Dicer-dependent heterochromatic small RNAs in the model diatom species Phaeodactylum tricornutum.

Diatoms are eukaryotic microalgae responsible for nearly half of the marine productivity. RNA interference (RNAi) is a mechanism of regulation of gene expression mediated by small RNAs (sRNAs) processed by the endoribonuclease Dicer (DCR). To date, the mechanism and physiological role of RNAi in diatoms are unknown. We mined diatom genomes and transcriptomes for key RNAi effectors and retraced their phylogenetic history. We generated DCR knockout lines in the model diatom species Phaeodactylum tricornutum and analyzed their mRNA and sRNA populations, repression-associated histone marks, and acclimatory response to nitrogen starvation. Diatoms presented a diversification of key RNAi effectors whose distribution across species suggests the presence of distinct RNAi pathways. P. tricornutum DCR was found to process 26-31-nt-long double-stranded sRNAs originating mostly from transposons covered by repression-associated epigenetic marks. In parallel, P. tricornutum DCR was necessary for the maintenance of the repression-associated histone marks H3K9me2/3 and H3K27me3. Finally, PtDCR-KO lines presented a compromised recovery post nitrogen starvation suggesting a role for P. tricornutum DCR in the acclimation to nutrient stress. Our study characterized the molecular function of the single DCR homolog of P. tricornutum suggesting an association between RNAi and heterochromatin maintenance in this model diatom species.

Complete genome sequence of a carlavirus identified in grapevine (Vitis sp) in Greece.

Using high-throughput sequencing, we identified a novel carlavirus sequence in a 28-year-old 'Kotsifali' grapevine sample collected in Heraklion (Crete, Greece). Using RT-PCR and 5'/3' RACE together with Sanger sequencing, the complete genome sequence of 8299 nt was confirmed and found to contain five open reading frames (ORFs) but to lack an ORF6, which is present in some members of the genus Carlavirus. The novel sequence is most similar to those of two carlaviruses infecting caper, and taking into account the ICTV nomenclature, we propose the name "grapevine carlavirus 1" for this new virus.

Viral Detection: Past, Present, and Future.

Viruses are essentially composed of a nucleic acid (segmented or not, DNA, or RNA) and a protein coat. Despite their simplicity, these small pathogens are responsible for significant economic and humanitarian losses that have had dramatic consequences in the course of human history. Since their discovery, scientists have developed different strategies to efficiently detect viruses, using all possible viral features. Viruses shape, proteins, and nucleic acid are used in viral detection. In this review, the development of these techniques, especially for plant and mammalian viruses, their strengths and weaknesses as well as the latest cutting-edge technologies that may be playing important roles in the years to come are described.

Chromatin dynamics during interphase and cell division: similarities and differences between model and crop plants.

Genetic information in the cell nucleus controls organismal development and responses to the environment, and finally ensures its own transmission to the next generations. To achieve so many different tasks, the genetic information is associated with structural and regulatory proteins, which orchestrate nuclear functions in time and space. Furthermore, plant life strategies require chromatin plasticity to allow a rapid adaptation to abiotic and biotic stresses. Here, we summarize current knowledge on the organization of plant chromatin and dynamics of chromosomes during interphase and mitotic and meiotic cell divisions for model and crop plants differing as to genome size, ploidy, and amount of genomic resources available. The existing data indicate that chromatin changes accompany most (if not all) cellular processes and that there are both shared and unique themes in the chromatin structure and global chromosome dynamics among species. Ongoing efforts to understand the molecular mechanisms involved in chromatin organization and remodeling have, together with the latest genome editing tools, potential to unlock crop genomes for innovative breeding strategies and improvements of various traits.

Isoprenoid biosynthesis in the diatom Haslea ostrearia.

Diatoms are eukaryotic, unicellular algae that are responsible for c. 20% of the Earth's primary production. Their dominance and success in contemporary oceans have prompted investigations on their distinctive metabolism and physiology. One metabolic pathway that remains largely unexplored in diatoms is isoprenoid biosynthesis, which is responsible for the production of numerous molecules with unique features. We selected the diatom species Haslea ostrearia because of its characteristic isoprenoid content and carried out a comprehensive transcriptomic analysis and functional characterization of the genes identified. We functionally characterized one farnesyl diphosphate synthase, two geranylgeranyl diphosphate synthases, one short-chain polyprenyl synthase, one bifunctional isopentenyl diphosphate isomerase - squalene synthase, and one phytoene synthase. We inferred the phylogenetic origin of these genes and used a combination of functional analysis and subcellular localization predictions to propose their physiological roles. Our results provide insight into isoprenoid biosynthesis in H. ostrearia and propose a model of the central steps of the pathway. This model will facilitate the study of metabolic pathways of important isoprenoids in diatoms, including carotenoids, sterols and highly branched isoprenoids.

Combined Activity of DCL2 and DCL3 Is Crucial in the Defense against Potato Spindle Tuber Viroid.

Viroids are self replicating non-coding RNAs capable of infecting a wide range of plant hosts. They do not encode any proteins, thus the mechanism by which they escape plant defenses remains unclear. RNAi silencing is a major defense mechanism against virus infections, with the four DCL proteins being principal components of the pathway. We have used Nicotiana benthamiana as a model to study Potato spindle tuber viroid infection. This viroid is a member of the Pospiviroidae family and replicates in the nucleus via an asymmetric rolling circle mechanism. We have created knock-down plants for all four DCL genes and their combinations. Previously, we showed that DCL4 has a positive effect on PSTVd infectivity since viroid levels drop when DCL4 is suppressed. Here, we show that PSTVd levels remain decreased throughout infection in DCL4 knockdown plants, and that simultaneous knockdown of DCL1, DCL2 or DCL3 together with DCL4 cannot reverse this effect. Through infection of plants suppressed for multiple DCLs we further show that a combined suppression of DCL2 and DCL3 has a major effect in succumbing plant antiviral defense. Based on our results, we further suggest that Pospoviroids may have evolved to be primarily processed by DCL4 as it seems to be a DCL protein with less detrimental effects on viroid infectivity. These findings pave the way to delineate the complexity of the relationship between viroids and plant RNA silencing response.

ERIL1, the plant homologue of ERI-1, is involved in the processing of chloroplastic rRNAs.

Proteins belonging to the enhancer of RNA interference-1 subfamily of 3'-5' exoribonucleases participate in divergent RNA pathways. They degrade small interfering RNAs (siRNAs), thus suppressing RNA interference, and are involved in the maturation of ribosomal RNAs and the degradation of histone messenger RNAs (mRNAs). Here, we report evidence for the role of the plant homologue of these proteins, which we termed ENHANCED RNA INTERFERENCE-1-LIKE-1 (ERIL1), in chloroplast function. In vitro assays with AtERIL1 proved that the conserved 3'-5' exonuclease activity is shared among all homologues studied. Confocal microscopy revealed that ERL1, a nucleus-encoded protein, is targeted to the chloroplast. To gain insight into its role in plants, we used Nicotiana benthamiana and Arabidopsis thaliana plants that constitutively overexpress or suppress ERIL1. In the mutant lines of both species we observed malfunctions in photosynthetic ability. Molecular analysis showed that ERIL1 participates in the processing of chloroplastic ribosomal RNAs (rRNAs). Lastly, our results suggest that the missexpression of ERIL1 may have an indirect effect on the microRNA (miRNA) pathway. Altogether our data point to an additional piece of the puzzle in the complex RNA metabolism of chloroplasts.

Dicer-Like 4 Is Involved in Restricting the Systemic Movement of Zucchini yellow mosaic virus in Nicotiana benthamiana.

Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Rosea1 to obtain recombinant clone ZYMV-Ros1. While in some plant-virus combinations Rosea1 induces accumulation of anthocyanins in infected tissues, ZYMV-Ros1 infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmentation in infected tissues of the model plant Nicotiana benthamiana. In this species, ZYMV-Ros1 multiplied efficiently in local inoculated tissue but only a few progeny particles established infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Ros1 local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Ros1 systemic accumulation was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Ros1 systemic accumulation dramatically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.

RNA silencing movement in plants.

Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant, respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.

A bromodomain-containing host protein mediates the nuclear importation of a satellite RNA of Cucumber mosaic virus.

Replication of the satellite RNA (satRNA) of Cucumber Mosaic Virus is dependent on replicase proteins of helper virus (HV). However, we recently demonstrated that like with Potato spindle tuber viroid (PSTVd), a satRNA associated with Cucumber Mosaic Virus strain Q (Q-satRNA) has the propensity to localize in the nucleus and generate multimers that subsequently serve as templates for HV-dependent replication. But the mechanism regulating the nuclear importation of Q-satRNA is unknown. Here we show that the nuclear importation of Q-satRNA is mediated by a bromodomain-containing host protein (BRP1), which is also apparently involved in the nuclear localization of PSTVd. A comparative analysis of nuclear and cytoplasmic fractions from Nicotiana benthamiana plants coinfected with Q-satRNA and its HV confirmed the association of Q-satRNA but not HV with the nuclear compartment. A combination of the MS2-capsid protein-based RNA tagging assay and confocal microscopy demonstrated that the nuclear localization of Q-satRNA was completely blocked in transgenic lines of Nicotiana benthamiana (ph5.2nb) that are defective in BRP1 expression. This defect, however, was restored when the ph5.2nb lines of N. benthamiana were trans-complemented by ectopically expressed BRP1. The binding specificity of BRP1 with Q-satRNA was confirmed in vivo and in vitro by coimmunoprecipitation and electrophoretic mobility shift assays, respectively. Finally, infectivity assays involving coexpression of Q-satRNA and its HV in wild-type and ph5.2nb lines of N. benthamiana accentuated a biological role for BRP1 in the Q-satRNA infection cycle. The significance of these results in relation to a possible evolutionary relationship to viroids is discussed.

Stress-related transcriptomic changes associated with GFP transgene expression and active transgene silencing in plants.

Plants respond to biotic and abiotic stress by activating and interacting with multiple defense pathways, allowing for an efficient global defense response. RNA silencing is a conserved mechanism of regulation of gene expression directed by small RNAs important in acquired plant immunity and especially virus and transgene repression. Several RNA silencing pathways in plants are crucial to control developmental processes and provide protection against abiotic and biotic stresses as well as invasive nucleic acids such as viruses and transposable elements. Various notable studies have shed light on the genes, small RNAs, and mechanisms involved in plant RNA silencing. However, published research on the potential interactions between RNA silencing and other plant stress responses is limited. In the present study, we tested the hypothesis that spreading and maintenance of systemic post-transcriptional gene silencing (PTGS) of a GFP transgene are associated with transcriptional changes that pertain to non-RNA silencing-based stress responses. To this end, we analyzed the structure and function of the photosynthetic apparatus and conducted whole transcriptome analysis in a transgenic line of Nicotiana benthamiana that spontaneously initiates transgene silencing, at different stages of systemic GFP-PTGS. In vivo analysis of chlorophyll a fluorescence yield and expression levels of key photosynthetic genes indicates that photosynthetic activity remains unaffected by systemic GFP-PTGS. However, transcriptomic analysis reveals that spreading and maintenance of GFP-PTGS are associated with transcriptional reprogramming of genes that are involved in abiotic stress responses and pattern- or effector-triggered immunity-based stress responses. These findings suggest that systemic PTGS may affect non-RNA-silencing-based defense pathways in N. benthamiana, providing new insights into the complex interplay between different plant stress responses.

Spotlight on Plant Bromodomain Proteins.

Bromodomain-containing proteins (BRD-proteins) are the "readers" of histone lysine acetylation, translating chromatin state into gene expression. They act alone or as components of larger complexes and exhibit diverse functions to regulate gene expression; they participate in chromatin remodeling complexes, mediate histone modifications, serve as scaffolds to recruit transcriptional regulators or act themselves as transcriptional co-activators or repressors. Human BRD-proteins have been extensively studied and have gained interest as potential drug targets for various diseases, whereas in plants, this group of proteins is still not well investigated. In this review, we aimed to concentrate scientific knowledge on these chromatin "readers" with a focus on Arabidopsis. We organized plant BRD-proteins into groups based on their functions and domain architecture and summarized the published work regarding their interactions, activity and diverse functions. Overall, it seems that plant BRD-proteins are indispensable components and fine-tuners of the complex network plants have built to regulate development, flowering, hormone signaling and response to various biotic or abiotic stresses. This work will facilitate the understanding of their roles in plants and highlight BRD-proteins with yet undiscovered functions.

Genealogical Analyses of 3 Cultivated and 1 Wild Specimen of Vitis vinifera from Greece.

Grapevine (Vitis vinifera) has been an important crop with considerable cultural and economic significance for over 2,500 years, and Greece has been an important entry point into Europe for lineages that were domesticated in Western Asia and the Caucasus. However, whole-genome-based investigation of the demographic history of Greek cultivars relative to other European lineages has only started recently. To understand how Greek cultivars relate to Eurasian domesticated and wild populations, we sequenced 3 iconic domesticated strains ('Xinomavro,' 'Agiorgitiko,' 'Mavrotragano') along with 1 wild accession (the vinetree of Pausanias-a historically important wild specimen) and analyzed their genomic diversity together with a large sample of publicly available domesticated and wild strains. We also reconstructed genealogies by leveraging the powerful tsinfer methodology which has not previously been used in this system. We show that cultivated strains from Greece differ genetically from other strains in Europe. Interestingly, all the 3 cultivated Greek strains clustered with cultivated and wild accessions from Transcaucasia, South Asia, and the Levant and are amongst the very few cultivated European strains belonging to this cluster. Furthermore, our results indicate that 'Xinomavro' shares close genealogical proximity with European elite cultivars such as 'Chardonnay,' 'Riesling,' and 'Gamay' but not 'Pinot.' Therefore, the proximity of 'Xinomavro' to Gouais/Heunisch Weiss is confirmed and the utility of ancestral recombination graph reconstruction approaches to study genealogical relationships in crops is highlighted.

PSTVd infection in Nicotiana benthamiana plants has a minor yet detectable effect on CG methylation.

Viroids are small circular RNAs infecting a wide range of plants. They do not code for any protein or peptide and therefore rely on their structure for their biological cycle. Observed phenotypes of viroid infected plants are thought to occur through changes at the transcriptional/translational level of the host. A mechanism involved in such changes is RNA-directed DNA methylation (RdDM). Till today, there are contradictory works about viroids interference of RdDM. In this study, we investigated the epigenetic effect of viroid infection in Nicotiana benthamiana plants. Using potato spindle tuber viroid (PSTVd) as the triggering pathogen and via bioinformatic analyses, we identified endogenous gene promoters and transposable elements targeted by 24 nt host siRNAs that differentially accumulated in PSTVd-infected and healthy plants. The methylation status of these targets was evaluated following digestion with methylation-sensitive restriction enzymes coupled with PCR amplification, and bisulfite sequencing. In addition, we used Methylation Sensitive Amplification Polymorphism (MSAP) followed by sequencing (MSAP-seq) to study genomic DNA methylation of 5-methylcytosine (5mC) in CG sites upon viroid infection. In this study we identified a limited number of target loci differentially methylated upon PSTVd infection. These results enhance our understanding of the epigenetic host changes as a result of pospiviroid infection.

A new microRNA target prediction tool identifies a novel interaction of a putative miRNA with CCND2.

Computational methods for miRNA target prediction vary in the algorithm used; and while one can state opinions about the strengths or weaknesses of each particular algorithm, the fact of the matter is that they fall substantially short of capturing the full detail of physical, temporal and spatial requirements of miRNA::target-mRNA interactions. Here, we introduce a novel miRNA target prediction tool called Targetprofiler that utilizes a probabilistic learning algorithm in the form of a hidden Markov model trained on experimentally verified miRNA targets. Using a large scale protein downregulation data set we validate our method and compare its performance to existing tools. We find that Targetprofiler exhibits greater correlation between computational predictions and protein downregulation and predicts experimentally verified miRNA targets more accurately than three other tools. Concurrently, we use primer extension to identify the mature sequence of a novel miRNA gene recently identified within a cancer associated genomic region and use Targetprofiler to predict its potential targets. Experimental verification of the ability of this small RNA molecule to regulate the expression of CCND2, a gene with documented oncogenic activity, confirms its functional role as a miRNA. These findings highlight the competitive advantage of our tool and its efficacy in extracting biologically significant results.

Detection of Viroid RNA and vd-siRNA in N. benthamiana Plants: Northern Blot Analyses for Viroid and vd-siRNAs.

Viroids are considered the most minimalistic group of pathogens. Despite their presumed inability to encode for proteins, viroids induce several diseases in plants of primary economic importance. The production of viroid derived siRNAs (vd-siRNAs) of 21-24 nt, accompanies viroid infections in plants and results from the activation of the RNA silencing mechanism and specifically the function of Dicer endonucleases. A comprehensive set of experiments for the study and thorough analysis of viroid-infected plants has been developed. Here we present a detailed experimental plan including optimized protocols for plant infection by agroinfiltration, RNA extraction, and northern blot hybridization for the detection of both viroid genomic RNA and vd-siRNAs.

RNA silencing pathways in plant development and defense.

RNA silencing refers to a conserved eukaryotic process and is regarded as one of the most important processes in plants, with the ability to regulate gene expression both transcriptionally and post-transcriptionally. Different classes of non-coding RNAs (ncRNAs) constitute key components of the RNA silencing pathways and play pivotal roles in modulating various biological processes as well as host-pathogen interactions. One of the most extensively studied classes of ncRNAs are the 20-24 nucleotide (nt) long microRNAs (miRNAs), which are core components of the endogenous gene silencing pathway. miRNAs act as negative regulators of endogenous gene expression through either mRNA-target cleavage, translational inhibition, or DNA methylation, and are inextricably linked to a plethora of developmental processes, such as leaf pattern formation as well as abiotic and biotic stress responses. In this review, we focus on the role of the RNA silencing pathways in the regulation of developmental processes as well as in the plant responses to biotic stress.

Revisiting the Non-Coding Nature of Pospiviroids.

Viroids are small, circular, highly structured pathogens that infect a broad range of plants, causing economic losses. Since their discovery in the 1970s, they have been considered as non-coding pathogens. In the last few years, the discovery of other RNA entities, similar in terms of size and structure, that were shown to be translated (e.g., cirRNAs, precursors of miRNA, RNA satellites) as well as studies showing that some viroids are located in ribosomes, have reignited the idea that viroids may be translated. In this study, we used advanced bioinformatic analysis, in vitro experiments and LC-MS/MS to search for small viroid peptides of the PSTVd. Our results suggest that in our experimental conditions, even though the circular form of PSTVd is found in ribosomes, no produced peptides were identified. This indicates that the presence of PSTVd in ribosomes is most probably not related to peptide production but rather to another unknown function that requires further study.

Phytobacterial type III effectors HopX1, HopAB1 and HopF2 enhance sense-post-transcriptional gene silencing independently of plant R gene-effector recognition.

Plant- and animal-pathogenic bacteria deploy a variable arsenal of type III effector proteins (T3EP) to manipulate host defense. Specific biochemical functions and molecular or subcellular targets have been demonstrated or proposed for a growing number of T3EP but remain unknown for the majority of them. Here, we show that transient expression of genes coding certain bacterial T3EP (HopAB1, HopX1, and HopF2), which did not elicit hypersensitive response (HR) in transgenic green fluorescent protein (GFP) Nicotiana benthamiana 16C line, enhanced the sense post-transcriptional gene silencing (S-PTGS) triggered by agrodelivery of a GFP-expressing cassette and the silencing enhancement could be blocked by two well-known viral silencing suppressors. Further analysis using genetic truncations and site-directed mutations showed that the receptor recognition domains of HopAB1 and HopX1 are not involved in enhancing silencing. Our studies provide new evidence that phytobacterial pathogen T3EP manipulate the plant small interfering RNA pathways by enhancing silencing efficiency in the absence of effector-triggered immunity signaling and suggest that phytopathogenic bacterial effectors affect host RNA silencing in yet other ways than previously described.

Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway.

Previously, we had shown that stable expression of a hairpin RNA sharing homology with the coat protein (CP) of the Cucumber mosaic virus (CMV) (hpRNA(CMV)) produced CMV resistant Nicotiana tabacum plants. However, only 17% of the hpRNA(CMV)-expressing plants generated substantial amounts of siRNAs that mediated CMV resistance (siRNAs(CMV)). Here, we demonstrate that the transcription of a hpRNA(CMV) per se is not sufficient to trigger cytoplasmic and nuclear RNAi. A multiple-transgene copy line showed a strong resistance phenotype. Segregation of individual copies revealed that in one locus, the transgene-produced hpRNA(CMV) transcript was processed into 21-nt and 24-nt siRNAs(CMV) and lines containing this locus were resistant. At a second locus, where the transgene was shown to be transcribed, no siRNAs(CMV) were produced and lines harbouring only this locus were susceptible. In addition, the second locus failed to trigger de novo RNA-directed DNA methylation (RdDM) in cis, of its cognate sequence. However, after being induced in trans, methylation in the transcribed region of the transgene was maintained in both CG and CHG residues. Sequence-specific maintenance of methylation in transcribed regions, as well as diverse RNA degradation pathways in plants are discussed in view of our observations.