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.

HSP90 canonical content organizes a molecular scaffold mechanism to progress flowering.

Highly interactive signaling processes constitute a set of parameters intertwining in a continuum mode to shape body formation and development. A sophisticated gene network is required to integrate environmental and endogenous cues in order to modulate flowering. However, the molecular mechanisms that coordinate the circuitries of flowering genes remain unclear. Here using complemented experimental approaches, we uncover the decisive and essential role of HEAT SHOCK PROTEIN 90 (HSP90) in restraining developmental noise to an acceptable limit. Localized depletion of HSP90 mRNAs in the shoot apex resulted in low penetrance of vegetative-to-reproductive phase transition and completely abolished flower formation. Extreme variation in expression of flowering genes was also observed in HSP90 mRNA-depleted transformed plants. Transient heat-shock treatments moderately increased HSP90 mRNA levels and rescued flower arrest. The offspring had a low, nevertheless noticeable failure to promote transition from vegetative into the reproductive phase and showed flower morphological heterogeneity. In floral tissues a moderate variation in HSP90 transcript levels and in the expression of flowering genes was detected. Key flowering proteins comprised clientele of the molecular chaperone demonstrating that the HSP90 is essential during vegetative-to-reproductive phase transition and flower development. Our results uncover that HSP90 consolidates a molecular scaffold able to arrange and organize flowering gene network and protein circuitry, and effectively counterbalance the extent to which developmental noise perturbs phenotypic traits.

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.

SERRATE, a miRNA biogenesis factor, affects viroid infection in Nicotiana benthamiana and Nicotiana tabacum.

Viroids are plant infecting, non - coding RNA molecules of economic importance. Potato spindle tuber viroid (PSTVd), the type species of Pospiviroidae family, has been shown to be affected by specific RNA silencing pathways. Dicer like 1 (DCL1), a key player in micro RNA (miRNA) pathway has been previously linked with PSTVd infectivity. In this report we aim to further dissect the interaction between the miRNA pathway and Pospiviroid virulence. We mainly focused on the Zinc-finger protein SERRATE (SE) a co-factor of DCL1 and core component of miRNA pathway. We generated Nicotiana tabacum and Nicotiana benthamiana SE knock-down plants exhibiting considerable miRNA reduction and strong phenotypic abnormalities. PSTVd infection of SE suppressed plants resulted in a significant viroid reduction, especially at the initial infection stages. This positive correlation between SE levels and viroid infectivity underlines its role in PSTVd life cycle and reveals the importance of the miRNA pathway upon viroid infection.