Publications by authors named "Michael J Axtell"

58 Publications

Mechanisms of Resistance and Virulence in Parasitic Plant-Host Interactions.

Plant Physiol 2020 Dec 18. Epub 2020 Dec 18.

Department of Biology, University of Virginia, Charlottesville, VA USA.

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.
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http://dx.doi.org/10.1093/plphys/kiaa064DOI Listing
December 2020

Expression and processing of polycistronic artificial microRNAs and trans-acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana.

Plant J 2021 Mar 2. Epub 2021 Mar 2.

Department of Biology, The Pennsylvania State University, University Park, PA, 16802, USA.

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.
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http://dx.doi.org/10.1111/tpj.15221DOI Listing
March 2021

MSH1-induced heritable enhanced growth vigor through grafting is associated with the RdDM pathway in plants.

Nat Commun 2020 10 22;11(1):5343. Epub 2020 Oct 22.

Departments of Biology and Plant Science, The Pennsylvania State University, University Park, PA, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-19140-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582163PMC
October 2020

Segregation of an MSH1 RNAi transgene produces heritable non-genetic memory in association with methylome reprogramming.

Nat Commun 2020 05 5;11(1):2214. Epub 2020 May 5.

Departments of Biology and Plant Science, The Pennsylvania State University, University Park, PA, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-16036-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7200659PMC
May 2020

Integrated annotations and analyses of small RNA-producing loci from 47 diverse plants.

Genome Res 2020 03 16;30(3):497-513. Epub 2020 Mar 16.

Department of Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

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.
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http://dx.doi.org/10.1101/gr.256750.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111516PMC
March 2020

Compensatory sequence variation between -species small RNAs and their target sites.

Elife 2019 12 17;8. Epub 2019 Dec 17.

Intercollege PhD Program in Plant Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, United States.

-species small regulatory RNAs (sRNAs) are delivered to host plants from diverse pathogens and parasites and can target host mRNAs. How -species sRNAs can be effective on diverse hosts has been unclear. Multiple species of the parasitic plant produce -species sRNAs that collectively target many host mRNAs. Confirmed target sites are nearly always in highly conserved, protein-coding regions of host mRNAs. -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, -species sRNAs may be able to successfully target multiple homologous mRNAs from diverse hosts.
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http://dx.doi.org/10.7554/eLife.49750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917502PMC
December 2019

Exchange of Small Regulatory RNAs between Plants and Their Pests.

Plant Physiol 2020 01 21;182(1):51-62. Epub 2019 Oct 21.

Department of Biology, Intercollege Ph.D. Program in Plant Biology, and Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802

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 -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, 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 species small RNA research in plants and discusses several outstanding questions for future research.
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http://dx.doi.org/10.1104/pp.19.00931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945882PMC
January 2020

Convergent horizontal gene transfer and cross-talk of mobile nucleic acids in parasitic plants.

Nat Plants 2019 09 22;5(9):991-1001. Epub 2019 Jul 22.

Intercollege Graduate Program in Plant Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA.

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.
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http://dx.doi.org/10.1038/s41477-019-0458-0DOI Listing
September 2019

Several phased siRNA annotation methods can frequently misidentify 24 nucleotide siRNA-dominated loci.

Plant Direct 2018 Dec 17;2(12):e00101. Epub 2018 Dec 17.

Genetics Ph.D. Program Huck Institutes of the Life Sciences The Pennsylvania State University University Park Pennsylvania.

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 () are well represented in all land plants to date. In contrast, 24 nt-dominated 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 loci. In this study, we use stringent methodologies in order to examine whether or not 24 nt-dominated loci also exist in . We find that highly expressed heterochromatic siRNAs were consistently misidentified as 24 nt-dominated loci using multiple detecting algorithms. We also find that is not found in , and it seems to have been lost in the last common ancestor of Brassicales. Altogether, our research highlights the potential issues with widely used detecting algorithms which may lead to false positives when trying to annotate new , especially 24 nt-dominated loci.
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http://dx.doi.org/10.1002/pld3.101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6508839PMC
December 2018

Molecular Dialog Between Parasitic Plants and Their Hosts.

Annu Rev Phytopathol 2019 08 21;57:279-299. Epub 2019 Jun 21.

School of Plant and Environmental Sciences, Virginia Tech, Blacksburg, Virginia 24061, USA; email:

Parasitic plants steal sugars, water, and other nutrients from host plants through a haustorial connection. Several species of parasitic plants such as witchweeds ( spp.) and broomrapes ( and 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.
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http://dx.doi.org/10.1146/annurev-phyto-082718-100043DOI Listing
August 2019

MicroRNAs in Plants: Key Findings from the Early Years.

Plant Cell 2019 06 29;31(6):1206-1207. Epub 2019 Apr 29.

The Pennsylvania State UniversityDepartment of Biology and Huck Institutes of the Life SciencesUniversity Park, Pennsylvania 16802

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http://dx.doi.org/10.1105/tpc.19.00310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588298PMC
June 2019

Small RNA warfare: exploring origins and function of trans-species microRNAs from the parasitic plant Cuscuta.

Curr Opin Plant Biol 2019 08 28;50:76-81. Epub 2019 Apr 28.

Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA; Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pbi.2019.03.014DOI Listing
August 2019

Second to None: Plant Secondary siRNAs as Defensive Agents against Phytophthora.

Authors:
Michael J Axtell

Cell Host Microbe 2019 01;25(1):7-9

Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.chom.2018.12.007DOI Listing
January 2019

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci.

Plant J 2018 06 13;94(6):1051-1063. Epub 2018 May 13.

Genetics PhD Program, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, 16802, USA.

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.
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http://dx.doi.org/10.1111/tpj.13919DOI Listing
June 2018

Revisiting Criteria for Plant MicroRNA Annotation in the Era of Big Data.

Plant Cell 2018 02 17;30(2):272-284. Epub 2018 Jan 17.

Donald Danforth Plant Science Center, St. Louis, Missouri 63132

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.
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http://dx.doi.org/10.1105/tpc.17.00851DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5868703PMC
February 2018

MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs.

Nature 2018 01;553(7686):82-85

Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.

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.
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http://dx.doi.org/10.1038/nature25027DOI Listing
January 2018

Lost in translation? microRNAs at the rough ER.

Authors:
Michael J Axtell

Trends Plant Sci 2017 04 17;22(4):273-274. Epub 2017 Mar 17.

Department of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802 USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.tplants.2017.03.002DOI Listing
April 2017

AGO4 is specifically required for heterochromatic siRNA accumulation at Pol V-dependent loci in Arabidopsis thaliana.

Plant J 2017 Apr 11;90(1):37-47. Epub 2017 Feb 11.

Intercollege Plant Biology Ph.D. Program, Huck Institutes of the Life Sciences, Penn State University, University Park, PA, 16802, USA.

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.
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http://dx.doi.org/10.1111/tpj.13463DOI Listing
April 2017

Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs.

Nucleic Acids Res 2016 09 20;44(15):7395-405. Epub 2016 May 20.

Intercollege Plant Biology PhD Program, Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, USA Department of Biology, Penn State University, University Park, PA 16802, USA

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.
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http://dx.doi.org/10.1093/nar/gkw457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009732PMC
September 2016

Improved Placement of Multi-mapping Small RNAs.

G3 (Bethesda) 2016 07 7;6(7):2103-11. Epub 2016 Jul 7.

Huck Institutes of the Life Sciences, Penn State University, Philadelphia 16802 Department of Biology, Penn State University, Philadelphia 16802

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.
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http://dx.doi.org/10.1534/g3.116.030452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938663PMC
July 2016

Genome-Wide Characterization of Maize Small RNA Loci and Their Regulation in the required to maintain repression6-1 (rmr6-1) Mutant and Long-Term Abiotic Stresses.

Plant Physiol 2016 Mar 8;170(3):1535-48. Epub 2016 Jan 8.

Department of Agronomy, Animals, Food, Natural Resources and Environment, University of Padova, Agripolis Viale dell'Università 16, 35020 Legnaro PD Italy (A.L., C.F., S.F., S.V.); andDepartment of Biology and Huck Institutes of the Life Sciences, Penn State University, University Park, Pennsylvania 16802 (A.L., M.J.A.)

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.
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http://dx.doi.org/10.1104/pp.15.01205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775107PMC
March 2016

Comparison of small RNA profiles in Nicotiana benthamiana and Solanum lycopersicum infected by polygonum ringspot tospovirus reveals host-specific responses to viral infection.

Virus Res 2016 Jan 30;211:38-45. Epub 2015 Sep 30.

Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virusres.2015.09.019DOI Listing
January 2016

More than meets the eye? Factors that affect target selection by plant miRNAs and heterochromatic siRNAs.

Curr Opin Plant Biol 2015 Oct 31;27:118-24. Epub 2015 Jul 31.

Plant Biology Ph.D. Program, Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, USA; Genetics Ph.D. Program, Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, USA; Department of Biology, Penn State University, University Park, PA 16802, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pbi.2015.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732885PMC
October 2015

Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants.

Plant Cell 2015 Aug 24;27(8):2148-62. Epub 2015 Jul 24.

Plant Biology PhD Program, Penn State University, University Park, Pennsylvania 16802 Huck Institutes of the Life Sciences, Penn State University, University Park, Pennsylvania 16802 Department of Biology, Penn State University, University Park, Pennsylvania 16802

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.
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http://dx.doi.org/10.1105/tpc.15.00228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568501PMC
August 2015

Small RNA profiles of wild-type and silencing suppressor-deficient tomato spotted wilt virus infected Nicotiana benthamiana.

Virus Res 2015 Oct 3;208:30-8. Epub 2015 Jun 3.

Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.virusres.2015.05.021DOI Listing
October 2015

Quantitating plant microRNA-mediated target repression using a dual-luciferase transient expression system.

Methods Mol Biol 2015 ;1284:287-303

Department of Biology, Pennsylvania State University, University Park, PA, 16802, USA.

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.
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http://dx.doi.org/10.1007/978-1-4939-2444-8_14DOI Listing
November 2015

Seeing the forest for the trees: annotating small RNA producing genes in plants.

Curr Opin Plant Biol 2014 Apr 15;18:87-95. Epub 2014 Mar 15.

Department of Biology, Penn State University, University Park, PA 16802, USA; Plant Biology Intercollegiate Ph.D. Program, Penn State University, University Park, PA 16802, USA; Huck Institutes of the Life Sciences, Penn State University, University Park, PA 16802, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pbi.2014.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4001702PMC
April 2014

A non-canonical plant microRNA target site.

Nucleic Acids Res 2014 Apr 21;42(8):5270-9. Epub 2014 Feb 21.

INRA, UMR1318, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France, AgroParisTech, Institut Jean-Pierre Bourgin, RD10, F-78000 Versailles, France and Department of Biology and Plant Biology Ph.D. Program, Pennsylvania State University, University Park, PA 16802, USA.

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.
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http://dx.doi.org/10.1093/nar/gku157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005643PMC
April 2014

Analysis of complementarity requirements for plant microRNA targeting using a Nicotiana benthamiana quantitative transient assay.

Plant Cell 2014 Feb 7;26(2):741-53. Epub 2014 Feb 7.

Department of Biology, Pensylvania State University, University Park, Pensylvania 16802.

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.
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http://dx.doi.org/10.1105/tpc.113.120972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967037PMC
February 2014

Long-range genomic enrichment, sequencing, and assembly to determine unknown sequences flanking a known microRNA.

PLoS One 2013 20;8(12):e83721. Epub 2013 Dec 20.

Integrative Biosciences PhD Program in Bioinformatics and Genomics, Huck Institutes of the Life Sciences, and Department of Biology, Pennsylvania State University, University Park, Pennsylvania, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083721PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869802PMC
March 2015