In plants, small non-coding RNAs (≈20-30 nt) play a major role in a gene regulation mechanism that controls development, maintains heterochromatin and defends against viruses. However, their possible role in cell division (mitosis and meiosis) still remains to be ascertained. ARGONAUTE (AGO) proteins are key players in the different small RNA (sRNA) pathways. Arabidopsis contains 10 AGO proteins belonging to three distinct phylogenetic clades based on amino acid sequence, namely: AGO1/AGO5/AGO10, AGO2/AGO3/AGO7, and AGO4/AGO6/AGO8/AGO9. To gain new insights into the role of AGO proteins, we have focused our attention on AGO2, AGO5, and AGO9 by means of the analysis of plants carrying mutations in the corresponding genes. AGO2 plays a role in the natural cis-antisense (nat-siRNA) pathway and is required for an efficient DNA repair. On the other hand, AGO5, involved in miRNA (microRNA)-directed target cleavage, and AGO9, involved in RNA-directed DNA methylation (RdDM), are highly enriched in germline. On these grounds, we have analyzed the effects of these proteins on the meiotic process and also on DNA repair. It was confirmed that AGO2 is involved in DNA repair. In ago2-1 the mean cell chiasma frequency in pollen mother cells (PMCs) was increased relative to the wild-type (WT). ago5-4 showed a delay in germination time and a slight decrease in fertility, however the meiotic process and chiasma levels were normal. Meiosis in PMCs of ago9-1 was characterized by a high frequency of chromosome interlocks from pachytene to metaphase I, but chiasma frequency and fertility were normal. Genotoxicity assays have confirmed that AGO9 is also involved in somatic DNA repair.

Plant and animal microRNA (miRNA) pathways share many analogous components, the ARGONAUTE (AGO) proteins being foremost among them. We sought to ascertain the degree of functional conservation shared by Homo sapiens ARGONAUTE 2 (HsAGO2) and Arabidopsis thaliana ARGONAUTE 1 (AtAGO1), which are the predominant AGO family members involved with miRNA activity in their respective species. Transgenic Arabidopsis plants expressing HsAGO2 were indistinguishable from counterparts over-expressing AtAGO1, each group exhibiting the morphological and molecular hallmarks of miRNA-pathway loss-of-function alleles. However, unlike AtAGO1, HsAGO2 was unable to rescue the ago1-27 allele. We conclude that, despite the evolutionary gulf between them, HsAGO2 is likely capable of interacting with some component/s of the Arabidopsis miRNA pathway, thereby perturbing its operation, although differences have arisen such that HsAGO2 alone is insufficient to confer efficient silencing of miRNA targets in planta.

Small RNA (sRNA)-mediated gene silencing represents a conserved regulatory mechanism controlling a wide diversity of developmental processes through interactions of sRNAs with proteins of the ARGONAUTE (AGO) family. On the basis of a large phylogenetic analysis that includes 206 AGO genes belonging to 23 plant species, AGO genes group into four clades corresponding to the phylogenetic distribution proposed for the ten family members of Arabidopsis thaliana. A primary analysis of the corresponding protein sequences resulted in 50 sequences of amino acids (blocks) conserved across their linear length. Protein members of the AGO4/6/8/9 and AGO1/10 clades are more conserved than members of the AGO5 and AGO2/3/7 clades. In addition to blocks containing components of the PIWI, PAZ, and DUF1785 domains, members of the AGO2/3/7 and AGO4/6/8/9 clades possess other consensus block sequences that are exclusive of members within these clades, suggesting unforeseen functional specialization revealed by their primary sequence. We also show that AGO proteins of animal and plant kingdoms share linear sequences of blocks that include motifs involved in posttranslational modifications such as those regulating AGO2 in humans and the PIWI protein AUBERGINE in Drosophila. Our results open possibilities for exploring new structural and functional aspects related to the evolution of AGO proteins within the plant kingdom, and their convergence with analogous proteins in mammals and invertebrates.

Argonaute 2 (AGO2)-mediated role in plant defense against fungal pathogens remains largely unknown. In this study, integrated miRNAome and transcriptome analysis employing ago2 mutant was performed to reveal AGO2-associated miRNAs and defense responses against the devastating necrotrophic phytopathogen Sclerotinia sclerotiorum. Both miRNAome and transcriptomes of S. sclerotiorum-inoculated ago2-1 mutant (ago2-Ss) and wild-type (WT-Ss) as well as mock-inoculated ago2-1 mutant (ago2) and wild-type (WT) Arabidopsis plants, were analyzed by sRNA and mRNA deep sequencing. Differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) of the comparisons WT-Ss/WT, ago2/WT, ago2-Ss/WT-Ss, and ago2-Ss/ago2 were identified. Furthermore, integration analysis for the DEMs and DEGs identified over 40 potential AGO2-dependent Sclerotinia sclerotiorum-responsive (ATSR) DEM-DEG pairs involving modulation of immune recognition, calcium flux, redox homeostasis, hormone accumulation and signaling, cell wall modification and metal ion homeostasis. Data-mining result indicated that most of the DEMs were bound with AGO2. Moreover, Arabidopsis mutant analysis demonstrated that three ROS and redox homeostatasis related DEGs of identified DEM-DEG pairs, GSTU2, GSTU5, and RBOHF contributed to the AGO2-mediated defense against S. sclerotiorum. This work provides genome-wide prediction of miRNA-target gene pairs that are potentially associated with the AGO2-dependent resistance against S. sclerotiorum.

RNA interference (RNAi) is a biological process in which small RNAs regulate gene silencing at the transcriptional or posttranscriptional level. The trigger for gene silencing is double-stranded RNA generated from an endogenous genomic locus or a foreign source, such as a transgene or virus. In addition to regulating endogenous gene expression, RNAi provides the mechanistic basis for small RNA-mediated communication between plant hosts and interacting pathogenic microbes, known as cross-kingdom RNAi. Two core protein components, Argonaute (AGO) and Dicer (DCL), are central to the RNAi machinery of eukaryotes. Plants encode for several copies of AGO and DCL genes; in Arabidopsis thaliana, the AGO protein family contains 10 members, and the DCL family contains four. Little is known about the conservation and specific roles of these proteins in monocotyledonous plants, which account for the most important food staples. Here, we utilized in silico tools to investigate the structure and related functions of AGO and DCL proteins from the model grass Brachypodium distachyon. Based on the presence of characteristic domains, 16 BdAGO- and 6 BdDCL-predicted proteins were identified. Phylogenetic analysis showed that both protein families were expanded in Brachypodium as compared with Arabidopsis. For BdDCL proteins, both plant species contain a single copy of DCL1 and DCL4; however, Brachypodium contains two copies each of DCL2 and DCL3. Members of the BdAGO family were placed in all three functional clades of AGO proteins previously described in Arabidopsis. The greatest expansion occurred in the AtAGO1/5/10 clade, which contains nine BdAGOs (BdAGO5/6/7/9/10/11/12/15/16). The catalytic tetrad of the AGO P-element-induced wimpy testis domain (PIWI), which is required for endonuclease activity, is conserved in most BdAGOs, with the exception of BdAGO1, which lacks the last D/H residue. Three-dimensional modeling of BdAGO proteins using tertiary structure prediction software supported the phylogenetic classification. We also predicted a provisional interactome network for BdAGOs, their localization within the cell, and organ/tissue-specific expression. Exploring the specifics of RNAi machinery proteins in a model grass species can serve as a proxy for agronomically important cereals such as barley and wheat, where the development of RNAi-based plant protection strategies is of great interest.

The specific recognition of miRNAs by Argonaute (AGO) proteins, the effector proteins of the RNA-induced silencing complex, constitutes the final step of the biogenesis of miRNAs and is crucial for their target interaction. In the genome of Arabidopsis thaliana (Ath), 10 different AGO proteins are encoded and the sorting decision, which miRNA associates with which AGO protein, was reported to depend exclusively on the identity of the 5'-sequence position of mature miRNAs. Hence, with only four different bases possible, a 5'-position-only sorting signal would not suffice to specifically target all 10 different AGOs individually or would suggest redundant AGO action. Alternatively, other and as of yet unidentified sorting signals may exist. We analyzed a dataset comprising 117 Ath-miRNAs with clear sorting preference to either AGO1, AGO2, or AGO5 as identified in co-immunoprecipitation experiments combined with sequencing. While mutual information analysis did not identify any other single position but the 5'-nucleotide to be informative for the sorting at sufficient statistical significance, significantly better than random classification results using Random Forests nonetheless suggest that additional positions and combinations thereof also carry information with regard to the AGO sorting. Positions 2, 6, 9, and 13 appear to be of particular importance. Furthermore, uracil bases at defined positions appear to be important for the sorting to AGO2 and AGO5, in particular. No predictive value was associated with miRNA length or base pair binding pattern in the miRNA:miRNA* duplex. From inspecting available AGO gene expression data in Arabidopsis, we conclude that the temporal and spatial expression profile may also contribute to the fine-tuning of miRNA sorting and function.

RNA silencing is an important mechanism to regulate gene expression and antiviral defense in plants. Nevertheless, RNA silencing machinery in the important oil crop Brassica napus and function in resistance to the devastating fungal pathogen Sclerotinia sclerotiorum are not well-understood. In this study, gene families of RNA silencing machinery in B. napus were identified and their role in resistance to S. sclerotiorum was revealed. Genome of the allopolyploid species B. napus possessed 8 Dicer-like (DCL), 27 Argonaute (AGO), and 16 RNA-dependent RNA polymerase (RDR) genes, which included almost all copies from its progenitor species B. rapa and B. oleracea and three extra copies of RDR5 genes, indicating that the RDR5 group in B. napus appears to have undergone further expansion through duplication during evolution. Moreover, compared with Arabidopsis, some AGO and RDR genes such as AGO1, AGO4, AGO9, and RDR5 had significantly expanded in these Brassica species. Twenty-one out of 51 DCL, AGO, and RDR genes were predicted to contain calmodulin-binding transcription activators (CAMTA)-binding site (CGCG box). S. sclerotiorum inoculation strongly induced the expression of BnCAMTA3 genes while significantly suppressed that of some CGCG-containing RNA silencing component genes, suggesting that RNA silencing machinery might be targeted by CAMTA3. Furthermore, Arabidopsis mutant analyses demonstrated that dcl4-2, ago9-1, rdr1-1, rdr6-11, and rdr6-15 mutants were more susceptible to S. sclerotiorum, while dcl1-9 was more resistant. Our results reveal the importance of RNA silencing in plant resistance to S. sclerotiorum and imply a new mechanism of CAMTA function as well as RNA silencing regulation.

Biotic stress in legume crops is one of the major threats to crop yield and productivity. Being sessile organisms, plants have evolved a myriad of mechanisms to combat different stresses imposed on them. One such mechanism, deciphered in the last decade, is small RNA (sRNA) mediated defense in plants. Small RNAs (sRNAs) have emerged as one of the major players in gene expression regulation in plants during developmental stages and under stress conditions. They are known to act both at transcriptional and post-transcriptional levels. Dicer-like (DCL), Argonaute (AGO), and RNA dependent RNA polymerase (RDR) constitute the major components of sRNA biogenesis machinery and are known to play a significant role in combating biotic and abiotic stresses. This study is, therefore, focused on identification and characterization of sRNA biogenesis proteins in three important legume crops, namely chickpea, pigeonpea, and groundnut. Phylogenetic analysis of these proteins between legume species classified them into distinct clades and suggests the evolutionary conservation of these genes across the members of Papillionidoids subfamily. Variable expression of sRNA biogenesis genes in response to the biotic stresses among the three legumes indicate the possible existence of specialized regulatory mechanisms in different legumes. This is the first ever study to understand the role of sRNA biogenesis genes in response to pathogen attacks in the studied legumes.

Quantitative proteomics strategies - which are playing important roles in the expanding field of plant molecular systems biology - are traditionally designated as either hypothesis driven or non-hypothesis driven. Many of these strategies aim to select individual peptide ions for tandem mass spectrometry (MS/MS), and to do this mixed hypothesis driven and non-hypothesis driven approaches are theoretically simple to implement. In-depth investigations into the efficacies of such approaches have, however, yet to be described. In this study, using combined samples of unlabeled and metabolically 15N-labeled Arabidopsis thaliana proteins, we investigate the mixed use of targeted data acquisition (TDA) and data dependent acquisition (DDA) - referred to as TDA/DDA - to facilitate both hypothesis driven and non-hypothesis driven quantitative data collection in individual LC-MS/MS experiments. To investigate TDA/DDA for hypothesis driven data collection, 7 miRNA target proteins of differing size and abundance were targeted using inclusion lists comprised of 1558 m/z values, using 3 different TDA/DDA experimental designs. In samples in which targeted peptide ions were of particularly low abundance (i.e., predominantly only marginally above mass analyser detection limits), TDA/DDA produced statistically significant increases in the number of targeted peptides identified (230 ± 8 versus 80 ± 3 for DDA; p = 1.1 × 10-3) and quantified (35 ± 3 versus 21 ± 2 for DDA; p = 0.038) per experiment relative to the use of DDA only. These expected improvements in hypothesis driven data collection were observed alongside unexpected improvements in non-hypothesis driven data collection. Untargeted peptide ions with m/z values matching those in inclusion lists were repeatedly identified and quantified across technical replicate TDA/DDA experiments, resulting in significant increases in the percentages of proteins repeatedly quantified in TDA/DDA experiments only relative to DDA experiments only (33.0 ± 2.6% versus 8.0 ± 2.7%, respectively; p = 0.011). These results were observed together with uncompromised broad-scale MS/MS data collection in TDA/DDA experiments relative to DDA experiments. Using our observations we provide guidelines for TDA/DDA method design for quantitative plant proteomics studies, and suggest that TDA/DDA is a broadly underutilized proteomics data acquisition strategy.

Over the last decade, several studies have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection. We have previously shown that targeted gene silencing mediated by an in planta expression of non-coding inhibitory double-stranded RNAs (dsRNAs) can protect host plants against various diseases with unprecedented efficiency. In addition to the generation of RNA-silencing (RNAi) signals in planta, plants can be protected from pathogens, and pests by spray-applied RNA-based biopesticides. Despite the striking efficiency of RNA-silencing-based technologies holds for agriculture, the molecular mechanisms underlying spray-induced gene silencing (SIGS) strategies are virtually unresolved, a requirement for successful future application in the field. Based on our previous work, we predict that the molecular mechanism of SIGS is controlled by the fungal-silencing machinery. In this study, we used SIGS to compare the silencing efficiencies of computationally-designed vs. manually-designed dsRNA constructs targeting ARGONAUTE and DICER genes of Fusarium graminearum (Fg). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from Fg infection and that the manual design of dsRNAs resulted in higher gene-silencing efficiencies than the tool-based design. Moreover, our results indicate the possibility of cross-kingdom RNA silencing in the Fg-barley interaction, a phenomenon in which sRNAs operate as effector molecules to induce gene silencing between species from different kingdoms, such as a plant host and their interacting pathogens.

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.

Small non-coding RNAs (smRNA) participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA) and short-interfering RNAs (siRNA) are generated from long double stranded RNA (dsRNA) that are cleaved into 20-24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL). One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO) proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in three legumes: Medicago truncatula, soybean (Glycine max) and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179, and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes, like AGO10 or DCL4, could not yet be detected in M. truncatula available genomic and expressed sequence (EST) databases. In contrast to Arabidopsis, an important gene diversification was observed in the three legume models (for DCL2, AGO4, AGO2, and AGO10) or specifically in soybean for DCL1 and DCL4. Functional significance of these variant isoforms may reflect peculiarities of smRNA biogenesis and functions in legumes.

Cross-kingdom small RNA (sRNA) silencing has recently emerged as a mechanism facilitating fungal colonization and disease development. Here we characterized RNAi pathways in Zymoseptoria tritici, a major fungal pathogen of wheat, and assessed their contribution to pathogenesis. Computational analysis of fungal sRNA and host mRNA sequencing datasets was used to define the global sRNA populations in Z. tritici and predict their mRNA targets in wheat. 389 in planta-induced sRNA loci were identified. sRNAs generated from some of these loci were predicted to target wheat mRNAs including those potentially involved in pathogen defense. However, molecular approaches failed to validate targeting of selected wheat mRNAs by fungal sRNAs. Mutant strains of Z. tritici carrying deletions of genes encoding key components of RNAi such as Dicer-like (DCL) and Argonaute (AGO) proteins were generated, and virulence bioassays suggested that these are dispensable for full infection of wheat. Nonetheless, our results did suggest the existence of non-canonical DCL-independent pathway(s) for sRNA biogenesis in Z. tritici. dsRNA targeting essential fungal genes applied in vitro or generated from an RNA virus vector in planta in a procedure known as HIGS (Host-Induced Gene Silencing) was ineffective in preventing Z. tritici growth or disease. We also demonstrated that Z. tritici is incapable of dsRNA uptake. Collectively, our data suggest that RNAi approaches for gene function analyses in this fungal species and potentially also as a control measure may not be as effective as has been demonstrated for some other plant pathogenic fungi.