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1.  Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis 
FEBS letters  2006;580(13):3117-3120.
HEN1-dependent methylation of the 3′-terminal nucleotide is a crucial step in plant microRNA (miRNA) biogenesis. Here we report that several viral RNA silencing suppressors (P1/HC-Pro, p21 and p19) inhibit miRNA methylation. These suppressors have distinct effects on different miRNAs. We also show that miRNA* is methylated in vivo in a suppressor-sensitive manner, suggesting that the viral proteins interfere with miRNA/miRNA* duplexes. p19 and p21 bind both methylated and unmethylated miRNA/miRNA* duplexes in vivo. These findings suggest miRNA/miRNA* as the in vivo substrates for the HEN1 miRNA methyltransferase and raise intriguing possibilities regarding the cellular location of miRNA methylation.
doi:10.1016/j.febslet.2006.04.063
PMCID: PMC5136478  PMID: 16678167
Viral RNA silencing suppressor; Methylation; microRNA/microRNA*; HEN1
2.  Antiviral Roles of Plant ARGONAUTES 
ARGONAUTES (AGOs) are the effector proteins functioning in eukaryotic RNA silencing pathways. AGOs associate with small RNAs and are programmed to target complementary RNA or DNA. Plant viruses induce a potent and specific antiviral RNA silencing host response in which AGOs play a central role. Antiviral AGOs associate with virus-derived small RNAs to repress complementary viral RNAs or DNAs, or with endogenous small RNAs to regulate host gene expression and promote antiviral defense. Here, we review recent progress towards understanding the roles of plant AGOs in antiviral defense. We also discuss the strategies that viruses have evolved to modulate, attenuate or suppress AGO antiviral functions.
doi:10.1016/j.pbi.2015.06.013
PMCID: PMC4618181  PMID: 26190744
3.  Small RNA-Based Antiviral Defense in the Phytopathogenic Fungus Colletotrichum higginsianum 
PLoS Pathogens  2016;12(6):e1005640.
Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5’U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism.
Author Summary
Colletotrichum sp. comprises a diverse group of fungal pathogens that attack over 3000 plant species worldwide. Understanding the underlying mechanisms that govern fungal development and pathogenicity may enable more effective and sustainable approaches to crop disease management and control. In most organisms, RNA silencing is an important mechanism to control endogenous and exogenous RNA. RNA silencing utilizes small regulatory molecules (small RNAs) produced by proteins called Dicer (DCL), and exercise their function though effector proteins named Argonaute (AGO). Here, we investigated the role of RNA silencing machinery in the fungus Colletotrichum higginsianum, by generating deletions in genes encoding RNA silencing components. Severe defects were observed in both conidiation and conidia morphology in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains. Analysis of transcripts and small RNAs revealed an uncharacterized dsRNA virus persistently infecting C. higginsianum. The virus was shown (1) to be de-repressed in the Δdcl1, Δdcl1Δdcl2 and Δago1 strains, and (2) to cause the conidiation and spore mutant phenotypes. Our results indicate that C. higginsianum employs RNA silencing as an antiviral mechanism to suppress viruses and their debilitating effects.
doi:10.1371/journal.ppat.1005640
PMCID: PMC4890784  PMID: 27253323
4.  Loss of CMD2‐mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis 
Molecular Plant Pathology  2016;17(7):1095-1110.
Summary
Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer‐preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)‐mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild‐type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2‐type varieties TME 3 and TME 7, but the CMD1‐type cultivar TMS 30572 and the CMD3‐type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2‐mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field‐level resistance in CMD2‐type cultivars presently grown by farmers in East Africa, where CMD pressure is high.
doi:10.1111/mpp.12353
PMCID: PMC5021159  PMID: 26662210
cassava; cassava mosaic disease; CMD2; geminivirus resistance; somaclonal variation; somatic embryogenesis; susceptible
5.  Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors 
The Plant Journal  2015;82(6):1061-1075.
Summary
Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost‐effective and large‐scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high‐throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390‐based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390‐based precursors that include distal stem–loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome‐wide transcriptome profiling combined with 5′‐RLM‐RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.
Significance Statement
A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost‐effective and large‐scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390‐based precursors including Arabidopsis thaliana MIR390a distal stem‐loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.
doi:10.1111/tpj.12835
PMCID: PMC4464980  PMID: 25809382
RNA silencing; artificial microRNA; MIRNA precursor; Brachypodium distachyon; monocot; Arabidopsis thaliana; technical advance
6.  Highly Specific Gene Silencing in a Monocot Species by Artificial MicroRNAs Derived From Chimeric MIRNA Precursors 
SUMMARY
Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5’-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.
doi:10.1111/tpj.12835
PMCID: PMC4464980  PMID: 25809382
RNA silencing; artificial microRNA; MIRNA precursor; Brachypodium distachyon; monocot; Arabidopsis thaliana
7.  Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants 
RNA & disease (Houston, Tex.)  2016;3(1):e1130.
Artificial small RNAs (sRNAs) are short ≈21-nt non-coding RNAs engineered to inactivate sequence complementary RNAs. In plants, they have been extensively used to silence cellular transcripts in gene function analyses and to target invading RNA viruses to induce resistance. Current artificial sRNA-based antiviral resistance in plants is mainly limited to a single virus, and is jeopardized by the emergence of mutations in the artificial sRNA target site or by the presence of co-infecting viruses. Hence, there is a need to further develop the artificial sRNA approach to generate more broad and durable antiviral resistance in plants. A recently developed toolbox allows for the time and cost-effective large-scale production of artificial sRNA constructs in plants. The toolbox includes the P-SAMS web tool for the automated design of artificial sRNAs, and a new generation of artificial microRNA and synthetic trans-acting small interfering RNA (syn-tasiRNA) vectors for direct cloning and high expression of artificial sRNAs. Here we describe how the simplicity and high-throughput capability of these new technologies should accelerate the study of artificial sRNA-based antiviral resistance in plants. In particular, we discuss the potential of the syn-tasiRNA approach as a promising strategy for developing more effective, durable and broad antiviral resistance in plants.
PMCID: PMC4768481  PMID: 26925463
small RNA; silencing; artificial microRNA; synthetic trans-acting small interfering RNA; plant virus; virus resistance
8.  Preparation of Multiplexed Small RNA Libraries From Plants 
Bio-protocol  2014;4(21):e1275.
High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3′ and 5′ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3′ PCR primers used here include unique 6-nucleotide sequences to allow for multiplexing up to 12 samples.
PMCID: PMC4675356  PMID: 26661568
9.  P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design 
Bioinformatics  2015;32(1):157-158.
Summary: The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications, P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both applications is wizard-assisted, and the job runtime is relatively short. Both applications output the sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for cloning into ‘B/c’ compatible vectors.
Availability and implementation: The P-SAMS website is available at http://p-sams.carringtonlab.org.
Contact: acarbonell@ibmcp.upv.es or nfahlgren@danforthcenter.org
doi:10.1093/bioinformatics/btv534
PMCID: PMC4681993  PMID: 26382195
10.  ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis 
Nature communications  2014;5:5468.
Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5′-terminal nucleotide is important for sRNA sorting into different AGOs. Here, we show that miRNA duplex structure also contributes to miRNA sorting. Base-pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favors miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modeling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base-pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2.
doi:10.1038/ncomms6468
PMCID: PMC4238042  PMID: 25406978
11.  Specific Argonautes Selectively Bind Small RNAs Derived from Potato Spindle Tuber Viroid and Attenuate Viroid Accumulation In Vivo 
Journal of Virology  2014;88(20):11933-11945.
ABSTRACT
The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5′-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart.
IMPORTANCE To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250 to 400 nt), infect and incite disease in plants. The accumulation in these plants of 21- to 24-nt viroid-derived small RNAs (vd-sRNAs) supports the notion that DCLs also target viroids but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed were associated with vd-sRNAs displaying the same properties (5′-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4, and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.
doi:10.1128/JVI.01404-14
PMCID: PMC4178711  PMID: 25100851
12.  Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection 
PLoS Pathogens  2015;11(3):e1004755.
In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.
Author Summary
RNA silencing is a primary, adaptive defense system against viruses in plants. Viruses have evolved counter-defensive mechanisms that inhibit RNA silencing through the activity of silencing suppressor proteins. Understanding how antiviral silencing is controlled, and how suppressor proteins function, is essential for understanding how plants normally resist viruses, why some viruses are highly virulent in different hosts, and how sustainable antiviral resistance strategies can be deployed in agricultural settings. We used a mutant version of Turnip mosaic virus lacking a functional silencing suppressor (HC-Pro) to understand the genetic requirements for resistance in the model plant Arabidopsis thaliana. We focused on ARGONAUTE proteins, which have long been hypothesized to bind short interfering RNAs (siRNAs) derived from virus genomes for use as sequence-specific guides to recognize and target viral RNA for degradation or repression. We demonstrated specialized antiviral roles for specific ARGONAUTES and showed that several can bind viral siRNAs from across the entire viral genome. However, ARGONAUTE proteins are only loaded with virus-derived siRNAs in the absence of HC-Pro, which we showed binds siRNAs from the viral genome. This indicates that several AGO proteins, which collectively are necessary for full anti-TuMV defense, need to properly load virus-derived siRNAs to execute their antiviral roles.
doi:10.1371/journal.ppat.1004755
PMCID: PMC4373807  PMID: 25806948
13.  Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon 
Genome Biology  2013;14(12):R145.
Background
The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon.
Results
B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner.
Conclusions
B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.
doi:10.1186/gb-2013-14-12-r145
PMCID: PMC4053937  PMID: 24367943
14.  Phytophthora Have Distinct Endogenous Small RNA Populations That Include Short Interfering and microRNAs 
PLoS ONE  2013;8(10):e77181.
In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.
doi:10.1371/journal.pone.0077181
PMCID: PMC3804510  PMID: 24204767
15.  The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification 
Current Biology  2012;22(10):881-890.
SUMMARY
Background
In nematodes, plants and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary siRNAs and the target mRNA leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood.
Results
From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10 and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, as well as other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage, but sensitive to high dosage of double-stranded RNAs (dsRNAs). We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6 and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways.
Conclusions
The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.
doi:10.1016/j.cub.2012.04.011
PMCID: PMC3371361  PMID: 22542102
rde-10; rde-11; RNAi; endogenous siRNAs; RNA silencing protein co-factors
16.  Virus-Derived Gene Expression and RNA Interference Vector for Grapevine 
Journal of Virology  2012;86(11):6002-6009.
The improvement of the agricultural and wine-making qualities of the grapevine (Vitis vinifera) is hampered by adherence to traditional varieties, the recalcitrance of this plant to genetic modifications, and public resistance to genetically modified organism (GMO) technologies. To address these challenges, we developed an RNA virus-based vector for the introduction of desired traits into grapevine without heritable modifications to the genome. This vector expresses recombinant proteins in the phloem tissue that is involved in sugar transport throughout the plant, from leaves to roots to berries. Furthermore, the vector provides a powerful RNA interference (RNAi) capability of regulating the expression of endogenous genes via virus-induced gene-silencing (VIGS) technology. Additional advantages of this vector include superb genetic capacity and stability, as well as the swiftness of technology implementation. The most significant applications of the viral vector include functional genomics of the grapevine and disease control via RNAi-enabled vaccination against pathogens or invertebrate pests.
doi:10.1128/JVI.00436-12
PMCID: PMC3372183  PMID: 22438553
17.  The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications 
PLoS Genetics  2011;7(11):e1002369.
Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.
Author Summary
Endogenous small interfering RNAs (siRNAs) are a class of small RNAs present in fungi, plants, and animals. Small RNAs, including microRNAs, are known to regulate the expression levels of genes, silence invading elements such as transposons, and act in cell division. However, the function of many endogenous siRNAs is unknown. We have found that the ERI-6/7 helicase is required for a subset of endogenous siRNAs present in the nematode Caenorhabditis elegans. The ERI-6/7 helicase acts in a pathway together with the Argonaute protein ERGO-1 to produce two types of siRNAs: a primary class of 26 nucleotides in length present in oocytes and embryos, and a class of 22 nucleotide siRNAs present in later stages of development. These siRNAs correspond to only about one hundred genes. Interestingly, we found that these genes fall into groups of genes that contain nearly identical DNA sequences. The sequences of these genes are not conserved in other organisms, not even in related nematodes. These results point to a potential function of these endogenous siRNAs: silencing of recently acquired, duplicated genes. Our work demonstrates a new role of small RNAs, different from known functions in transposon silencing and regulation of gene expression.
doi:10.1371/journal.pgen.1002369
PMCID: PMC3213143  PMID: 22102828
18.  The Arabidopsis lyrata genome sequence and the basis of rapid genome size change 
Nature genetics  2011;43(5):476-481.
We present the 207 Mb genome sequence of the outcrosser Arabidopsis lyrata, which diverged from the self-fertilizing species A. thaliana about 10 million years ago. It is generally assumed that the much smaller A. thaliana genome, which is only 125 Mb, constitutes the derived state for the family. Apparent genome reduction in this genus can be partially attributed to the loss of DNA from large-scale rearrangements, but the main cause lies in the hundreds of thousands of small deletions found throughout the genome. These occurred primarily in non-coding DNA and transposons, but protein-coding multi-gene families are smaller in A. thaliana as well. Analysis of deletions and insertions still segregating in A. thaliana indicates that the process of DNA loss is ongoing, suggesting pervasive selection for a smaller genome.
doi:10.1038/ng.807
PMCID: PMC3083492  PMID: 21478890
19.  GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences 
PLoS ONE  2011;6(10):e25279.
GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts.
doi:10.1371/journal.pone.0025279
PMCID: PMC3188579  PMID: 21998647
20.  Transcription Factors in Light and Circadian Clock Signaling Networks Revealed by Genomewide Mapping of Direct Targets for Neurospora White Collar Complex ▿† 
Eukaryotic Cell  2010;9(10):1549-1556.
Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.
doi:10.1128/EC.00154-10
PMCID: PMC2950426  PMID: 20675579
21.  Identification of genes required for de novo DNA methylation in Arabidopsis 
Epigenetics  2011;6(3):344-354.
De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.
doi:10.4161/epi.6.3.14242
PMCID: PMC3092683  PMID: 21150311
DNA methylation; Arabidopsis; de novo; genetic screen; whole-genome sequencing
22.  Unique Functionality of 22 nt miRNAs in Triggering RDR6-Dependent siRNA Biogenesis from Target Transcripts in Arabidopsis 
RNA interference pathways may involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNA (miRNA). Here, Arabidopsis thaliana secondary siRNA from mRNA, and trans-acting siRNA, are shown to be triggered through initial targeting by 22 nt miRNA that associate with AGO1. In contrast to canonical 21 nt miRNA, 22 nt miRNA primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in production of 21 nt forms of miR173, miR472 and miR828. Both 21 and 22 nt forms associated with AGO1 and guided accurate slicer activity, but only 22 nt miRNA were competent to trigger RDR6-dependent siRNA from target RNA. These data suggest that AGO1 functions differentially with 21 and 22 nt miRNA to engage the RDR6-associated amplification apparatus.
doi:10.1038/nsmb.1866
PMCID: PMC2916640  PMID: 20562854
23.  Distinct Argonaute-mediated 22G-RNA pathways direct genome surveillance in the C. elegans germline 
Molecular cell  2009;36(2):231-244.
Summary
Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that in the germ-line, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germ-line nuage structures called P-granules. WAGO-1 silences certain genes, transposons, pseudogenes and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest novel regulatory functions for small RNAs.
doi:10.1016/j.molcel.2009.09.020
PMCID: PMC2776052  PMID: 19800275
24.  Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO 
PLoS Pathogens  2010;6(6):e1000962.
Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.
Author Summary
Plant viral pathogens cause an estimated US$60 billion loss in crop yields worldwide each year. Potyviruses, accounting for ∼30% of known plant viruses, include many agriculturally important viruses. Despite their importance, the cell-to-cell spread of potyviruses remains poorly understood. Previous studies have shown that at early time points of infection, the virus-encoded CI protein, one of 11 known potyviral proteins, is associated with cone-shaped structures at plasmodesmata (PD) and is involved in viral cell-to-cell movement. In this paper, we show that a newly identified potyviral protein, P3N-PIPO, is a PD-located protein and directs the CI protein to PD, facilitating the deposition of the cone-shaped structures of CI at PD by interacting with CI protein. We demonstrate that the mutant of CI, which impairs potyviral cell-to-cell movement, loses its ability to accumulate at PD. We further reveal that P3N-PIPO utilizes the secretory pathway rather than the actomyosin motility system for trafficking to PD. Taken together, the data presented in this study suggest that CI and P3N-PIPO coordinates the formation of conical structure at PD for potyviral cell-to-cell spread.
doi:10.1371/journal.ppat.1000962
PMCID: PMC2891837  PMID: 20585568
25.  Genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs in Arabidopsis 
Nucleic Acids Research  2010;38(9):3081-3093.
Flowering is the primary trait affected by ambient temperature changes. Plant microRNAs (miRNAs) are small non-coding RNAs playing an important regulatory role in plant development. In this study, to elucidate the mechanism of flowering-time regulation by small RNAs, we identified six ambient temperature-responsive miRNAs (miR156, miR163, miR169, miR172, miR398 and miR399) in Arabidopsis via miRNA microarray and northern hybridization analyses. We also determined the expression profile of 120 unique miRNA loci in response to ambient temperature changes by miRNA northern hybridization analysis. The expression of the ambient temperature-responsive miRNAs and their target genes was largely anticorrelated at two different temperatures (16 and 23°C). Interestingly, a lesion in short vegetative phase (SVP), a key regulator within the thermosensory pathway, caused alteration in the expression of miR172 and a subset of its target genes, providing a link between a thermosensory pathway gene and miR172. The miR172-overexpressing plants showed a temperature-independent early flowering phenotype, suggesting that modulation of miR172 expression leads to temperature insensitivity. Taken together, our results suggest a genetic framework for flowering-time regulation by ambient temperature-responsive miRNAs under non-stress temperature conditions.
doi:10.1093/nar/gkp1240
PMCID: PMC2875011  PMID: 20110261

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