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Trans -species RNA interference (RNAi) occurs naturally when small RNAs (sRNAs) silence genes in species different from their origin. This phenomenon has been observed between plants and various organisms including fungi, animals and other plant species. Understanding the mechanisms used in natural cases of trans- species RNAi, such as sRNA processing and movement, will enable more effective development of crop protection methods using host-induced gene silencing (HIGS). Recent progress has been made in understanding the mechanisms of cell-to-cell and long-distance movement of sRNAs within individual plants. This increased understanding of endogenous plant sRNA movement may be translatable to trans -species sRNA movement. Here, we review diverse cases of natural trans- species RNAi focusing on current theories regarding intercellular and long-distance sRNA movement. We also touch on trans -species sRNA evolution, highlighting its research potential and its role in improving the efficacy of HIGS. [ABSTRACT FROM AUTHOR]

Author(s): Saima Shahid [1, 2]; Gunjune Kim [3]; Nathan R. Johnson [1, 2]; Eric Wafula [2]; Feng Wang [1, 2]; Ceyda Coruh [1, 2]; Vivian Bernal-Galeano [3]; Tamia Phifer [4]; [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.10.040 Byline: J. Graham Ruby (1)(2), Calvin Jan (1)(2), Christopher Player (1), Michael J. Axtell (1), William Lee (3), Chad Nusbaum (3), Hui Ge (1), David P. Bartel (1)(2) Abstract: We sequenced [approximately equal to]400,000 small RNAs from Caenorhabditis elegans. Another 18 microRNA (miRNA) genes were identified, thereby extending to 112 our tally of confidently identified miRNA genes in C. elegans. Also observed were thousands of endogenous siRNAs generated by RNA-directed RNA polymerases acting preferentially on transcripts associated with spermatogenesis and transposons. In addition, a third class of nematode small RNAs, called 21U-RNAs, was discovered. 21U-RNAs are precisely 21 nucleotides long, begin with a uridine 5'-monophosphate but are diverse in their remaining 20 nucleotides, and appear modified at their 3'-terminal ribose. 21U-RNAs originate from more than 5700 genomic loci dispersed in two broad regions of chromosome IV -- primarily between protein-coding genes or within their introns. These loci share a large upstream motif that enables accurate prediction of additional 21U-RNAs. The motif is conserved in other nematodes, presumably because of its importance for producing these diverse, autonomously expressed, small RNAs (dasRNAs). Author Affiliation: (1) Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA (2) Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA (3) Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA Article History: Received 16 June 2006; Revised 23 September 2006; Accepted 27 October 2006 Article Note: (miscellaneous) Published: December 14, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.09.032 Byline: Michael J. Axtell (1)(2), Calvin Jan (1)(2), Ramya Rajagopalan (1)(2), David P. Bartel (1)(2) Abstract: In Arabidopsis, microRNA-directed cleavage can define one end of RNAs that then generate phased siRNAs. However, most miRNA-targeted RNAs do not spawn siRNAs, suggesting the existence of additional determinants within those that do. We find that in moss, phased siRNAs arise from regions flanked by dual miR390 cleavage sites. AtTAS3, an siRNA locus important for development and conserved among higher plants, also has dual miR390 complementary sites. Both sites bind miR390 in vitro and are functionally required in Arabidopsis, but cleavage is undetectable at the 5' site -- demonstrating that noncleavable sites can be functional in plants. Phased siRNAs also emanate from the bounded regions of every Arabidopsis gene with two known microRNA/siRNA complementary sites, but only rarely from genes with single sites. Therefore, two 'hits,' -- often, but not always, two cleavage events -- constitute a conserved trigger for siRNA biogenesis, a finding with implications for recognition and silencing of aberrant RNA. Author Affiliation: (1) Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA (2) Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA Article History: Received 21 March 2006; Revised 7 July 2006; Accepted 25 September 2006 Article Note: (miscellaneous) Published: November 2, 2006

SUMMARY: Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach, as they have the advantage of producing just a single functional small RNA, which can be designed for high target specificity and low off‐target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans‐acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA‐ and tasiRNA‐based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. All analyses used transiently expressed transgenes delivered by infiltration of leaves with Agrobacterium tumefacians. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5′‐most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3′‐end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single‐target assays such as RNA blots or real‐time polymerase chain reaction, is also discussed. Significance Statement: Several strategies for multiplexed gene silencing using artificial microRNAs or tasiRNAs have been described. We find that many result in imprecise processing, and thus low accumulation of the intended small RNAs. Our findings highlight the importance of small RNA sequencing to analyze gene silencing experiments fully, and the need for continued methodological development of these methods. [ABSTRACT FROM AUTHOR]

Parasitic plants pose a major biotic threat to plant growth and development and lead to losses in crop productivity of billions of USD annually. By comparison with "normal" autotrophic plants, parasitic plants live a heterotrophic lifestyle and rely on water, solutes and to a greater (holoparasitic plants) or lesser extent (hemiparasitic plants) on sugars from other host plants. Most hosts are unable to detect an infestation by plant parasites or unable to fend off these parasitic invaders. However, a few hosts have evolved defense strategies to avoid infestation or protect themselves actively post-attack often leading to full or partial resistance. Here, we review the current state of our understanding of the defense strategies to plant parasitism used by host plants with emphasis on the active molecular resistance mechanisms. Furthermore, we outline the perspectives and the potential of future studies that will be indispensable to develop and breed resistant crops. [ABSTRACT FROM AUTHOR]

Although the central dogma still holds true generally, our understanding has expanded the concept to encompass reverse transcription of RNA to DNA, regulatory functions of noncoding RNAs, and epigenetic modifications. If DNA is the playbook of life, then RNA, in all its forms, is the company of actors that brings life into being. [Extracted from the article]

• The parasitic plant Cuscuta exchanges diverse macromolecules with its hosts. • Trans- species microRNAs from Cuscuta regulate host genes. • The genes encoding these microRNAs could originate from horizontal-gene transfer events. • Extracellular vesicles are one possible mechanism for how trans- species microRNAs are delivered to the host. Parasitic plants make direct contact with their host's vasculature. In parasitism by Cuscuta , RNA and other macromolecules regularly move between host and parasite. Recently, trans- species microRNA from Cuscuta have been shown to functionally target host genes which have essential roles in host defense. Known pathways for the evolution of microRNAs, and the prevalence of horizontal gene transfer events in the Cuscuta lineage, hint that trans- species microRNAs could originate from captured host genes. It is unknown how the delivery of microRNAs from the parasite to the host takes place. One exciting possibility is through apoplastic export using extracellular vesicles, a process which has recently been shown to transport select small RNAs in plants and fungi. These discoveries represent the initial findings of what may be a widespread mechanism of interactions between species. [ABSTRACT FROM AUTHOR]

Parasitic plants steal sugars, water, and other nutrients from host plants through a haustorial connection. Several species of parasitic plants such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) are major biotic constraints to agricultural production. Parasitic plants are understudied compared with other major classes of plant pathogens, but the recent availability of genomic and transcriptomic data has accelerated the rate of discovery of the molecular mechanisms underpinning plant parasitism. Here, we review the current body of knowledge of how parasitic plants sense host plants, germinate, form parasitic haustorial connections, and suppress host plant immune responses. Additionally, we assess whether parasitic plants fit within the current paradigms used to understand the molecular mechanisms of microbial plant–pathogen interactions. Finally, we discuss challenges facing parasitic plant research and propose the most urgent questions that need to be answered to advance our understanding of plant parasitism. [ABSTRACT FROM AUTHOR]

Summary: Plant small RNAs (sRNAs) modulate key physiological mechanisms through post‐transcriptional and transcriptional silencing of gene expression. Small RNAs fall into two major categories: those are reliant on RNA‐dependent RNA polymerases (RDRs) for biogenesis and those that are not. Known RDR1/2/6‐dependent sRNAs include phased and repeat‐associated short interfering RNAs, while known RDR1/2/6‐independent sRNAs are primarily microRNAs (miRNA) and other hairpin‐derived sRNAs. In this study we produced and analyzed sRNA‐seq libraries from rdr1/rdr2/rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on RDR1, ‐2, or ‐6 function, casting doubt on their classification. We also found 38 RDR1/2/6‐independent sRNA loci that are not MIRNAs or otherwise hairpin‐derived, and did not fit into other known paradigms for sRNA biogenesis. These 38 sRNA‐producing loci have as‐yet‐undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein‐coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types of sRNA in Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

In plants, 24 nucleotide long heterochromatic si RNAs (het-si RNAs) transcriptionally regulate gene expression by RNA-directed DNA methylation (Rd DM). The biogenesis of most het-si RNAs depends on the plant-specific RNA polymerase IV (Pol IV), and ARGONAUTE4 ( AGO4) is a major het-si RNA effector protein. Through genome-wide analysis of sRNA-seq data sets, we found that AGO4 is required for the accumulation of a small subset of het-si RNAs. The accumulation of AGO4-dependent het-si RNAs also requires several factors known to participate in the effector portion of the Rd DM pathway, including RNA POLYMERASE V ( POL V), DOMAINS REARRANGED METHYLTRANSFERASE 2 ( DRM2) and SAWADEE HOMEODOMAIN HOMOLOGUE 1 ( SHH1). Like many AGO proteins, AGO4 is an endonuclease that can 'slice' RNAs. We found that a slicing-defective AGO4 was unable to fully recover AGO4-dependent het-si RNA accumulation from ago4 mutant plants. Collectively, our data suggest that AGO4-dependent si RNAs are secondary si RNAs dependent on the prior activity of the Rd DM pathway at certain loci. [ABSTRACT FROM AUTHOR]

Abstract: Highly parallel sequencing of cDNA derived from endogenous small RNAs (small RNA-seq) is a key method that has accelerated understanding of regulatory small RNAs in eukaryotes. Eukaryotic regulatory small RNAs, which include microRNAs (miRNAs), short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs), typically derive from the processing of longer precursor RNAs. Alignment of small RNA-seq data to a reference genome allows the inference of the longer precursor and thus the annotation of small RNA producing genes. ShortStack is a program that was developed to comprehensively analyze reference-aligned small RNA-seq data, and output detailed and useful annotations of the causal small RNA-producing genes. Here, we provide a step-by-step tutorial of ShortStack usage with the goal of introducing new users to the software and pointing out some common pitfalls. [Copyright &y& Elsevier]

Conserved plant microRNAs (miRNAs) modulate important biological processes but little is known about conserved cis-regulatory elements (CREs) surrounding MIRNA genes. We developed a solution-based targeted genomic enrichment methodology to capture, enrich, and sequence flanking genomic regions surrounding conserved MIRNA genes with a locked-nucleic acid (LNA)-modified, biotinylated probe complementary to the mature miRNA sequence. Genomic DNA bound by the probe is captured by streptavidin-coated magnetic beads, amplified, sequenced and assembled de novo to obtain genomic DNA sequences flanking MIRNA locus of interest. We demonstrate the sensitivity and specificity of this enrichment methodology in Arabidopsis thaliana to enrich targeted regions spanning 10–20 kb surrounding known MIR166 and MIR165 loci. Assembly of the sequencing reads successfully recovered all targeted loci. While further optimization for larger, more complex genomes is needed, this method may enable determination of flanking genomic DNA sequence surrounding a known core (like a conserved mature miRNA) from multiple species that currently don't have a full genome assembly available. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures, and their accurate processing is required for the production of functional miRNAs. Here, using the pri-miR-166 family in Arabidopsis thaliana as a paradigm, we report the crucial role of pri-miRNA terminal loops in miRNA biogenesis. We found that multibranched terminal loops in pri-miR-166s substantially suppress miR-166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal loop-branched pri-miRNAs can be processed by Dicer-like 1 (DCL1) complexes bidirectionally from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In both cases, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 bp from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. These results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complex secondary structures. [ABSTRACT FROM AUTHOR]

Regulatory small RNAs, which range in size from 20 to 24 nucleotides are ubiquitous components of endogenous plant transcriptomes, as well as common responses to exogenous viral infections and introduced double-stranded RNA (dsRNA). Endogenous small RNAs derive from the processing of helical RNA precursors and can be categorized into several groups based on differences in biogenesis and function. A major distinction can be observed between small RNAs derived from single-stranded precursors with a hairpin structure [referred to here as hairpin RNAs (hpRNAs)] and those derived from dsRNA precursors [small interfering RNAs (siRNAs)]. hpRNAs in plants can be divided into two secondary groups: microRNAs and those that are not microRNAs. The currently known siRNAs fall mostly into one of three secondary groups: heterochromatic siRNAs, secondary siRNAs, and natural anti-sense transcript siRNAs. Tertiary subdivisions can be identified within many of the secondary classifications as well. Comparisons between the different classes of plant small RNAs help to illuminate key goals future research. [ABSTRACT FROM AUTHOR]

Dicer-Like1 (DCL1), an RNaseIII endonuclease, and Hyponastic Leaves1 (HYL1), a double-stranded RNA-binding protein, are core components of the plant microRNA (miRNA) biogenesis machinery, hyl1 null mutants accumulate low levels of miRNAs and display pleiotropic developmental phenotypes. We report the identification of five new hyl1 suppressor mutants, all of which are alleles of DCL1. These new alleles affect either the helicase or the RNaseIIIa domains of DCL1, highlighting the critical functions of these domains. Biochemical analysis of the DCL1 suppressor variants reveals that they process the primary transcript (pri-miRNA) more efficiently than wild-type DCL1, with both higher Kcat and lower Km values. The DCL1 variants largely rescue wild-type miRNA accumulation levels in vivo, but do not rescue the MIRNA processing precision defects of the hyl1 null mutant. In vitro, the helicase domain confers ATP dependence on DCL1-catalyzed MIRNA processing, attenuates DCL1 cleavage activity, and is required for precise MIRNA processing of some substrates. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. Currently, miRBase (version 13) lists 142 and 353 miRNAs from Arabidopsis and rice ( Oryza sativa), respectively. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. In contrast with the well-characterized miRNA targets that are cleaved in Arabidopsis, only a few such targets have been confirmed in rice. To identify small RNA targets in rice, we applied the ‘degradome sequencing’ approach, which globally identifies the remnants of small RNA-directed target cleavage by sequencing the 5′ ends of uncapped RNAs. One hundred and sixty targets of 53 miRNA families (24 conserved and 29 rice-specific) and five targets of TAS3-small interfering RNAs (siRNAs) were identified. Surprisingly, an additional conserved target for miR398, which has not been reported so far, has been validated. Besides conserved homologous transcripts, 23 non-conserved genes for nine conserved miRNAs and 56 genes for 29 rice-specific miRNAs were also identified as targets. Besides miRNA targets, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA-producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants. [ABSTRACT FROM AUTHOR]

Summary: MicroRNAs (miRNAs) are excised from hairpin structures within primary miRNAs (pri-miRNAs). Most animal pri-miRNAs are processed by two cleavages, the first at a loop-distal site ∼11 nucleotides (nt) from the end of the hairpin and the second ∼22 nt beyond the first . To identify RNA structural determinants of miRNA processing in plants, we analyzed the functional consequences of changing the secondary structure of the lower (loop-distal), middle (miRNA:miRNA∗), and upper (loop-proximal) stems of the hairpin in two different pri-miRNAs. Closing bulges immediately below the loop-distal cleavage sites increased the accumulation of accurately cleaved precursor miRNAs but decreased the abundance of the mature miRNAs. A pri-miRNA variant with an unpaired lower stem was not processed, and variants with a perfectly paired middle or upper stem were processed normally. Bioinformatic analysis of pri-miRNA structures, together with physical mapping of initial cleavage sites and in vitro processing of pri-miRNA, reveals that the first, loop-distal cleavage is often at a distance of ∼15 nt from an unpaired region. Hence, a common determinant of the rate and location of the initial pri-miRNA cleavage is an imperfectly base-paired duplex of ∼15 nt between the miRNA:miRNA∗ duplex and either a less structured region of the lower stem or its end. [Copyright &y& Elsevier]

Endogenous 24 nt short interfering RNAs (siRNAs), derived mostly from intergenic and repetitive genomic regions, constitute a major class of endogenous small RNAs in flowering plants. Accumulation of Arabidopsis thaliana 24 nt siRNAs requires the Dicer family member DCL3, and clear homologs of DCL3 exist in both flowering and non-flowering plants. However, the absence of a conspicuous 24 nt peak in the total RNA populations of several non-flowering plants has raised the question of whether this class of siRNAs might, in contrast to the ancient 21 nt microRNAs (miRNAs) and 21-22 nt trans-acting siRNAs (tasiRNAs), be an angiosperm-specific innovation. Analysis of non-miRNA, non-tasiRNA hotspots of small RNA production within the genome of the moss Physcomitrella patens revealed multiple loci that consistently produced a mixture of 21-24 nt siRNAs with a peak at 23 nt. These Pp23SR loci were significantly enriched in transposon content, depleted in overlap with annotated genes, and typified by dense concentrations of the 5-methyl cytosine (5 mC) DNA modification. Deep sequencing of small RNAs from two independent Ppdcl3 mutants showed that the P. patens DCL3 homolog is required for the accumulation of 22-24 nt siRNAs, but not 21 nt siRNAs, at Pp23SR loci. The 21 nt component of Pp23SR-derived siRNAs was also unaffected by a mutation in the RNA-dependent RNA polymerase mutant Pprdr6. Transcriptome-wide, Ppdcl3 mutants failed to accumulate 22-24 nt small RNAs from repetitive regions while transcripts from two abundant families of long terminal repeat (LTR) retrotransposon-associated reverse transcriptases were upregulated. Ppdcl3 mutants also displayed an acceleration of leafy gametophore production, suggesting that repetitive siRNAs may play a role in the development of P. patens. We conclude that intergenic/repeat-derived siRNAs are indeed a broadly conserved, distinct class of small regulatory RNAs within land plants. [ABSTRACT FROM AUTHOR]