New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis

[EN] Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are used for small RNA-based, specific gene silencing or knockdown in plants. Current methods to generate amiRNA or syn-tasiRNA constructs are not well adapted for cost-effective, large-scale production or for multiplexing to specifically suppress multiple targets. Here, we describe simple, fast, and cost-effective methods with high-throughput capability to generate amiRNA and multiplexed syn-tasiRNA constructs for efficient gene silencing in Arabidopsis (Arabidopsis thaliana) and other plant species. amiRNA or syn-tasiRNA inserts resulting from the annealing of two overlapping and partially complementary oligonucleotides are ligated directionally into a zero background BsaI/ccdB-based expression vector. BsaI/ccdB vectors for amiRNA or syn-tasiRNA cloning and expression contain a modified version of Arabidopsis MIR390a or TAS1c precursors, respectively, in which a fragment of the endogenous sequence was substituted by a ccdB cassette flanked by two BsaI sites. Several amiRNA and syn-tasiRNA sequences designed to target one or more endogenous genes were validated in transgenic plants that (1) exhibited the expected phenotypes predicted by loss of target gene function, (2) accumulated high levels of accurately processed amiRNAs or syn-tasiRNAs, and (3) had reduced levels of the corresponding target RNAs.This work was supported by the National Science Foundation (grant nos. MCB-0956526 and MCB-1231726), the National Institutes of Health (grant no. AI043288), the Japan Society for the Promotion of Science (postdoctoral fellowship to A.T.), and the National Institute of Food and Agriculture (postdoctoral fellowship no. MOW-2012-01361 to N.F.)Carbonell, A.; Takeda, A.; Fahlgren, N.; Johnson, SC.; Cuperus, JT.; Carrington, JC. (2014). New generation of artificial microRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. PLANT PHYSIOLOGY. 165(1):15-29. https://doi.org/10.1104/pp.113.234989S1529165

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants

[EN] In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.We thank Goretti Nguyen for excellent technical assistance. A. C. was supported by a postdoctoral fellowship from the Ministerio de Ciencia e Innovacion (BMC-2008-0188). H.G.-R. was the recipient of a Helen Hay Whitney Postdoctoral fellowship (F-972). 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Specificity of ARGONAUTE7-miR390 Interaction and Dual Functionality in TAS3 Trans-Acting siRNA Formation

SummaryTrans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5′ adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5′ nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps

Identification of MIR390a precursor processing defective mutants in Arabidopsis by direct genome sequencing

Transacting siRNA (tasiRNA) biogenesis in Arabidopsis is initiated by microRNA (miRNA) –guided cleavage of primary transcripts. In the case of TAS3 tasiRNA formation, ARGONAUTE7 (AGO7)– miR390 complexes interact with primary transcripts at two sites, resulting in recruitment of RNA-DEPENDENT RNA POLYMERASE6 for dsRNA biosynthesis. An extensive screen for Arabidopsis mutants with specific defects in TAS3 tasiRNA biogenesis or function was done. This yielded numerous ago7 mutants, one dcl4 mutant, and two mutants that accumulated low levels of miR390. A direct genome sequencing-based approach to both map and rapidly identify one of the latter mutant alleles was developed. This revealed a G-to-A point mutation (mir390a-1) that was calculated to stabilize a relatively nonpaired region near the base of the MIR390a foldback, resulting in misprocessing of the miR390/ miR390* duplex and subsequent reduced TAS3 tasiRNA levels. Directed substitutions, as well as analysis of variation at paralogous miR390-generating loci (MIR390a and MIR390b), indicated that base pair properties and nucleotide identity within a region 4–6 bases below the miR390/miR390* duplex region contributed to the efficiency and accuracy of precursor processing

Functional Analysis of Three Arabidopsis

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses

Phytophthora Have Distinct Endogenous Small RNA Populations That Include Short Interfering and microRNAs

In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work

Vision, challenges and opportunities for a Plant Cell Atlas

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.</jats:p

Evolution and Functional Diversification of MIRNA Genes

MicroRNAs (miRNAs) are small regulatory RNAs found in diverse eukaryotic lineages. In plants, a minority of annotated MIRNA gene families are conserved between plant families, while the majority are family- or species-specific, suggesting that most known MIRNA genes arose relatively recently in evolutionary time. Given the high proportion of young MIRNA genes in plant species, new MIRNA families are likely spawned and then lost frequently. Unlike highly conserved, ancient miRNAs, young miRNAs are often weakly expressed, processed imprecisely, lack targets, and display patterns of neutral variation, suggesting that young MIRNA loci tend to evolve neutrally. Genome-wide analyses from several plant species have revealed that variation in miRNA foldback expression, structure, processing efficiency, and miRNA size have resulted in the unique functionality of MIRNA loci and resulting miRNAs. Additionally, some miRNAs have evolved specific properties and functions that regulate other transcriptional or posttranscriptional silencing pathways. The evolution of miRNA processing and functional diversity underscores the dynamic nature of miRNA-based regulation in complex regulatory networks

Effects of sequence motifs in the yeast 3′ untranslated region determined from massively parallel assays of random sequences

Abstract Background The 3′ untranslated region (UTR) plays critical roles in determining the level of gene expression through effects on activities such as mRNA stability and translation. Functional elements within this region have largely been identified through analyses of native genes, which contain multiple co-evolved sequence features. Results To explore the effects of 3′ UTR sequence elements outside of native sequence contexts, we analyze hundreds of thousands of random 50-mers inserted into the 3′ UTR of a reporter gene in the yeast Saccharomyces cerevisiae. We determine relative protein expression levels from the fitness of transformants in a growth selection. We find that the consensus 3′ UTR efficiency element significantly boosts expression, independent of sequence context; on the other hand, the consensus positioning element has only a small effect on expression. Some sequence motifs that are binding sites for Puf proteins substantially increase expression in the library, despite these proteins generally being associated with post-transcriptional downregulation of native mRNAs. Our measurements also allow a systematic examination of the effects of point mutations within efficiency element motifs across diverse sequence backgrounds. These mutational scans reveal the relative in vivo importance of individual bases in the efficiency element, which likely reflects their roles in binding the Hrp1 protein involved in cleavage and polyadenylation. Conclusions The regulatory effects of some 3′ UTR sequence features, like the efficiency element, are consistent regardless of sequence context. In contrast, the consequences of other 3′ UTR features appear to be strongly dependent on their evolved context within native genes