a long standing question. It has been suggested that PSTVd-derived small RNAs (sRNAs) could direct RNA silencing of a targeted host gene(s) resulting in symptom development. To test this, we expressed PSTVd sequences as artificial microRNAs (amiRNAs) in Nicotiana tabacum and Nicotiana benthamiana. One amiRNA, amiR46 that corresponds to sequences within the PSTVd virulence modulating region (VMR), induced abnormal phenotypes in both Nicotiana species that closely resemble those displayed by PSTVd infected plants. In N. tabacum amiR46 plants, phenotype severity correlated with amiR46 accumulation and expression down-regulation of the bioinformatically-identified target gene, a Nicotiana soluble inorganic pyrophosphatase (siPPase). Taken together, our phenotypic and molecular analyses suggest that disease symptom development in Nicotiana species following PSTVd infection results from sRNA-directed RNA silencing of the host gene, siPPase.

Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III-like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2'-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg(2+) was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn(2+). Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.