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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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Small regulatory RNAs can move between organisms and regulate gene expression in the recipient. Whether the trans-species small RNAs being exported are distinguished from the normal endogenous small RNAs of the source organism is not known. The parasitic plant Cuscuta campestris (dodder) produces many microRNAs that specifically accumulate at the host-parasite interface, several of which have trans-species activity. We found that induction of C. campestris interface-induced microRNAs is similar regardless of host species and occurs in C. campestris haustoria produced in the absence of any host. The loci-encoding C. campestris interface-induced microRNAs are distinguished by a common cis-regulatory element. This element is identical to a conserved upstream sequence element (USE) used by plant small nuclear RNA loci. The properties of the interface-induced microRNA primary transcripts strongly suggest that they are produced via U6-like transcription by RNA polymerase III. The USE promotes accumulation of interface-induced miRNAs (IIMs) in a heterologous system. This promoter element distinguishes C. campestris IIM loci from other plant small RNAs. Our data suggest that C. campestris IIMs are produced in a manner distinct from canonical miRNAs. All confirmed C. campestris microRNAs with documented trans-species activity are interface-induced and possess these features. We speculate that RNA polymerase III transcription of IIMs may allow these miRNAs to be exported to hosts., Competing Interests: Conflict of interest statement: S.M. and S.G. were employees of New England Biolabs, Inc. New England Biolabs is a manufacturer and vendor of molecular biology reagents, including several enzymes and buffers used in this study. This affiliation does not affect the authors’ impartiality, adherence to journal standards and policies, or availability of data. C.H., J.H., and M.J.A. declare no conflicts of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Epigenomics is the study of molecular signatures associated with discrete regions within genomes, many of which are important for a wide range of nuclear processes. The ability to profile the epigenomic landscape associated with genes, repetitive regions, transposons, transcription, differential expression, cis-regulatory elements, and 3D chromatin interactions has vastly improved our understanding of plant genomes. However, many epigenomic and single-cell genomic assays are challenging to perform in plants, leading to a wide range of data quality issues; thus, the data require rigorous evaluation prior to downstream analyses and interpretation. In this commentary, we provide considerations for the evaluation of plant epigenomics and single-cell genomics data quality with the aim of improving the quality and utility of studies using those data across diverse plant species., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

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., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© American Society of Plant Biologists 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plants transmit signals long distances, as evidenced in grafting experiments that create distinct rootstock-scion junctions. Noncoding small RNA is a signaling molecule that is graft transmissible, participating in RNA-directed DNA methylation; but the meiotic transmissibility of graft-mediated epigenetic changes remains unclear. Here, we exploit the MSH1 system in Arabidopsis and tomato to introduce rootstock epigenetic variation to grafting experiments. Introducing mutations dcl2, dcl3 and dcl4 to the msh1 rootstock disrupts siRNA production and reveals RdDM targets of methylation repatterning. Progeny from grafting experiments show enhanced growth vigor relative to controls. This heritable enhancement-through-grafting phenotype is RdDM-dependent, involving 1380 differentially methylated genes, many within auxin-related gene pathways. Growth vigor is associated with robust root growth of msh1 graft progeny, a phenotype associated with auxin transport based on inhibitor assays. Large-scale field experiments show msh1 grafting effects on tomato plant performance, heritable over five generations, demonstrating the agricultural potential of epigenetic variation.

MSH1 is a plant-specific protein. RNAi suppression of MSH1 results in phenotype variability for developmental and stress response pathways. Segregation of the RNAi transgene produces non-genetic msh1 'memory' with multi-generational inheritance. First-generation memory versus non-memory comparison, and six-generation inheritance studies, identifies gene-associated, heritable methylation repatterning. Genome-wide methylome analysis integrated with RNAseq and network-based enrichment studies identifies altered circadian clock networks, and phytohormone and stress response pathways that intersect with circadian control. A total of 373 differentially methylated loci comprising these networks are sufficient to discriminate memory from nonmemory full sibs. Methylation inhibitor 5-azacytidine diminishes the differences between memory and wild type for growth, gene expression and methylation patterning. The msh1 reprogramming is dependent on functional HISTONE DEACETYLASE 6 and methyltransferase MET1, and transition to memory requires the RNA-directed DNA methylation pathway. This system of phenotypic plasticity may serve as a potent model for defining accelerated plant adaptation during environmental change.

Plant endogenous small RNAs (sRNAs) are important regulators of gene expression. There are two broad categories of plant sRNAs: microRNAs (miRNAs) and endogenous short interfering RNAs (siRNAs). MicroRNA loci are relatively well-annotated but compose only a small minority of the total sRNA pool; siRNA locus annotations have lagged far behind. Here, we used a large data set of published and newly generated sRNA sequencing data (1333 sRNA-seq libraries containing more than 20 billion reads) and a uniform bioinformatic pipeline to produce comprehensive sRNA locus annotations of 47 diverse plants, yielding more than 2.7 million sRNA loci. The two most numerous classes of siRNA loci produced mainly 24- and 21-nucleotide (nt) siRNAs, respectively. Most often, 24-nt-dominated siRNA loci occurred in intergenic regions, especially at the 5'-flanking regions of protein-coding genes. In contrast, 21-nt-dominated siRNA loci were most often derived from double-stranded RNA precursors copied from spliced mRNAs. Genic 21-nt-dominated loci were especially common from disease resistance genes, including from a large number of monocots. Individual siRNA sequences of all types showed very little conservation across species, whereas mature miRNAs were more likely to be conserved. We developed a web server where our data and several search and analysis tools are freely accessible., (© 2020 Lunardon et al.; Published by Cold Spring Harbor Laboratory Press.)

Regulatory small RNAs are well known as antiviral agents, regulators of gene expression, and defenders of genome integrity in plants. Several studies over the last decade have also shown that some small RNAs are exchanged between plants and their pathogens and parasites. Naturally occurring trans -species small RNAs are used by host plants to silence mRNAs in pathogens. These gene-silencing events are thought to be detrimental to the pathogen and beneficial to the host. Conversely, trans- species small RNAs from pathogens and parasites are deployed to silence host mRNAs; these events are thought to be beneficial for the pests. The natural ability of plants to exchange small RNAs with invading eukaryotic organisms can be exploited to provide disease resistance. This review gives an overview of the current state of trans- species small RNA research in plants and discusses several outstanding questions for future research., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Trans -species small regulatory RNAs (sRNAs) are delivered to host plants from diverse pathogens and parasites and can target host mRNAs. How trans -species sRNAs can be effective on diverse hosts has been unclear. Multiple species of the parasitic plant Cuscuta produce trans -species sRNAs that collectively target many host mRNAs. Confirmed target sites are nearly always in highly conserved, protein-coding regions of host mRNAs. Cuscuta trans -species sRNAs can be grouped into superfamilies that have variation in a three-nucleotide period. These variants compensate for synonymous-site variation in host mRNAs. By targeting host mRNAs at highly conserved protein-coding sites, and simultaneously expressing multiple variants to cover synonymous-site variation, Cuscuta trans -species sRNAs may be able to successfully target multiple homologous mRNAs from diverse hosts., Competing Interests: NJ, Cd, MA No competing interests declared, (© 2019, Johnson et al.)

Horizontal gene transfer (HGT), the movement and genomic integration of DNA across species boundaries, is commonly associated with bacteria and other microorganisms, but functional HGT (fHGT) is increasingly being recognized in heterotrophic parasitic plants that obtain their nutrients and water from their host plants through direct haustorial feeding. Here, in the holoparasitic stem parasite Cuscuta, we identify 108 transcribed and probably functional HGT events in Cuscuta campestris and related species, plus 42 additional regions with host-derived transposon, pseudogene and non-coding sequences. Surprisingly, 18 Cuscuta fHGTs were acquired from the same gene families by independent HGT events in Orobanchaceae parasites, and the majority are highly expressed in the haustorial feeding structures in both lineages. Convergent retention and expression of HGT sequences suggests an adaptive role for specific additional genes in parasite biology. Between 16 and 20 of the transcribed HGT events are inferred as ancestral in Cuscuta based on transcriptome sequences from species across the phylogenetic range of the genus, implicating fHGT in the successful radiation of Cuscuta parasites. Genome sequencing of C. campestris supports transfer of genomic DNA-rather than retroprocessed RNA-as the mechanism of fHGT. Many of the C. campestris genes horizontally acquired are also frequent sources of 24-nucleotide small RNAs that are typically associated with RNA-directed DNA methylation. One HGT encoding a leucine-rich repeat protein kinase overlaps with a microRNA that has been shown to regulate host gene expression, suggesting that HGT-derived parasite small RNAs may function in the parasite-host interaction. This study enriches our understanding of HGT by describing a parasite-host system with unprecedented gene exchange that points to convergent evolution of HGT events and the functional importance of horizontally transferred coding and non-coding sequences.

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.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The eukaryotic plant pathogen Phytophthora encodes conserved effector proteins to eliminate host secondary siRNAs. In this issue of Cell Host & Microbe, Hou et al. (2019) report that reduction in secondary siRNA levels renders the host hypersusceptible to Phytophthora and plant secondary siRNAs likely serve as trans-species defensive molecules against pathogens., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Small RNAs regulate key physiological functions in land plants. Small RNAs can be divided into two categories: microRNAs (miRNAs) and short interfering RNAs (siRNAs); siRNAs are further subdivided into transposon/repetitive region-localized heterochromatic siRNAs and phased siRNAs (phasiRNAs). PhasiRNAs are produced from the miRNA-mediated cleavage of a Pol II RNA transcript; the miRNA cleavage site provides a defined starting point from which phasiRNAs are produced in a distinctly phased pattern. 21-22 nucleotide (nt)-dominated phasiRNA-producing loci ( PHAS ) are well represented in all land plants to date. In contrast, 24 nt-dominated PHAS loci are known to be encoded only in monocots and are generally restricted to male reproductive tissues. Currently, only one miRNA (miR2275) is known to trigger the production of these 24 nt-dominated PHAS loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt-dominated PHAS loci also exist in Arabidopsis thaliana . We find that highly expressed heterochromatic siRNAs were consistently misidentified as 24 nt-dominated PHAS loci using multiple PHAS- detecting algorithms. We also find that MIR2275 is not found in A. thaliana , and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used PHAS- detecting algorithms which may lead to false positives when trying to annotate new PHAS , especially 24 nt-dominated loci.

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., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

MicroRNAs (miRNAs) are ∼21-nucleotide-long regulatory RNAs that arise from endonucleolytic processing of hairpin precursors. Many function as essential posttranscriptional regulators of target mRNAs and long noncoding RNAs. Alongside miRNAs, plants also produce large numbers of short interfering RNAs (siRNAs), which are distinguished from miRNAs primarily by their biogenesis (typically processed from long double-stranded RNA instead of single-stranded hairpins) and functions (typically via roles in transcriptional regulation instead of posttranscriptional regulation). Next-generation DNA sequencing methods have yielded extensive data sets of plant small RNAs, resulting in many miRNA annotations. However, it has become clear that many miRNA annotations are questionable. The sheer number of endogenous siRNAs compared with miRNAs has been a major factor in the erroneous annotation of siRNAs as miRNAs. Here, we provide updated criteria for the confident annotation of plant miRNAs, suitable for the era of "big data" from DNA sequencing. The updated criteria emphasize replication and the minimization of false positives, and they require next-generation sequencing of small RNAs. We argue that improved annotation systems are needed for miRNAs and all other classes of plant small RNAs. Finally, to illustrate the complexities of miRNA and siRNA annotation, we review the evolution and functions of miRNAs and siRNAs in plants., (© American Society of Plant Biologists. All rights reserved.)

Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bidirectional movement of viruses, proteins and mRNAs between host and parasite, but the functional effects of these movements are not known. Here we show that Cuscuta campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana. Many of these miRNAs are 22 nucleotides in length. Plant miRNAs of this length are uncommon, and are associated with amplification of target silencing through secondary short interfering RNA (siRNA) production. Several A. thaliana mRNAs are targeted by 22-nucleotide C. campestris miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation. Hosts with mutations in two of the loci that encode target mRNAs supported significantly higher growth of C. campestris. The same miRNAs that are expressed and active when C. campestris parasitizes A. thaliana are also expressed and active when it infects Nicotiana benthamiana. Homologues of target mRNAs from many other plant species also contain the predicted target sites for the induced C. campestris miRNAs. These data show that C. campestris miRNAs act as trans-species regulators of host-gene expression, and suggest that they may act as virulence factors during parasitism.

MicroRNAs (miRNAs) function to post-transcriptionally regulate target RNAs, including long non-coding RNAs and mRNAs. A recent study demonstrates that Arabidopsis miRNAs are enriched at the rough endoplasmic reticulum (ER). This enrichment is a surprise, given that most known miRNA targets are not expected to be translated at the rough ER., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

In plants, 24 nucleotide long heterochromatic siRNAs (het-siRNAs) transcriptionally regulate gene expression by RNA-directed DNA methylation (RdDM). The biogenesis of most het-siRNAs depends on the plant-specific RNA polymerase IV (Pol IV), and ARGONAUTE4 (AGO4) is a major het-siRNA 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-siRNAs. The accumulation of AGO4-dependent het-siRNAs also requires several factors known to participate in the effector portion of the RdDM 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-siRNA accumulation from ago4 mutant plants. Collectively, our data suggest that AGO4-dependent siRNAs are secondary siRNAs dependent on the prior activity of the RdDM pathway at certain loci., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3' modifications (truncations and non-templated tailing) of plant miRNAs when the 2'-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella Elevated rates of non-templated nucleotides were observed at the 3' ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. 'Off-sized' small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3'-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3'-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3' nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3'-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

High-throughput sequencing of small RNAs (sRNA-seq) is a popular method used to discover and annotate microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), and Piwi-associated RNAs (piRNAs). One of the key steps in sRNA-seq data analysis is alignment to a reference genome. sRNA-seq libraries often have a high proportion of reads that align to multiple genomic locations, which makes determining their true origins difficult. Commonly used sRNA-seq alignment methods result in either very low precision (choosing an alignment at random), or sensitivity (ignoring multi-mapping reads). Here, we describe and test an sRNA-seq alignment strategy that uses local genomic context to guide decisions on proper placements of multi-mapped sRNA-seq reads. Tests using simulated sRNA-seq data demonstrated that this local-weighting method outperforms other alignment strategies using three different plant genomes. Experimental analyses with real sRNA-seq data also indicate superior performance of local-weighting methods for both plant miRNAs and heterochromatic siRNAs. The local-weighting methods we have developed are implemented as part of the sRNA-seq analysis program ShortStack, which is freely available under a general public license. Improved genome alignments of sRNA-seq data should increase the quality of downstream analyses and genome annotation efforts., (Copyright © 2016 Johnson et al.)

Endogenous small RNAs (sRNAs) contribute to gene regulation and genome homeostasis, but their activities and functions are incompletely known. The maize genome has a high number of transposable elements (TEs; almost 85%), some of which spawn abundant sRNAs. We performed sRNA and total RNA sequencing from control and abiotically stressed B73 wild-type plants and rmr6-1 mutants. RMR6 encodes the largest subunit of the RNA polymerase IV complex and is responsible for accumulation of most 24-nucleotide (nt) small interfering RNAs (siRNAs). We identified novel MIRNA loci and verified miR399 target conservation in maize. RMR6-dependent 23-24 nt siRNA loci were specifically enriched in the upstream region of the most highly expressed genes. Most genes misregulated in rmr6-1 did not show a significant correlation with loss of flanking siRNAs, but we identified one gene supporting existing models of direct gene regulation by TE-derived siRNAs. Long-term drought correlated with changes of miRNA and sRNA accumulation, in particular inducing down-regulation of a set of sRNA loci in the wild-typeleaf., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Viral small RNAs (vsRNAs) are one of the key elements involved in RNA silencing-based defense against viruses in plants. We analyzed the vsRNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot virus (PolRSV) (Tospovirus, Bunyaviridae). VsRNAs were abundant in both hosts, but a different size profile was observed, with an abundance peak at 21 in N. benthamiana and at 22 nt in tomato. VsRNAs mapping to the PolRSV L genomic segment were under-represented in both hosts, while S and M segments were differentially and highly targeted in N. benthamiana and tomato, respectively. Differences in preferential targeting of single ORFs were observed, with over-representation of NSs ORF-derived reads in N. benthamiana. Intergenic regions (IGRs)-mapping vsRNAs were under-represented, while enrichment of vsRNAs reads mapping to the NSs positive sense strand was observed in both hosts. Comparison with a previous study on tomato spotted wilt virus (TSWV) under the same experimental conditions, showed that the relative accumulation of PolRSV-specific and endogenous sRNAs was similar to the one observed for silencing suppressor-deficient TSWV strains, suggesting possible different properties of PolRSV NSs silencing suppressor compared to that of TSWV., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Tospoviruses are plant-infecting viruses belonging to the family Bunyaviridae. We used a collection of wild-type, phylogenetically distinct tomato spotted wilt virus isolates and related silencing-suppressor defective mutants to study the effects on the small RNA (sRNA) accumulation during infection of Nicotiana benthamiana. Our data showed that absence of a functional silencing suppressor determined a marked increase of the total amount of viral sRNAs (vsRNAs), and specifically of the 21 nt class. We observed a common under-representation of vsRNAs mapping to the intergenic region of S and M genomic segments, and preferential mapping of the reads against the viral sense open reading frames, with the exception of the NSs gene. The NSs-mutant strains showed enrichment of NSm-derived vsRNA compared to the expected amount based on gene size. Analysis of 5' terminal nucleotide preference evidenced a significant enrichment in U for the 21 nt- and in A for 24 nt-long endogenous sRNAs in all the samples. Hotspot analysis revealed a common abundant accumulation of reads at the 5' end of the L segment, mostly in the antiviral sense, for the NSs-defective isolates, suggesting that absence of the silencing suppressor can influence preferential targeting of the viral genome., (Copyright © 2015 Elsevier B.V. All rights reserved.)

MicroRNAs, which target mRNAs for post-transcriptional regulation, and heterochromatic siRNAs, which target chromatin causing DNA methylation, make up the majority of the endogenous regulatory small RNA pool in most plant specimens. They both function to guide Argonaute proteins to targeted nucleic acids on the basis of complementarity. Recent work on plant miRNA-target interactions has clarified the general ''rules' of complementarity, while also providing several intriguing exceptions to these rules. In addition, emerging evidence suggests that several factors besides miRNA-target complementarity affect plant miRNA function. For heterochromatic siRNAs, recent work has made progress towards comprehensively identifying potential target regions, but numerous fundamental questions remain to be answered., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs., (© 2015 American Society of Plant Biologists. All rights reserved.)

microRNA (miRNA) mediated repression of target genes plays essential roles in a variety of functions in plants. An easy-to-use method that can effectively validate functional miRNA-target interactions in plants thus is of particular interest. Here, we describe an Agrobacterium tumefaciens-mediated in vivo assay utilizing a dual-luciferase reporter system. With this method, the strength of miRNA-mediated target repression can be quantified at both the mRNA (via qRT-PCR) and protein (via dual-luciferase assay) levels quickly and accurately.

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 © 2013 Elsevier Inc. All rights reserved.)

Plant microRNAs (miRNAs) typically form near-perfect duplexes with their targets and mediate mRNA cleavage. Here, we describe an unconventional miRNA target of miR398 in Arabidopsis, an mRNA encoding the blue copper-binding protein (BCBP). BCBP mRNA carries an miR398 complementary site in its 5'-untranslated region (UTR) with a bulge of six nucleotides opposite to the 5' region of the miRNA. Despite the disruption of a target site region thought to be especially critical for function, BCBP mRNAs are cleaved by ARGONAUTE1 between nucleotides 10th and 11th, opposite to the miRNA, like conventional plant target sites. Levels of BCBP mRNAs are inversely correlated to levels of miR398 in mutants lacking the miRNA, or transgenic plants overexpressing it. Introducing two mutations that disrupt the miRNA complementarity around the cleavage site renders the target cleavage-resistant. The BCBP site functions outside of the context of the BCBP mRNA and does not depend on 5'-UTR location. Reducing the bulge does not interfere with miR398-mediated regulation and completely removing it increases the efficiency of the slicing. Analysis of degradome data and target predictions revealed that the miR398-BCBP interaction seems to be rather unique. Nevertheless, our results imply that functional target sites with non-perfect pairings in the 5' region of an ancient conserved miRNA exist in plants.

A key goal in genomics is the complete annotation of the expressed regions of the genome. In plants, substantial portions of the genome make regulatory small RNAs produced by Dicer-Like (DCL) proteins and utilized by Argonaute (AGO) proteins. These include miRNAs and various types of endogenous siRNAs. Small RNA-seq, enabled by cheap and fast DNA sequencing, has produced an enormous volume of data on plant miRNA and siRNA expression in recent years. In this review, we discuss recent progress in using small RNA-seq data to produce stable and reliable annotations of miRNA and siRNA genes in plants. In addition, we highlight key goals for the future of small RNA gene annotation in plants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) guide RNA-induced silencing complexes to target RNAs based on miRNA-target complementarity. Using a dual-luciferase based sensor system in Nicotiana benthamiana, we quantitatively assessed the relationship between miRNA-target complementarity and silencing efficacy measured at both the RNA and protein levels, using several conserved miRNAs and their known target sites from Arabidopsis thaliana. We found that naturally occurring sites have variable efficacies attributable to their complementarity patterns. We also observed that sites with a few mismatches to the miRNA 3' regions, which are common in plants, are often equally effective and sometimes more effective than perfectly matched sites. By contrast, mismatches to the miRNA 5' regions strongly reduce or eliminate repression efficacy but are nonetheless present in several natural sites, suggesting that in some cases, suboptimal miRNA efficacies are either tolerated or perhaps selected for. Central mismatches fully abolished repression efficacy in our system, but such sites then became effective miRNA target mimics. Complementarity patterns that are functional in animals (seed sites, 3'-supplementary sites, and centered sites) did not reliably confer repression, regardless of context (3'-untranslated region or open reading frame) or measurement type (RNA or protein levels). Overall, these data provide a robust and empirical foundation for understanding, predicting, and designing functional miRNA target sites in plants.

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.

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.

Small RNA sequencing allows genome-wide discovery, categorization, and quantification of genes producing regulatory small RNAs. Many tools have been described for annotation and quantification of microRNA loci (MIRNAs) from small RNA-seq data. However, in many organisms and tissue types, MIRNA genes comprise only a small fraction of all small RNA-producing genes. ShortStack is a stand-alone application that analyzes reference-aligned small RNA-seq data and performs comprehensive de novo annotation and quantification of the inferred small RNA genes. ShortStack's output reports multiple parameters of direct relevance to small RNA gene annotation, including RNA size distributions, repetitiveness, strandedness, hairpin-association, MIRNA annotation, and phasing. In this study, ShortStack is demonstrated to perform accurate annotations and useful descriptions of diverse small RNA genes from four plants (Arabidopsis, tomato, rice, and maize) and three animals (Drosophila, mice, and humans). ShortStack efficiently processes very large small RNA-seq data sets using modest computational resources, and its performance compares favorably to previously described tools. Annotation of MIRNA loci by ShortStack is highly specific in both plants and animals. ShortStack is freely available under a GNU General Public License.

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 antisense 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 for future research.

microRNA156 (miR156) affects developmental timing in flowering plants. miR156 and its target relationships with members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family appear universally conserved in land plants, but the specific functions of miR156 outside of flowering plants are unknown. We find that miR156 promotes a developmental change from young filamentous protonemata to leafy gametophores in the moss Physcomitrella patens, opposite to its role as an inhibitor of development in flowering plants. P. patens miR156 also influences accumulation of trans-acting small interfering RNAs (tasiRNAs) dependent upon a second ancient microRNA, miR390. Both miR156 and miR390 directly target a single major tasiRNA primary transcript. Inhibition of miR156 function causes increased miR390-triggered tasiRNA accumulation and decreased accumulation of tasiRNA targets. Overexpression of miR390 also caused a slower formation of gametophores, elevated miR390-triggered tasiRNA accumulation, and reduced level of tasiRNA targets. We conclude that a gene regulatory network controlled by miR156, miR390, and their targets controls developmental change in P. patens. The broad outlines and regulatory logic of this network are conserved in flowering plants, albeit with some modifications. Partially conserved small RNA networks thus influence developmental timing in plants with radically different body plans.

Trans-acting small interfering RNAs (ta-siRNAs) are plant-specific siRNAs released from TAS precursor transcripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased processing. Like microRNAs (miRNAs), ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens (PpDCL4) is essential for the biogenesis of 21 nucleotide (nt) ta-siRNAs. In ΔPpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. ΔPpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in ΔPpDCL3/ΔPpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the ΔPpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TAS6. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci.

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 K(cat) and lower K(m) 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.

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.

MicroRNAs are pervasive in both plants and animals, but many aspects of their biogenesis, function and evolution differ. We reveal how these differences contribute to characteristic features of microRNA evolution in the two kingdoms., (© 2011 BioMed Central Ltd)

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.

Twenty-one-nucleotide microRNAs (miRNAs) and 24-nucleotide Pol IV-dependent small interfering RNAs (p4-siRNAs) are the most abundant types of small RNAs in angiosperms. Some miRNAs are well conserved among different plant lineages, whereas others are less conserved, and it is not clear whether less-conserved miRNAs have the same functionality as the well conserved ones. p4-siRNAs are broadly produced in the Arabidopsis genome, sometimes from active hot spot loci, but it is unknown whether individual p4-siRNA hot spots are retained as hot spots between plant species. In this study, we compare small RNAs in two closely related species (Arabidopsis thaliana and Arabidopsis lyrata) and find that less-conserved miRNAs have high rates of divergence in MIRNA hairpin structures, mature miRNA sequences, and target-complementary sites in the other species. The fidelity of miRNA biogenesis from many less-conserved MIRNA hairpins frequently deteriorates in the sister species relative to the species of first discovery. We also observe that p4-siRNA occupied loci have a slight tendency to be retained as p4-siRNA loci between species, but the most active A. lyrata p4-siRNA hot spots are generally not syntenic to the most active p4-siRNA hot spots of A. thaliana. Altogether, our findings indicate that many MIRNAs and most p4-siRNA hot spots are rapidly changing and evolutionarily transient within the Arabidopsis genus.

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 approximately 11 nucleotides (nt) from the end of the hairpin and the second approximately 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 approximately 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 approximately 15 nt between the miRNA:miRNA(*) duplex and either a less structured region of the lower stem or its end., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Short, interfering RNAs (siRNAs) arise from the processing of long double-stranded RNA (dsRNA) by Dicer enzymes. Dicers generate siRNA duplexes by successive hydrolysis of both strands of the dsRNA phosphodiester backbone at positions determined by measuring 21-24 nucleotides from an exposed dsRNA terminus. Therefore, a population of dsRNAs with precisely identical termini will produce siRNA spaced in regular, 21-24-nucleotide intervals. This chapter presents an easily customized and generally applicable strategy for identifying loci which produce the "phased" siRNAs diagnostic of such processing. Given the input of a large set of expressed small RNAs and of the corresponding genome or transcriptome from which the small RNAs are derived, the methodology produces a ranking of user-defined loci with respect to their likely production of phased siRNAs. Top ranked loci are candidates for further computational and biological analyses.

Expression profiling of the 5' ends of uncapped mRNAs ("degradome" sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative regulatory proteins. Remnants of MIRNA hairpin processing also populated the degradome data and indicated an unusual "loop-first" mode of precise processing for the MIR319 gene family. Precise loop-first processing was confirmed for native Physcomitrella, rice, and Arabidopsis MIR319 hairpins, as well as an Arabidopsis artificial MIRNA (aMIRNA) based upon a MIR319 backbone. MIR319 is thus a conserved exception to the general rule of loop-last processing of MIRNA hairpins. Loop-first MIR319 processing may contribute to the high efficacy of a widely used MIR319-based strategy for aMIRNA production in plants.

Unlabelled: MicroRNAs (miRNAs) are approximately 20- to 22-nt long endogenous RNA sequences that play a critical role in the regulation of gene expression in eukaryotic genomes. Confident identification of miRNA targets is vital to understand their functions. Currently available computational algorithms for miRNA target prediction have diverse degrees of sensitivity and specificity and as a consequence each predicted target generally requires experimental confirmation. miRNAs and other small RNAs that direct endonucleolytic cleavage of target mRNAs produce diagnostic uncapped, polyadenylated mRNA fragments. Degradome sequencing [also known as PARE (parallel analysis of RNA ends) and GMUCT (genome-wide mapping of uncapped transcripts)] samples the 5'-ends of uncapped mRNAs and can be used to discover in vivo miRNA targets independent of computational predictions. Here, we describe a generalizable computational pipeline, CleaveLand, for the detection of cleaved miRNA targets from degradome data. CleaveLand takes as input degradome sequences, small RNAs and an mRNA database and outputs small RNA targets. CleaveLand can thus be applied to degradome data from any species provided a set of mRNA transcripts and a set of query miRNAs or other small RNAs are available., Availability: The code and documentation for CleaveLand is freely available under a GNU license at http://www.bio.psu.edu/people/faculty/Axtell/AxtellLab/Software.html