Integrated Genome-Scale Analysis and Northern Blot Detection of Retrotransposon siRNAs Across Plant Species

Böhrer, Marcel; Rymen, Bart; Himber, Christophe; Gerbaud, Aude; Pflieger, David; Laudencia‐Chingcuanco, Debbie; Cartwright, Amy; Vogel, John P.; Sibout, Richard; Blevins, Todd

doi:10.1007/978-1-0716-0712-1_23

Cited by 4 publications

(13 citation statements)

References 65 publications

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“…Brachypodium distachyon natural accessions used in this study comprised Bd21, Bd21-3, Cm18, Cb23, ABR2, Bd29-1 BdTR13c, RON2 and Arn1. Because Pol IV is known to play an important role in LTR-RT silencing in plants (Stonaker et al ., 2009; Ito et al ., 2011; Ferrafiat et al ., 2019), we also included the sodium azide mutagenized pol IV mutant line NaN74 ( Bd nrpd1-1 ) (Dalmais et al ., 2013; Böhrer et al ., 2020), the T-DNA insertion pol IV mutant line JJJ18557 Nr31 (Bragg et al ., 2012) Bd nrpd1-2 (-/-) and a corresponding sibling, outcrossed control line Bd NRPD1 (+/+) in the background of the natural accession Bd21-3. For in vitro experiments, seeds were soaked for 4 h in tap water and, without damaging the embryo, the lemma was carefully peeled off.…”

Section: Methodsmentioning

confidence: 99%

“…For the small RNA blot analysis, 200 µg of each total RNA were size-fractionated using the RNeasy Midi Kit (QIAGEN), as described previously (Böhrer et al ., 2020). Low molecular weight (LMW, <200 nt) RNAs are not bound by the silica membrane of the columns and were isolated from the collected flow-through and wash aliquots.…”

Section: Methodsmentioning

confidence: 99%

“…To account for differences in sequencing depth, the obtained coverage for 2-LTR-junction spanning reads was normalized with the total coverage obtained with bedtools (v 2. For the small RNA blot analysis, 200 µg of each total RNA were size-fractionated using the RNeasy Midi Kit (QIAGEN), as described previously (Böhrer et al, 2020) . Low molecular weight (LMW, <200 nt) RNAs are not bound by the silica membrane of the columns and were isolated from the collected flow-through and wash aliquots.…”

Section: Analysis Of Mobilome-seqmentioning

confidence: 99%

“…The pivotal role of Pol IV in producing TE-specific 24-nt siRNAs for RNA-directed DNA methylation has been demonstrated in many plant species including A. thaliana (Ito et al, 2011) , rice (Xu et al, 2020) and for the Alesia family (RLC_BdisC010) in B. distachyon (Böhrer et al, 2020) . To confirm that the increased production of 2-LTR eccDNA circles of HOPPLA in mutants deficient in B. distachyon NRPD1 is correlated with a depletion of 24 nt siRNAs, we performed a small RNA blot including To complete their life cycle, reverse transcribed extrachromosomal copies of LTR-RTs have to 270 integrate into the host genome (Schulman, 2013) .…”

Section: Mutant Plantsmentioning

confidence: 99%

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Transposition ofHOPPLAin siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility inBrachypodium distachyon

Thieme,

Minadakis,

Himber

et al. 2023

Preprint

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Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual roles in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grassBrachypodium distachyonto characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations inB. distachyonnatural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transposition activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility inB. distachyonand reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Analysis Of Mobilome-seqmentioning

confidence: 99%

Section: Mutant Plantsmentioning

confidence: 99%

See 2 more Smart Citations

Transposition ofHOPPLAin siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility inBrachypodium distachyon

Thieme,

Minadakis,

Himber

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Another limitation is the fact that the target sequence may include nucleotide substitutions and/or indels in transcripts produced from different copies. In such case, northern blotting seems to be a good complementary method, as it may reveal the size distribution of TE-derived transcripts, including full length TEs ( Böhrer et al., 2020 ).…”

Section: Detection Of Te-derived Transcriptsmentioning

confidence: 99%

A review of strategies used to identify transposition events in plant genomes

et al. 2022

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Transposable elements (TEs) were initially considered redundant and dubbed ‘junk DNA’. However, more recently they were recognized as an essential element of genome plasticity. In nature, they frequently become active upon exposition of the host to stress conditions. Even though most transposition events are neutral or even deleterious, occasionally they may happen to be beneficial, resulting in genetic novelty providing better fitness to the host. Hence, TE mobilization may promote adaptability and, in the long run, act as a significant evolutionary force. There are many examples of TE insertions resulting in increased tolerance to stresses or in novel features of crops which are appealing to the consumer. Possibly, TE-driven de novo variability could be utilized for crop improvement. However, in order to systematically study the mechanisms of TE/host interactions, it is necessary to have suitable tools to globally monitor any ongoing TE mobilization. With the development of novel potent technologies, new high-throughput strategies for studying TE dynamics are emerging. Here, we present currently available methods applied to monitor the activity of TEs in plants. We divide them on the basis of their operational principles, the position of target molecules in the process of transposition and their ability to capture real cases of actively transposing elements. Their possible theoretical and practical drawbacks are also discussed. Finally, conceivable strategies and combinations of methods resulting in an improved performance are proposed.

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CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing

Felgines,

Rymen,

Martins

et al. 2023

Preprint

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Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a “one CLSY per Pol IV” model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopypol ivnull mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

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Integrated Genome-Scale Analysis and Northern Blot Detection of Retrotransposon siRNAs Across Plant Species

Cited by 4 publications

References 65 publications

Transposition ofHOPPLAin siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility inBrachypodium distachyon

Transposition ofHOPPLAin siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility inBrachypodium distachyon

A review of strategies used to identify transposition events in plant genomes

CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing

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