In Arabidopsis thaliana, RNA-Induced Silencing Complex-Loading of MicroRNAs Plays a Minor Regulatory Role During Photomorphogenesis Except for miR163.

The shift of dark-grown seedlings to the light leads to substantial reprogramming of gene expression, which results in dramatic developmental changes (referred to as de-etiolation or photomorphogenesis). MicroRNAs (miRNAs) regulate most steps of plant development, thus miRNAs might play important role in transcriptional reprogramming during de-etiolation. Indeed, miRNA biogenesis mutants show aberrant de-etiolation. Previous works showed that the total miRNA expression pattern (total miRNAome) is only moderately altered during photomorphogenesis. However, a recent study has shown that plant miRNAs are present in two pools, biologically active miRNAs loaded to RISC (RNA-induced silencing complex-loaded) form while inactive miRNAs accumulate in duplex form upon organ formation. To test if RISC-loading efficiency is changed during photomorphogenesis. we compared the total miRNAome and the RISC-loaded miRNAome of dark-grown and de-etiolated Arabidopsis thaliana seedlings. miRNA sequencing has revealed that although regulated RISC-loading is involved in the control of active miRNAome formation during de-etiolation, this effect is moderate. The total miRNAomes and the RISC-loaded miRNAomes of dark-grown and de-etiolated plants are similar indicating that most miRNAs are loaded onto RISC with similar efficiency in dark and light. Few miRNAs were loaded onto RISC with different efficiency and one miRNA, miR163, was RISC-loaded much more effectively in light than in dark. Thus, our results suggest that although RISC-loading contributes significantly to the control of the formation of organ-specific active miRNA pools, it plays a limited role in the regulation of active miRNA pool formation during de-etiolation. Regulated RISC-loading strongly modifies the expression of miRNA163, could play a role in the fine-tuning of a few other miRNAs, and do not modify the expression of most miRNAs.

Elongation factor TFIIS is essential for heat stress adaptation in plants.

Elongation factor TFIIS (transcription factor IIS) is structurally and biochemically probably the best characterized elongation cofactor of RNA polymerase II. However, little is known about TFIIS regulation or its roles during stress responses. Here, we show that, although TFIIS seems unnecessary under optimal conditions in Arabidopsis, its absence renders plants supersensitive to heat; tfIIs mutants die even when exposed to sublethal high temperature. TFIIS activity is required for thermal adaptation throughout the whole life cycle of plants, ensuring both survival and reproductive success. By employing a transcriptome analysis, we unravel that the absence of TFIIS makes transcriptional reprogramming sluggish, and affects expression and alternative splicing pattern of hundreds of heat-regulated transcripts. Transcriptome changes indirectly cause proteotoxic stress and deterioration of cellular pathways, including photosynthesis, which finally leads to lethality. Contrary to expectations of being constantly present to support transcription, we show that TFIIS is dynamically regulated. TFIIS accumulation during heat occurs in evolutionary distant species, including the unicellular alga Chlamydomonas reinhardtii, dicot Brassica napus and monocot Hordeum vulgare, suggesting that the vital role of TFIIS in stress adaptation of plants is conserved.

The role of RST1 and RIPR proteins in plant RNA quality control systems.

To keep mRNA homeostasis, the RNA degradation, quality control and silencing systems should act in balance in plants. Degradation of normal mRNA starts with deadenylation, then deadenylated transcripts are degraded by the SKI-exosome 3'-5' and/or XRN4 5'-3' exonucleases. RNA quality control systems identify and decay different aberrant transcripts. RNA silencing degrades double-stranded transcripts and homologous mRNAs. It also targets aberrant and silencing prone transcripts. The SKI-exosome is essential for mRNA homeostasis, it functions in normal mRNA degradation and different RNA quality control systems, and in its absence silencing targets normal transcripts. It is highly conserved in eukaryotes, thus recent reports that the plant SKI-exosome is associated with RST1 and RIPR proteins and that, they are required for SKI-exosome-mediated decay of silencing prone transcripts were unexpected. To clarify whether RST1 and RIPR are essential for all SKI-exosome functions or only for the elimination of silencing prone transcripts, degradation of different reporter transcripts was studied in RST1 and RIPR inactivated Nicotiana benthamiana plants. As RST1 and RIPR, like the SKI-exosome, were essential for Non-stop and No-go decay quality control systems, and for RNA silencing- and minimum ORF-mediated decay, we propose that RST1 and RIPR are essential components of plant SKI-exosome supercomplex.

Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3'UTR intron-mediated NMD in Neurospora crassa.

Eukaryotic release factor 1 (eRF1) is a translation termination factor that binds to the ribosome at stop codons. The expression of eRF1 is strictly controlled, since its concentration defines termination efficiency and frequency of translational readthrough. Here, we show that eRF1 expression in Neurospora crassa is controlled by an autoregulatory circuit that depends on the specific 3'UTR structure of erf1 mRNA. The stop codon context of erf1 promotes readthrough that protects the mRNA from its 3'UTR-induced nonsense-mediated mRNA decay (NMD). High eRF1 concentration leads to inefficient readthrough, thereby allowing NMD-mediated erf1 degradation. We propose that eRF1 expression is controlled by similar autoregulatory circuits in many fungi and seed plants and discuss the evolution of autoregulatory systems of different translation termination factors.

Bulbous perennials precisely detect the length of winter and adjust flowering dates.

In order to identify the most relevant environmental parameters that regulate flowering time of bulbous perennials, first flowering dates of 329 taxa over 33 yr are correlated with monthly and daily mean values of 16 environmental parameters (such as insolation, precipitation, temperature, soil water content, etc.) spanning at least 1 yr back from flowering. A machine learning algorithm is deployed to identify the best explanatory parameters because the problem is strongly prone to overfitting for traditional methods: if the number of parameters is the same or greater than the number of observations, then a linear model can perfectly fit the dependent variable (observations). Surprisingly, the best proxy of flowering date fluctuations is the daily snow depth anomaly, which cannot be a signal itself, however it should be related to some integrated temperature signal. Moreover, daily snow depth anomaly as proxy performs much better than mean soil temperature preceding the flowering, the best monthly explanatory parameter. Our findings support the existence of complicated temperature sensing mechanisms operating on different timescales, which is a prerequisite to precisely observe the length and severity of the winter season and translate for example, 'lack of snow' information to meaningful internal signals related to phenophases.

Nectar- and stigma exudate-specific expression of an acidic chitinase could partially protect certain apple cultivars against fire blight disease.

MAIN CONCLUSION: Certain apple cultivars accumulate to high levels in their nectar and stigma exudate an acidic chitinase III protein that can protect against pathogens including fire blight disease causing Erwinia amylovora. To prevent microbial infections, flower nectars and stigma exudates contain various antimicrobial compounds. Erwinia amylovora, the causing bacterium of the devastating fire blight apple disease, is the model pathogen that multiplies in flower secretions and infects through the nectaries. Although Erwinia-resistant apples are not available, certain cultivars are tolerant. It was reported that in flower infection assay, the 'Freedom' cultivar was Erwinia tolerant, while the 'Jonagold' cultivar was susceptible. We hypothesized that differences in the nectar protein compositions lead to different susceptibility. Indeed, we found that an acidic chitinase III protein (Machi3-1) selectively accumulates to very high levels in the nectar and the stigma exudate of the 'Freedom' cultivar. We show that three different Machi3-1 alleles exist in apple cultivars and that only the 5B-Machi3-1 allele expresses the Machi3-1 protein in the nectar and the stigma exudate. We demonstrate that the 5B-Machi3-1 allele was introgressed from the Malus floribunda 821 clone into different apple cultivars including the 'Freedom'. Our data suggest that MYB-binding site containing repeats of the 5B-Machi3-1 promoter is responsible for the strong nectar- and stigma exudate-specific expression. As we found that in vitro, the Machi3-1 protein impairs growth and biofilm formation of Erwinia at physiological concentration, we propose that the Machi3-1 protein could partially protect 5B-Machi3-1 allele containing cultivars against Erwinia by inhibiting the multiplication and biofilm formation of the pathogen in the stigma exudate and in the nectar.

The No-go decay system degrades plant mRNAs that contain a long A-stretch in the coding region.

RNA quality control systems identify and degrade aberrant mRNAs, thereby preventing the accumulation of faulty proteins. Non-stop decay (NSD) and No-go decay (NGD) are closely related RNA quality control systems that act during translation. NSD degrades mRNAs lacking a stop codon, while NGD recognizes and decays mRNAs that contain translation elongation inhibitory structures. NGD has been intensively studied in yeast and animals but it has not been described in plants yet. In yeast, NGD is induced if the elongating ribosome is stalled by a strong inhibitory structure. Then, the mRNA is cleaved by an unknown nuclease and the cleavage fragments are degraded. Here we show that NGD also operates in plant. We tested several potential NGD cis-elements and found that in plants, unlike in yeast, only long A-stretches induce NGD. These long A-stretches trigger endonucleolytic cleavage, and then the 5' fragments are degraded in a Pelota-, HBS1- and SKI2- dependent manner, while XRN4 eliminates the 3' fragment. We also show that plant NGD operates gradually, the longer the A-stretch, the more efficient the cleavage. Our data suggest that mechanistically NGD is conserved in eukaryotes, although the NGD inducing cis-elements could be different. Moreover, we found that Arabidopsis AtPelota1 functions in both NGD and NSD, while AtPelota2 represses these quality control systems. The function of plant NGD will be discussed.

The nonstop decay and the RNA silencing systems operate cooperatively in plants.

Translation-dependent mRNA quality control systems protect the protein homeostasis of eukaryotic cells by eliminating aberrant transcripts and stimulating the decay of their protein products. Although these systems are intensively studied in animals, little is known about the translation-dependent quality control systems in plants. Here, we characterize the mechanism of nonstop decay (NSD) system in Nicotiana benthamiana model plant. We show that plant NSD efficiently degrades nonstop mRNAs, which can be generated by premature polyadenylation, and stop codon-less transcripts, which are produced by endonucleolytic cleavage. We demonstrate that in plants, like in animals, Pelota, Hbs1 and SKI2 proteins are required for NSD, supporting that NSD is an ancient and conserved eukaryotic quality control system. Relevantly, we found that NSD and RNA silencing systems cooperate in plants. Plant silencing predominantly represses target mRNAs through endonucleolytic cleavage in the coding region. Here we show that NSD is required for the elimination of 5' cleavage product of mi- or siRNA-guided silencing complex when the cleavage occurs in the coding region. We also show that NSD and nonsense-mediated decay (NMD) quality control systems operate independently in plants.

Expression of the eRF1 translation termination factor is controlled by an autoregulatory circuit involving readthrough and nonsense-mediated decay in plants.

When a ribosome reaches a stop codon, the eukaryotic Release Factor 1 (eRF1) binds to the A site of the ribosome and terminates translation. In yeasts and plants, both over- and underexpression of eRF1 lead to altered phenotype indicating that eRF1 expression should be strictly controlled. However, regulation of eRF1 level is still poorly understood. Here we show that expression of plant eRF1 is controlled by a complex negative autoregulatory circuit, which is based on the unique features of the 3΄untranslated region (3΄UTR) of the eRF1-1 transcript. The stop codon of the eRF1-1 mRNA is in a translational readthrough promoting context, while its 3΄UTR induces nonsense-mediated decay (NMD), a translation termination coupled mRNA degradation mechanism. We demonstrate that readthrough partially protects the eRF1-1 mRNA from its 3΄UTR induced NMD, and that elevated eRF1 levels inhibit readthrough and stimulate NMD. Thus, high eRF1 level leads to reduced eRF1-1 expression, as weakened readthrough fails to protect the eRF1-1 mRNA from the more intense NMD. This eRF1 autoregulatory circuit might serve to finely balance general translation termination efficiency.

Identification of Nicotiana benthamiana microRNAs and their targets using high throughput sequencing and degradome analysis.

BACKGROUND: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots. RESULTS: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity. CONCLUSIONS: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.

Phosphorylation of the N- and C-terminal UPF1 domains plays a critical role in plant nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is an essential quality control system that degrades aberrant transcripts containing premature termination codons and regulates the expression of several normal transcripts. Targets for NMD are selected during translational termination. If termination is slow, the UPF1 NMD factor binds the eRF3 protein of the termination complex and then recruits UPF2 and UPF3. Consequently, the UPF1-2-3 NMD complex induces SMG7-mediated degradation of the target mRNA. It is unknown how formation of the NMD complex and transcript degradation are linked in plants. Previously we have shown that the N- and C-terminal domains of UPF1 act redundantly and that the N-terminal domain is phosphorylated. To clarify the role of UPF1 phosphorylation in plant NMD, we generated UPF1 mutants and analyzed their phosphorylation status and the NMD competency of the mutants. We show that although several residues in the N-terminal domain of UPF1 are phosphorylated, only three phosphorylated amino acids, S3, S13 and T29, play a role in NMD. Moreover, we found that the C-terminal domain consists of redundant S/TQ-rich segments and that S1076 is involved in NMD. All NMD-relevant phosphorylation sites were in the S/TQ context. Co-localization and fluorescence resonance energy transfer-fluorescence lifetime imaging assays suggest that N-terminal and probably also C-terminal phosphorylated S/TQ residues are the binding platform for SMG7. Our data support the hypothesis that phosphorylation of UPF1 connects NMD complex formation and the SMG7-mediated target transcript degradation steps of NMD. SMG7 binds the phosphorylated S/TQ sites of the UPF1 component of the NMD complex, and then it induces the degradation of the NMD target.

Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3'untranslated region (UTR). In yeasts, unusually long 3'UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3'UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3'UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.

The late steps of plant nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and degrades mRNAs containing premature termination codons (PTCs). If translation terminates at a PTC, the UPF1 NMD factor binds the terminating ribosome and recruits UPF2 and UPF3 to form a functional NMD complex, which triggers the rapid decay of the PTC-containing transcript. Although NMD deficiency is seedling lethal in plants, the mechanism of plant NMD remains poorly understood. To understand how the formation of the NMD complex leads to transcript decay we functionally mapped the UPF1 and SMG7 plant NMD factors, the putative key players of NMD target degradation. Our data indicate that the cysteine-histidine-rich (CH) and helicase domains of UPF1 are only essential for the early steps of NMD, whereas the heavily phosphorylated N- and C-terminal regions play a redundant but essential role in the target transcript degradation steps of NMD. We also show that both the N- and the C-terminal regions of SMG7 are essential for NMD. The N terminus contains a phosphoserine-binding domain that is required for the early steps of NMD, whereas the C terminus is required to trigger the degradation of NMD target transcripts. Moreover, SMG7 is a P-body component that can also remobilize UPF1 from the cytoplasm into processing bodies (P bodies). We propose that the N- and C-terminal phosphorylated regions of UPF1 recruit SMG7 to the functional NMD complex, and then SMG7 transports the PTC-containing transcripts into P bodies for degradation.

Long-range interactions in nonsense-mediated mRNA decay are mediated by intrinsically disordered protein regions.

In nonsense-mediated mRNA decay (NMD), large protein complexes cooperate to trigger degradation of mRNA with a premature termination codon. Due to the extreme variation in the size and topology of its mRNA substrate, the structural underpinning of the fidelity of NMD is little understood. Based on bioinformatic predictions, we suggest that fly-casting mechanisms enabled by long disordered regions in NMD complexes are exploited for the underlying effective long-range communication.

Functional analysis of the grapevine paralogs of the SMG7 NMD factor using a heterolog VIGS-based gene depletion-complementation system.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that identifies and eliminates transcripts having a premature translation termination codon (PTC). NMD is also involved in the control of several wild-type mRNAs. The NMD core machinery consists of three highly conserved NMD factors (UPF1, UPF2 and UPF3) and at least one less conserved 14-3-3-like domain containing protein (SMG7). A PTC is identified by UPF factors, and then SMG7 triggers rapid transcript decay. UPF factors are generally encoded by a single gene, whereas SMG7 has duplicated several times during evolution. Recently it was reported that the plant SMG7 is autoregulated through NMD and that SMG7 has two relatively divergent paralogs in dicots, SMG7 and SMG7L. In mammals all three SMG7 related genes (SMG5, SMG6 and SMG7) are essential in NMD, so we hypothesized that in plants the SMG7 and SMG7L duplicates may also play distinct roles in NMD. To test this possibility, we have analyzed the evolution and the function of plant SMG7 homologs. We show that SMG7L is not required for plant NMD. Interestingly, we found that the grapevine and poplar genomes contain two quite divergent SMG7 paralogs which may have derived from an ancient duplication event. Using heterolog depletion/complementation assays we demonstrate that both grapevine SMG7 copies retained the complete NMD activity and both of them are under NMD control, whilst SMG7L has lost NMD activity and NMD control.

Plant upstream ORFs can trigger nonsense-mediated mRNA decay in a size-dependent manner.

Nonsense-mediated decay (NMD) is a quality control mechanism that identifies and degrades aberrant mRNAs containing premature termination codons (PTC). NMD also regulates the expression of many wild-type genes. In plants, NMD identifies a stop codon as a PTC and initiates the rapid degradation of the transcript if the 3'untranslated region (UTR) is unusually long or if it harbors an intron. Approximately 20% of plant transcripts have an upstream ORF (uORF) in the 5'UTR. In theory, if a uORF is translated, the 3'UTR downstream of the uORF will be long and harbor introns, thus these transcripts might be degraded by NMD. Therefore, if uORFs can trigger NMD, uORF containing transcripts would be a major group of NMD regulated wild-type plant mRNAs. The aim of this study was to clarify whether plant uORFs could activate NMD. Here we demonstrate that plant uORFs induce NMD in a size-dependent manner, a 50 amino acid (aa) long uORF triggered NMD efficiently, whereas similar but shorter (31 and 15 aa long) uORFs failed to activate NMD response. We have found that only ~2% of annotated Arabidopsis genes contain a first uORF that is longer than 35 aa, thus we propose that NMD regulates only a small fraction of uORF containing transcripts. However, as mRNAs having uORF that is longer than the critical size are strongly overrepresented within the up-regulated transcripts of NMD deficient plants, it is likely that this subset of natural NMD targets induces NMD because of containing a relatively long translatable uORF.

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

Both introns and long 3'-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that identifies and eliminates aberrant mRNAs containing a premature termination codon (PTC). Although, key trans-acting NMD factors, UPF1, UPF2 and UPF3 are conserved in yeast and mammals, the cis-acting NMD elements are different. In yeast, short specific sequences or long 3'-untranslated regions (3'-UTRs) render an mRNA subject to NMD, while in mammals' 3'-UTR located introns trigger NMD. Plants also possess an NMD system, although little is known about how it functions. We have elaborated an agroinfiltration-based transient NMD assay system and defined the cis-acting elements that mediate plant NMD. We show that unusually long 3'-UTRs or the presence of introns in the 3'-UTR can subject mRNAs to NMD. These data suggest that both long 3'-UTR-based and intron-based PTC definition operated in the common ancestors of extant eukaryotes (stem eukaryotes) and support the theory that intron-based NMD facilitated the spreading of introns in stem eukaryotes. We have also identified plant UPF1 and showed that tethering of UPF1 to either the 5'- or 3'-UTR of an mRNA results in reduced transcript accumulation. Thus, plant UPF1 might bind to mRNA in a late, irreversible phase of NMD.

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing.

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.

Aureusvirus P14 is an efficient RNA silencing suppressor that binds double-stranded RNAs without size specificity.

RNA silencing is a conserved eukaryotic gene regulatory system in which sequence specificity is determined by small RNAs. Plant RNA silencing also acts as an antiviral mechanism; therefore, viral infection requires expression of a silencing suppressor. The mechanism and the evolution of silencing suppression are still poorly understood. Tombusvirus open reading frame (ORF) 5-encoded P19 is a size-selective double-stranded RNA (dsRNA) binding protein that suppresses silencing by sequestering double-stranded small interfering RNAs (siRNAs), the specificity determinant of the antiviral silencing system. To better understand the evolution of silencing suppression, we characterized the suppressor of the type member of Aureusviruses, the closest relatives of the genus Tombusvirus. We show that the Pothos latent virus (PoLV) ORF 5-encoded P14 is an efficient suppressor of both virus- and transgene-induced silencing. Findings that in vitro P14 binds dsRNAs and double-stranded siRNAs without obvious size selection suggest that P14, unlike P19, can suppress silencing by sequestering both long dsRNA and double-stranded siRNA components of the silencing machinery. Indeed, P14 prevents the accumulation of hairpin transcript-derived siRNAs, indicating that P14 inhibits inverted repeat-induced silencing by binding the long dsRNA precursors of siRNAs. However, viral siRNAs accumulate to high levels in PoLV-infected plants; therefore, P14 might inhibit virus-induced silencing by sequestering double-stranded siRNAs. Finally, sequence analyses suggest that P14 and P19 suppressors diverged from an ancient dsRNA binding suppressor that evolved as a nested protein within the common ancestor of aureusvirus-tombusvirus movement proteins.