Publications by authors named "David Baulcombe"

121 Publications

The small RNA locus map for Chlamydomonas reinhardtii.

PLoS One 2020 19;15(11):e0242516. Epub 2020 Nov 19.

Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom.

Small (s)RNAs play crucial roles in the regulation of gene expression and genome stability across eukaryotes where they direct epigenetic modifications, post-transcriptional gene silencing, and defense against both endogenous and exogenous viruses. It is known that Chlamydomonas reinhardtii, a well-studied unicellular green algae species, possesses sRNA-based mechanisms that are distinct from those of land plants. However, definition of sRNA loci and further systematic classification is not yet available for this or any other algae. Here, using data-driven machine learning approaches including Multiple Correspondence Analysis (MCA) and clustering, we have generated a comprehensively annotated and classified sRNA locus map for C. reinhardtii. This map shows some common characteristics with higher plants and animals, but it also reveals distinct features. These results are consistent with the idea that there was diversification in sRNA mechanisms after the evolutionary divergence of algae from higher plant lineages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0242516PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676726PMC
January 2021

Viral Fitness Determines the Magnitude of Transcriptomic and Epigenomic Reprograming of Defense Responses in Plants.

Mol Biol Evol 2020 07;37(7):1866-1881

Instituto de Biología Integrativa de Sistemas (I2SysBio), Consejo Superior de Investigaciones Científicas (CSIC)-Universitat de València, Paterna, Valencia, Spain.

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host.
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http://dx.doi.org/10.1093/molbev/msaa091DOI Listing
July 2020

Transposon age and non-CG methylation.

Nat Commun 2020 03 6;11(1):1221. Epub 2020 Mar 6.

Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, UK.

Silencing of transposable elements (TEs) is established by small RNA-directed DNA methylation (RdDM). Maintenance of silencing is then based on a combination of RdDM and RNA-independent mechanisms involving DNA methyltransferase MET1 and chromodomain DNA methyltransferases (CMTs). Involvement of RdDM, according to this model should decrease with TE age but here we show a different pattern in tomato and Arabidopsis. In these species the CMTs silence long terminal repeat (LTR) transposons in the distal chromatin that are younger than those affected by RdDM. To account for these findings we propose that, after establishment of primary RdDM as in the original model, there is an RNA-independent maintenance phase involving CMTs followed by secondary RdDM. This progression of epigenetic silencing in the gene-rich distal chromatin is likely to influence the transcriptome either in cis or in trans depending on whether the mechanisms are RNA-dependent or -independent.
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http://dx.doi.org/10.1038/s41467-020-14995-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060349PMC
March 2020

Extensive recombination challenges the utility of Sugarcane mosaic virus phylogeny and strain typing.

Sci Rep 2019 12 27;9(1):20067. Epub 2019 Dec 27.

University of Cambridge, Department of Plant Sciences, Cambridge, CB2 3EA, United Kingdom.

Sugarcane mosaic virus (SCMV) is distributed worldwide and infects three major crops: sugarcane, maize, and sorghum. The impact of SCMV is increased by its interaction with Maize chlorotic mottle virus which causes the synergistic maize disease maize lethal necrosis. Here, we characterised maize lethal necrosis-infected maize from multiple sites in East Africa, and found that SCMV was present in all thirty samples. This distribution pattern indicates that SCMV is a major partner virus in the East African maize lethal necrosis outbreak. Consistent with previous studies, our SCMV isolates were highly variable with several statistically supported recombination hot- and cold-spots across the SCMV genome. The recombination events generate conflicting phylogenetic signals from different fragments of the SCMV genome, so it is not appropriate to group SCMV genomes by simple similarity.
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http://dx.doi.org/10.1038/s41598-019-56227-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934591PMC
December 2019

Environmental and epigenetic regulation of Rider retrotransposons in tomato.

PLoS Genet 2019 09 16;15(9):e1008370. Epub 2019 Sep 16.

The Sainsbury Laboratory, University of Cambridge, Cambridge, United Kingdom.

Transposable elements in crop plants are the powerful drivers of phenotypic variation that has been selected during domestication and breeding programs. In tomato, transpositions of the LTR (long terminal repeat) retrotransposon family Rider have contributed to various phenotypes of agronomical interest, such as fruit shape and colour. However, the mechanisms regulating Rider activity are largely unknown. We have developed a bioinformatics pipeline for the functional annotation of retrotransposons containing LTRs and defined all full-length Rider elements in the tomato genome. Subsequently, we showed that accumulation of Rider transcripts and transposition intermediates in the form of extrachromosomal DNA is triggered by drought stress and relies on abscisic acid signalling. We provide evidence that residual activity of Rider is controlled by epigenetic mechanisms involving siRNAs and the RNA-dependent DNA methylation pathway. Finally, we demonstrate the broad distribution of Rider-like elements in other plant species, including crops. Our work identifies Rider as an environment-responsive element and a potential source of genetic and epigenetic variation in plants.
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http://dx.doi.org/10.1371/journal.pgen.1008370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6762207PMC
September 2019

Distinct roles of Argonaute in the green alga Chlamydomonas reveal evolutionary conserved mode of miRNA-mediated gene expression.

Sci Rep 2019 07 31;9(1):11091. Epub 2019 Jul 31.

Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, United Kingdom.

The unicellular green alga Chlamydomonas reinhardtii is evolutionarily divergent from higher plants, but has a fully functional silencing machinery including microRNA (miRNA)-mediated translation repression and mRNA turnover. However, distinct from the metazoan machinery, repression of gene expression is primarily associated with target sites within coding sequences instead of 3'UTRs. This feature indicates that the miRNA-Argonaute (AGO) machinery is ancient and the primary function is for post transcriptional gene repression and intermediate between the mechanisms in the rest of the plant and animal kingdoms. Here, we characterize AGO2 and 3 in Chlamydomonas, and show that cytoplasmically enriched Cr-AGO3 is responsible for endogenous miRNA-mediated gene repression. Under steady state, mid-log phase conditions, Cr-AGO3 binds predominantly miR-C89, which we previously identified as the predominant miRNA with effects on both translation repression and mRNA turnover. In contrast, the paralogue Cr-AGO2 is nuclear enriched and exclusively binds to 21-nt siRNAs. Further analysis of the highly similar Cr-AGO2 and Cr-AGO 3 sequences (90% amino acid identity) revealed a glycine-arginine rich N-terminal extension of ~100 amino acids that, given previous work on unicellular protists, may associate AGO with the translation machinery. Phylogenetic analysis revealed that this glycine-arginine rich N-terminal extension is present outside the animal kingdom and is highly conserved, consistent with our previous proposal that miRNA-mediated CDS-targeting operates in this green alga.
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http://dx.doi.org/10.1038/s41598-019-47415-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668577PMC
July 2019

How Virus Resistance Provided a Mechanistic Foundation for RNA Silencing.

Plant Cell 2019 07 8;31(7):1395-1396. Epub 2019 May 8.

Department of Plant Science University of CambridgeCambridge CB2 3EA, United Kingdom

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http://dx.doi.org/10.1105/tpc.19.00348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635879PMC
July 2019

Maternal small RNAs mediate spatial-temporal regulation of gene expression, imprinting, and seed development in .

Proc Natl Acad Sci U S A 2019 02 28;116(7):2761-2766. Epub 2019 Jan 28.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712;

seed development involves maternal small interfering RNAs (siRNAs) that induce RNA-directed DNA methylation (RdDM) through the -mediated pathway. To investigate their biological functions, we characterized siRNAs in the endosperm and seed coat that were separated by laser-capture microdissection (LCM) in reciprocal genetic crosses with an mutant. We also monitored the spatial-temporal activity of the -mediated pathway on seed development using the AGO4:GFP::AGO4 (promoter:GFP::protein) reporter and promoter:GUS sensors of siRNA-mediated silencing. From these approaches, we identified four distinct groups of siRNA loci dependent on or independent of the maternal allele in the endosperm or seed coat. A group of maternally expressed -siRNA loci targets endosperm-preferred genes, including those encoding AGAMOUS-LIKE (AGL) transcription factors. Using translational promoter:AGL::GUS constructs as sensors, we demonstrate that spatial and temporal expression patterns of these genes in the endosperm are regulated by the -mediated pathway irrespective of complete silencing () or incomplete silencing () of these target genes. Moreover, altered expression of these siRNA-targeted genes affects seed size. We propose that the corresponding maternal siRNAs could account for parent-of-origin effects on the endosperm in interploidy and hybrid crosses. These analyses reconcile previous studies on siRNAs and imprinted gene expression during seed development.
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http://dx.doi.org/10.1073/pnas.1807621116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377484PMC
February 2019

Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato.

Proc Natl Acad Sci U S A 2019 02 24;116(7):2755-2760. Epub 2019 Jan 24.

Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom;

Nucleotide binding site leucine-rich repeat (NLR) proteins of the plant innate immune system are negatively regulated by the miR482/2118 family miRNAs that are in a distinct 22-nt class of miRNAs with a double mode of action. First, they cleave the target RNA, as with the canonical 21-nt miRNAs, and second, they trigger secondary siRNA production using the target RNA as a template. Here, we address the extent to which the miR482/2118 family affects expression of NLR mRNAs and disease resistance. We show that structural differences of miR482/2118 family members in tomato () are functionally significant. The predicted target of the miR482 subfamily is a conserved motif in multiple NLR mRNAs, whereas for miR2118b, it is a noncoding RNA target formed by rearrangement of several different NLR genes. From RNA sequencing and degradome data in lines expressing short tandem target mimic (STTM) RNAs of miR482/2118, we confirm the different targets of these miRNAs. The effect on NLR mRNA accumulation is slight, but nevertheless, the tomato STTM lines display enhanced resistance to infection with the oomycete and bacterial pathogens. These data implicate an RNA cascade of miRNAs and secondary siRNAs in the regulation of NLR RNAs and show that the encoded NLR proteins have a role in quantitative disease resistance in addition to dominant gene resistance that has been well characterized elsewhere. We also illustrate the use of STTM RNA in a biotechnological approach for enhancing quantitative disease resistance in highly bred cultivars.
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http://dx.doi.org/10.1073/pnas.1814380116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377479PMC
February 2019

miRNA-Mediated Regulation of Synthetic Gene Circuits in the Green Alga Chlamydomonas reinhardtii.

ACS Synth Biol 2019 02 24;8(2):358-370. Epub 2019 Jan 24.

Department of Plant Sciences , University of Cambridge , Cambridge CB2 3EA , United Kingdom.

MicroRNAs (miRNAs), small RNA molecules of 20-24 nts, have many features that make them useful tools for gene expression regulation-small size, flexible design, target predictability, and action at a late stage of the gene expression pipeline. In addition, their role in fine-tuning gene expression can be harnessed to increase robustness of synthetic gene networks. In this work, we apply a synthetic biology approach to characterize miRNA-mediated gene expression regulation in the unicellular green alga Chlamydomonas reinhardtii. This characterization is then used to build tools based on miRNAs, such as synthetic miRNAs, miRNA-responsive 3'UTRs, miRNA decoys, and self-regulatory loops. These tools will facilitate the engineering of gene expression for new applications and improved traits in this alga.
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http://dx.doi.org/10.1021/acssynbio.8b00393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396871PMC
February 2019

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

ACS Synth Biol 2018 09 5;7(9):2074-2086. Epub 2018 Sep 5.

Institut de Biologie Physico-Chimique, UMR 8226 , CNRS, Sorbonne Université , Paris , France.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.
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http://dx.doi.org/10.1021/acssynbio.8b00251DOI Listing
September 2018

A novel DCL2-dependent miRNA pathway in tomato affects susceptibility to RNA viruses.

Genes Dev 2018 09 27;32(17-18):1155-1160. Epub 2018 Aug 27.

Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, United Kingdom.

Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The 2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.
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http://dx.doi.org/10.1101/gad.313601.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120711PMC
September 2018

Towards annotating the plant epigenome: the Arabidopsis thaliana small RNA locus map.

Sci Rep 2018 04 20;8(1):6338. Epub 2018 Apr 20.

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, United Kingdom.

Based on 98 public and internal small RNA high throughput sequencing libraries, we mapped small RNAs to the genome of the model organism Arabidopsis thaliana and defined loci based on their expression using an empirical Bayesian approach. The resulting loci were subsequently classified based on their genetic and epigenetic context as well as their expression properties. We present the results of this classification, which broadly conforms to previously reported divisions between transcriptional and post-transcriptional gene silencing small RNAs, and to PolIV and PolV dependencies. However, we are able to demonstrate the existence of further subdivisions in the small RNA population of functional significance. Moreover, we present a framework for similar analyses of small RNA populations in all species.
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http://dx.doi.org/10.1038/s41598-018-24515-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910406PMC
April 2018

Paramutation-like features of multiple natural epialleles in tomato.

BMC Genomics 2018 03 20;19(1):203. Epub 2018 Mar 20.

Department of Plant Sciences, University of Cambridge, Downing Site, Cambridge, CB2 3EA, UK.

Background: Freakish and rare or the tip of the iceberg? Both phrases have been used to refer to paramutation, an epigenetic drive that contravenes Mendel's first law of segregation. Although its underlying mechanisms are beginning to unravel, its understanding relies only on a few examples that may involve transgenes or artificially generated epialleles.

Results: By using DNA methylation of introgression lines as an indication of past paramutation, we reveal that the paramutation-like properties of the H06 locus in hybrids of Solanum lycopersicum and a range of tomato relatives and cultivars depend on the timing of sRNA production and conform to an RNA-directed mechanism. In addition, by scanning the methylomes of tomato introgression lines for shared regions of differential methylation that are absent in the S. lycopersicum parent, we identify thousands of candidate regions for paramutation-like behaviour. The methylation patterns for a subset of these regions segregate with non Mendelian ratios, consistent with secondary paramutation-like interactions to variable extents depending on the locus.

Conclusion: Together these results demonstrate that paramutation-like epigenetic interactions are common for natural epialleles in tomato, but vary in timing and penetrance.
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http://dx.doi.org/10.1186/s12864-018-4590-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859443PMC
March 2018

Maize chlorotic mottle virus exhibits low divergence between differentiated regional sub-populations.

Sci Rep 2018 01 19;8(1):1173. Epub 2018 Jan 19.

University of Cambridge, Plant Sciences, Cambridge, CB2 3EA, United Kingdom.

Maize chlorotic mottle virus has been rapidly spreading around the globe over the past decade. The interactions of maize chlorotic mottle virus with Potyviridae viruses causes an aggressive synergistic viral condition - maize lethal necrosis, which can cause total yield loss. Maize production in sub-Saharan Africa, where it is the most important cereal, is threatened by the arrival of maize lethal necrosis. We obtained maize chlorotic mottle virus genome sequences from across East Africa and for the first time from Ecuador and Hawaii, and constructed a phylogeny which highlights the similarity of Chinese to African isolates, and Ecuadorian to Hawaiian isolates. We used a measure of clustering, the adjusted Rand index, to extract region-specific SNPs and coding variation that can be used for diagnostics. The population genetics analysis we performed shows that the majority of sequence diversity is partitioned between populations, with diversity extremely low within China and East Africa.
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http://dx.doi.org/10.1038/s41598-018-19607-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5775324PMC
January 2018

Endogenous miRNA in the green alga Chlamydomonas regulates gene expression through CDS-targeting.

Nat Plants 2017 Oct 2;3(10):787-794. Epub 2017 Oct 2.

Department of Plant Sciences, University of Cambridge, Cambridge, CB2 3EA, UK.

MicroRNAs (miRNAs) are 21-24-nucleotide RNAs present in many eukaryotes that regulate gene expression as part of the RNA-induced silencing complex. The sequence identity of the miRNA provides the specificity to guide the silencing effector Argonaute (AGO) protein to target mRNAs via a base-pairing process . The AGO complex promotes translation repression and/or accelerated decay of this target mRNA . There is overwhelming evidence both in vivo and in vitro that translation repression plays a major role . However, there has been controversy about which of these three mechanisms is more significant in vivo, especially when effects of miRNA on endogenous genes cannot be faithfully represented by reporter systems in which, at least in metazoans, the observed repression vastly exceeds that typically observed for endogenous mRNAs . Here, we provide a comprehensive global analysis of the evolutionarily distant unicellular green alga Chlamydomonas reinhardtii to quantify the effects of miRNA on protein synthesis and RNA abundance. We show that, similar to metazoan steady-state systems, endogenous miRNAs in Chlamydomonas can regulate gene expression both by destabilization of the mRNA and by translational repression. However, unlike metazoan miRNA where target site utilization localizes mainly to 3' UTRs, in Chlamydomonas utilized target sites lie predominantly within coding regions. These results demonstrate the evolutionarily conserved mode of action for miRNAs, but details of the mechanism diverge between the plant and metazoan kingdoms.
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http://dx.doi.org/10.1038/s41477-017-0024-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662147PMC
October 2017

Epigenetic and Genetic Contributions to Adaptation in Chlamydomonas.

Mol Biol Evol 2017 09;34(9):2285-2306

Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom.

Epigenetic modifications, such as DNA methylation or histone modifications, can be transmitted between cellular or organismal generations. However, there are no experiments measuring their role in adaptation, so here we use experimental evolution to investigate how epigenetic variation can contribute to adaptation. We manipulated DNA methylation and histone acetylation in the unicellular green alga Chlamydomonas reinhardtii both genetically and chemically to change the amount of epigenetic variation generated or transmitted in adapting populations in three different environments (salt stress, phosphate starvation, and high CO2) for two hundred asexual generations. We find that reducing the amount of epigenetic variation available to populations can reduce adaptation in environments where it otherwise happens. From genomic and epigenomic sequences from a subset of the populations, we see changes in methylation patterns between the evolved populations over-represented in some functional categories of genes, which is consistent with some of these differences being adaptive. Based on whole genome sequencing of evolved clones, the majority of DNA methylation changes do not appear to be linked to cis-acting genetic mutations. Our results show that transgenerational epigenetic effects play a role in adaptive evolution, and suggest that the relationship between changes in methylation patterns and differences in evolutionary outcomes, at least for quantitative traits such as cell division rates, is complex.
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http://dx.doi.org/10.1093/molbev/msx166DOI Listing
September 2017

Improved Denaturation of Small RNA Duplexes and Its Application for Northern Blotting.

Methods Mol Biol 2017 ;1580:1-6

School of Biological Sciences, University of Edinburgh, Edinburgh, UK.

Small RNAs (sRNAs) are short (18-30 nucleotide) noncoding RNA molecules, which control gene expression and pathogen response in eukaryotes. They are associated with and guide nucleases to target nucleic acids by nucleotide base pairing. We found that current techniques for small RNA detection are adversely affected by the presence of complementary RNA. Thus we established FDF-PAGE (fully denaturing formaldehyde polyacrylamide gel electrophoresis), which dramatically improves denaturation efficiency and subsequently the detection of sequestered sRNAs.
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http://dx.doi.org/10.1007/978-1-4939-6866-4_1DOI Listing
February 2018

DNA Methylation Signatures of the Plant Chromomethyltransferases.

PLoS Genet 2016 Dec 20;12(12):e1006526. Epub 2016 Dec 20.

Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom.

DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G). By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation hides spatial and functional partitioning of different methylation pathways and is incomplete. CG methylation (mCG) is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and tomato and throughout the chromosomes of maize and rice. In A. thaliana the biased representation of mCCG in heterochromatin is related to specificities of H3K9 methyltransferase SUVH family members. At CHH motifs there is an over-representation of different variant forms of mCHH that, similarly to mCCG hypomethylation, is partitioned into the pericentric regions of the two dicots but dispersed in the monocot chromosomes. The over-represented mCHH motifs in A. thaliana associate with specific types of transposon including both class I and II elements. At mCHH the contextual bias is due to the involvement of various chromomethyltransferases whereas the context-independent CHH methylation in A. thaliana and tomato is mediated by the RNA-directed DNA methylation process that is most active in the gene-rich euchromatin. This analysis therefore reveals that the sequence context of the methylome of plant genomes is informative about the mechanisms associated with maintenance of methylation and the overlying chromatin structure.
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http://dx.doi.org/10.1371/journal.pgen.1006526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5221884PMC
December 2016

Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays.

Methods Mol Biol 2017 ;1456:127-139

PSL Research University, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, Ecole NormaleSupérieure, 46 rue d'Ulm, Paris, F75005, France.

Epigenetic response to stress in plants involves changes in DNA methylation, histone modifications, and expression of small noncoding RNAs (sRNA). Here we present the method of analysis of differential expression of sRNA populations using DNA tiling arrays. sRNA extracted from Arabidopsis thaliana plants exposed to pathogen elicitor or control plants were reverse-transcribed into cDNAs, and subsequently hybridized after labeling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by massive parallel sequence signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in sRNA abundance.
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http://dx.doi.org/10.1007/978-1-4899-7708-3_11DOI Listing
January 2018

Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs.

Genome Res 2016 Apr 11;26(4):519-29. Epub 2016 Mar 11.

Department of Plant Sciences, University of Cambridge CB2 3EA, Cambridge CB2 3EA, United Kingdom.

We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNA-mediated RNA silencing in the model algal species,Chlamydomonas reinhardtii Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon- and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNA-targeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes.
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http://dx.doi.org/10.1101/gr.199703.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4817775PMC
April 2016

SLTAB2 is the paramutated SULFUREA locus in tomato.

J Exp Bot 2016 04 8;67(9):2655-64. Epub 2016 Mar 8.

Department of Plant Sciences, University of Cambridge, Cambridge, UK

The sulfurea (sulf) allele is a silent epigenetic variant of a tomato (Solanum lycopersicum) gene affecting pigment production. It is homozygous lethal but, in a heterozygote sulf/+, the wild-type (wt) allele undergoes silencing so that the plants exhibit chlorotic sectors. This transfer of the silenced state between alleles is termed paramutation and is best characterized in maize. To understand the mechanism of paramutation we mapped SULF to the orthologue SLTAB2 of an Arabidopsis gene that, consistent with the pigment deficiency, is involved in the translation of photosystem I. Paramutation of SLTAB2 is linked to an increase in DNA methylation and the production of small interfering RNAs at its promoter. Virus-induced gene silencing of SLTAB2 phenocopies sulf, consistent with the possibility that siRNAs mediate the paramutation of SULFUREA Unlike the maize systems, the paramutagenicity of sulf is not, however, associated with repeated sequences at the region of siRNA production or DNA methylation.
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http://dx.doi.org/10.1093/jxb/erw096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861014PMC
April 2016

The P1N-PISPO trans-Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor.

J Virol 2016 Jan 20;90(7):3543-57. Epub 2016 Jan 20.

Center for Research in Agricultural Genomics, CSIC-IRTA-UAB-UB, Cerdanyola del Vallès, Barcelona, Spain

Unlabelled: The positive-sense RNA genome of Sweet potato feathery mottle virus (SPFMV) (genus Potyvirus, family Potyviridae) contains a large open reading frame (ORF) of 3,494 codons translatable as a polyprotein and two embedded shorter ORFs in the -1 frame: PISPO, of 230 codons, and PIPO, of 66 codons, located in the P1 and P3 regions, respectively. PISPO is specific to some sweet potato-infecting potyviruses, while PIPO is present in all potyvirids. In SPFMV these two extra ORFs are preceded by conserved G2A6 motifs. We have shown recently that a polymerase slippage mechanism at these sites could produce transcripts bringing these ORFs in frame with the upstream polyprotein, thus leading to P1N-PISPO and P3N-PIPO products (B. Rodamilans, A. Valli, A. Mingot, D. San Leon, D. B. Baulcombe, J. J. Lopez-Moya, and J.A. Garcia, J Virol 89:6965-6967, 2015, doi:10.1128/JVI.00337-15). Here, we demonstrate by liquid chromatography coupled to mass spectrometry that both P1 and P1N-PISPO are produced during viral infection and coexist in SPFMV-infected Ipomoea batatas plants. Interestingly, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, our results reveal a further layer of complexity of the RNA silencing suppression activity within the Potyviridae family.

Importance: Gene products of potyviruses include P1, HCPro, P3, 6K1, CI, 6K2, VPg/NIaPro, NIb, and CP, all derived from the proteolytic processing of a large polyprotein, and an additional P3N-PIPO product, with the PIPO segment encoded in a different frame within the P3 cistron. In sweet potato feathery mottle virus (SPFMV), another out-of-frame element (PISPO) was predicted within the P1 region. We have shown recently that a polymerase slippage mechanism can generate the transcript variants with extra nucleotides that could be translated into P1N-PISPO and P3N-PIPO. Now, we demonstrate by mass spectrometry analysis that P1N-PISPO is indeed produced in SPFMV-infected plants, in addition to P1. Interestingly, while in other potyviruses the suppressor of RNA silencing is HCPro, we show here that P1N-PISPO exhibited this activity in SPFMV, revealing how the complexity of the gene content could contribute to supply this essential function in members of the Potyviridae family.
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http://dx.doi.org/10.1128/JVI.02360-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794657PMC
January 2016

Mobile small RNAs regulate genome-wide DNA methylation.

Proc Natl Acad Sci U S A 2016 Feb 19;113(6):E801-10. Epub 2016 Jan 19.

Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037

RNA silencing at the transcriptional and posttranscriptional levels regulates endogenous gene expression, controls invading transposable elements (TEs), and protects the cell against viruses. Key components of the mechanism are small RNAs (sRNAs) of 21-24 nt that guide the silencing machinery to their nucleic acid targets in a nucleotide sequence-specific manner. Transcriptional gene silencing is associated with 24-nt sRNAs and RNA-directed DNA methylation (RdDM) at cytosine residues in three DNA sequence contexts (CG, CHG, and CHH). We previously demonstrated that 24-nt sRNAs are mobile from shoot to root in Arabidopsis thaliana and confirmed that they mediate DNA methylation at three sites in recipient cells. In this study, we extend this finding by demonstrating that RdDM of thousands of loci in root tissues is dependent upon mobile sRNAs from the shoot and that mobile sRNA-dependent DNA methylation occurs predominantly in non-CG contexts. Mobile sRNA-dependent non-CG methylation is largely dependent on the DOMAINS REARRANGED METHYLTRANSFERASES 1/2 (DRM1/DRM2) RdDM pathway but is independent of the CHROMOMETHYLASE (CMT)2/3 DNA methyltransferases. Specific superfamilies of TEs, including those typically found in gene-rich euchromatic regions, lose DNA methylation in a mutant lacking 22- to 24-nt sRNAs (dicer-like 2, 3, 4 triple mutant). Transcriptome analyses identified a small number of genes whose expression in roots is associated with mobile sRNAs and connected to DNA methylation directly or indirectly. Finally, we demonstrate that sRNAs from shoots of one accession move across a graft union and target DNA methylation de novo at normally unmethylated sites in the genomes of root cells from a different accession.
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http://dx.doi.org/10.1073/pnas.1515072113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4760824PMC
February 2016

Chlorophyll Content Assay to Quantify the Level of Necrosis Induced by Different R Gene/Elicitor Combinations after Transient Expression.

Bio Protoc 2015 Dec;5(23)

Plant Sciences Department, Cambridge University, Cambridge, UK.

This assay can be used to rapidly and accurately quantify levels of leaf necrosis induced after transient expression of R genes and elicitor combinations (Harris 2013). It is based on the inverse correlation between level of necrosis and chlorophyll content in leaf tissue. It is adapted from the calculations described by (Strain , 1971).
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http://dx.doi.org/10.21769/BioProtoc.1670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5524178PMC
December 2015

Two classes of short interfering RNA in RNA silencing.

EMBO J 2015 Oct 19;34(20):2590. Epub 2015 Aug 19.

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http://dx.doi.org/10.15252/embj.201570050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4609188PMC
October 2015

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.

RNA 2015 Oct 18;21(10):1731-45. Epub 2015 Aug 18.

Division of Virology, Department of Pathology, University of Cambridge, Cambridge CB2 1QP, United Kingdom.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.
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http://dx.doi.org/10.1261/rna.052548.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574750PMC
October 2015

VIGS, HIGS and FIGS: small RNA silencing in the interactions of viruses or filamentous organisms with their plant hosts.

Curr Opin Plant Biol 2015 Aug 3;26:141-6. Epub 2015 Aug 3.

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK. Electronic address:

Recent evidence indicates two-way traffic of silencing RNA between filamentous organisms and their plant hosts. There are also indications that suppressors of RNA silencing are transferred from filamentous organisms into host plant cells where they influence the innate immune system. Here I use virus disease as a template for interpretation of RNA silencing in connection with filamentous organisms and infected plant cells. I propose that host plant interactions of these organisms are influenced by RNA silencing networks in which there are: small interfering RNAs from the host that are transported into the filamentous organism and vice versa; silencing suppressors from the organism that are transported into the host; endogenous small interfering RNAs and micro RNAs that target components of the innate immune system or endogenous suppressors of the innate immune system.
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http://dx.doi.org/10.1016/j.pbi.2015.06.007DOI Listing
August 2015