The m6A reader ECT1 drives mRNA sequestration to dampen salicylic acid-dependent stress responses in Arabidopsis.

N 6-methyladenosine (m6A) is a common epitranscriptional mRNA modification in eukaryotes. Thirteen putative m6A readers, mostly annotated as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) proteins, have been identified in Arabidopsis (Arabidopsis thaliana), but few have been characterized. Here, we show that the Arabidopsis m6A reader ECT1 modulates salicylic acid (SA)-mediated plant stress responses. ECT1 undergoes liquid-liquid phase separation in vitro, and its N-terminal prion-like domain is critical for forming in vivo cytosolic biomolecular condensates in response to SA or bacterial pathogens. Fluorescence-activated particle sorting coupled with quantitative PCR analyses unveiled that ECT1 sequesters SA-induced m6A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses. Consistent with this finding, ECT1 overexpression promotes bacterial multiplication in plants. Collectively, our findings unequivocally link ECT1-associated cytosolic condensates to SA-dependent plant stress responses, advancing the current understanding of m6A readers and the SA signaling network.

Cooperative role of AtRsmD and AtRimM proteins in modification and maturation of 16S rRNA in plastids.

Chloroplast pre-ribosomal RNA (rRNA) undergoes maturation, which is critical for ribosome assembly. While the central and auxiliary factors in rRNA maturation have been elucidated in bacteria, their mode of action remains largely unexplored in chloroplasts. We now reveal chloroplast-specific factors involved in 16S rRNA maturation, Arabidopsis thaliana orthologs of bacterial RsmD methyltransferase (AtRsmD) and ribosome maturation factor RimM (AtRimM). A forward genetic screen aimed to find suppressors of the Arabidopsis yellow variegated 2 (var2) mutant defective in photosystem II quality control found a causal nonsense mutation in AtRsmD. The substantially impaired 16S rRNA maturation and translation due to the mutation rescued the leaf variegation phenotype by lowering the levels of chloroplast-encoded proteins, including photosystem II core proteins, in var2. The subsequent co-immunoprecipitation coupled with mass spectrometry analyses and bimolecular fluorescence complementation assay found that AtRsmD interacts with AtRimM. Consistent with their interaction, loss of AtRimM also considerably impairs 16S rRNA maturation with decelerated m2 G915 modification in 16S rRNA catalyzed by AtRsmD. The atrimM mutation also rescued var2 mutant phenotypes, corroborating the functional interplay between AtRsmD and AtRimM towards modification and maturation of 16S rRNA and chloroplast proteostasis. The maturation and post-transcriptional modifications of rRNA are critical to assembling ribosomes responsible for protein translation. Here, we revealed that the cooperative regulation of 16S rRNA m2 G915 modifications by AtRsmD methyltransferase and ribosome assembly factor AtRimM contributes to 16S rRNA maturation, ribosome assembly, and proteostasis in chloroplasts.

Cosuppression of AtGELP22 and AtGELP23, two ubiquitinated target proteins of RING E3 ligase AtAIRP5, increases tolerance to drought stress in Arabidopsis.

AtAIRP5 RING E3 ubiquitin ligase was recently identified as a positive regulator of the abscisic acid (ABA)-mediated drought stress response by stimulating the degradation of serine carboxypeptidase-like 1. Here, we identified GDSL-type esterase/lipase 22 (AtGELP22) and AtGELP23 as additional interacting partners of AtAIRP5. Yeast two-hybrid, pull-down, co-immunoprecipitation, and ubiquitination analyses verified that AtGELP22 and AtGELP23 are ubiquitinated target proteins of AtAIRP5. AtGELP22 and AtGELP23 were colocalized with AtAIRP5 to punctate-like structures in the cytosolic fraction, in which PYK10 and NAI2, two ER body marker proteins, are localized. T-DNA insertion atgelp22 and atgelp23 single knockout mutant plants showed phenotypes indistinguishable from those of wild-type plants under ABA treatment. In contrast, RNAi-mediated cosuppression of AtGELP22 and AtGELP23 resulted in hypersensitive ABA-mediated stomatal movements and higher tolerance to drought stress than that of the single mutant and wild-type plants. Taken together, our results suggest that the putative GDSL-type esterases/lipases AtGELP22 and AtGELP23 act as redundant negative regulators of the ABA-mediated drought stress response in Arabidopsis.

E3 ligase AtAIRP5/GARU regulates drought stress response by stimulating SERINE CARBOXYPEPTIDASE-LIKE1 turnover.

Ubiquitination is a major mechanism of eukaryotic posttranslational protein turnover that has been implicated in abscisic acid (ABA)-mediated drought stress response. Here, we isolated T-DNA insertion mutant lines in which ABA-insensitive RING protein 5 (AtAIRP5) was suppressed, resulting in hyposensitive ABA-mediated germination compared to wild-type Arabidopsis (Arabidopsis thaliana) plants. A homology search revealed that AtAIRP5 is identical to gibberellin (GA) receptor RING E3 ubiquitin (Ub) ligase (GARU), which downregulates GA signaling by degrading the GA receptor GID1, and thus AtAIRP5 was renamed AtAIRP5/GARU. The atairp5/garu knockout progeny were impaired in ABA-dependent stomatal closure and were markedly more susceptible to drought stress than wild-type plants, indicating a positive role for AtAIRP5/GARU in the ABA-mediated drought stress response. Yeast two-hybrid, pull-down, target ubiquitination, and in vitro and in planta degradation assays identified serine carboxypeptidase-like1 (AtSCPL1), which belongs to the clade 1A AtSCPL family, as a ubiquitinated target protein of AtAIRP5/GARU. atscpl1 single and atairp5/garu-1 atscpl1-2 double mutant plants were more tolerant to drought stress than wild-type plants in an ABA-dependent manner, suggesting that AtSCPL1 is genetically downstream of AtAIRP5/GARU. After drought treatment, the endogenous ABA levels in atscpl1 and atairp5/garu-1 atscpl1-2 mutant leaves were higher than those in wild-type and atairp5/garu leaves. Overall, our results suggest that AtAIRP5/GARU RING E3 Ub ligase functions as a positive regulator of the ABA-mediated drought response by promoting the degradation of AtSCPL1.

PLANT NATRIURETIC PEPTIDE A and Its Putative Receptor PNP-R2 Antagonize Salicylic Acid-Mediated Signaling and Cell Death.

The plant stress hormone salicylic acid (SA) participates in local and systemic acquired resistance, which eventually leads to whole-plant resistance to bacterial pathogens. However, if SA-mediated signaling is not appropriately controlled, plants incur defense-associated fitness costs such as growth inhibition and cell death. Despite its importance, to date only a few components counteracting the SA-primed stress responses have been identified in Arabidopsis (Arabidopsis thaliana). These include other plant hormones such as jasmonic acid and abscisic acid, and proteins such as LESION SIMULATING DISEASE1, a transcription coregulator. Here, we describe PLANT NATRIURETIC PEPTIDE A (PNP-A), a functional analog to vertebrate atrial natriuretic peptides, that appears to antagonize the SA-mediated plant stress responses. While loss of PNP-A potentiates SA-mediated signaling, exogenous application of synthetic PNP-A or overexpression of PNP-A significantly compromises the SA-primed immune responses. Moreover, we identify a plasma membrane-localized receptor-like protein, PNP-R2, that interacts with PNP-A and is required to initiate the PNP-A-mediated intracellular signaling. In summary, our work identifies a peptide and its putative cognate receptor as counteracting both SA-mediated signaling and SA-primed cell death in Arabidopsis.

Phase separation of chromatin and small RNA pathways in plants.

Gene expression can be modulated by epigenetic mechanisms, including chromatin modifications and small regulatory RNAs. These pathways are unevenly distributed within a cell and usually take place in specific intracellular regions. Unfortunately, the fundamental driving force and biological relevance of such spatial differentiation is largely unknown. Liquid-liquid phase separation (LLPS) is a natural propensity of demixing liquid phases and has been recently suggested to mediate the formation of biomolecular condensates that are relevant to diverse cellular processes. LLPS provides a mechanistic explanation for the self-assembly of subcellular structures by which the efficiency and specificity of certain cellular reactions are achieved. In plants, LLPS has been observed for several key factors in the chromatin and small RNA pathways. For example, the formation of facultative and obligate heterochromatin involves the LLPS of multiple relevant factors. In addition, phase separation is observed in a set of proteins acting in microRNA biogenesis and the small interfering RNA pathway. In this Focused Review, we highlight and discuss the recent findings regarding phase separation in the epigenetic mechanisms of plants.

Crystal Structures of the Plant Phospholipase A1 Proteins Reveal a Unique Dimerization Domain.

Phospholipase is an enzyme that hydrolyzes various phospholipid substrates at specific ester bonds and plays important roles such as membrane remodeling, as digestive enzymes, and the regulation of cellular mechanism. Phospholipase proteins are divided into following the four major groups according to the ester bonds they cleave off: phospholipase A1 (PLA1), phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD). Among the four phospholipase groups, PLA1 has been less studied than the other phospholipases. Here, we report the first molecular structures of plant PLA1s: AtDSEL and CaPLA1 derived from Arabidopsis thaliana and Capsicum annuum, respectively. AtDSEL and CaPLA1 are novel PLA1s in that they form homodimers since PLAs are generally in the form of a monomer. The dimerization domain at the C-terminal of the AtDSEL and CaPLA1 makes hydrophobic interactions between each monomer, respectively. The C-terminal domain is also present in PLA1s of other plants, but not in PLAs of mammals and fungi. An activity assay of AtDSEL toward various lipid substrates demonstrates that AtDSEL is specialized for the cleavage of sn-1 acyl chains. This report reveals a new domain that exists only in plant PLA1s and suggests that the domain is essential for homodimerization.

The N-Terminal UND Motif of the Arabidopsis U-Box E3 Ligase PUB18 Is Critical for the Negative Regulation of ABA-Mediated Stomatal Movement and Determines Its Ubiquitination Specificity for Exocyst Subunit Exo70B1.

The Arabidopsis thaliana U-box E3 ligases PUB18/PUB19 and PUB22/PUB23 are negative regulators of drought stress responses. PUB18/PUB19 regulate the drought stress response in an abscisic acid (ABA)-dependent manner, whereas PUB22/PUB23 regulate this response in an ABA-independent manner. A major structural difference between PUB18/PUB19 and PUB22/PUB23 is the presence of the UND (U-box N-terminal domain). Here, we focused on elucidating the molecular mechanism that mediates the functional difference between PUB18 and PUB22 and found that the UNDPUB18 was critically involved in the negative regulation of ABA-mediated stomatal movements. Exo70B1, a subunit of the exocyst complex, was identified as a target of PUB18, whereas Exo70B2 was a substrate of PUB22. However, the ∆UND-PUB18 derivative failed to ubiquitinate Exo70B1, but ubiquitinated Exo70B2. By contrast, the UNDPUB18-PUB22 chimeric protein ubiquitinated Exo70B1 instead of Exo70B2, suggesting that the ubiquitination specificities of PUB18 and PUB22 to Exo70B1 and Exo70B2, respectively, are dependent on the presence or absence of the UNDPUB18 motif. The ABA-insensitive phenotypes of the pub18 pub19 exo70b1 triple mutant were reminiscent of those of exo70b1 rather than pub18 pub19, indicating that Exo70B1 functions downstream of PUB18. Overall, our results suggest that the UNDPUB18 motif is crucial for the negative regulation of ABA-dependent stomatal movement and for determination of its ubiquitination specificity to Exo70B1.

The proper localization of RESPONSIVE TO DESICCATION 20 in lipid droplets depends on their biogenesis induced by STRESS-RELATED PROTEINS in vegetative tissues.

Arabidopsis LD surface proteins, SRPs are found only in higher plants and are important for LD biogenesis and abiotic stress signaling. However, the cellular mechanism of SRPs is still unclear. To investigate molecular functions of SRPs, we used tobacco transient expression system. Transient expression of SRPs was sufficient and synergistic for LD biogenesis, and SRPs participated in the formation step of LD in tobacco leaves. RESPONSIVE TO DESICCATION 20 (RD20), a known LD-localizing peroxygenase, localized to LD in the presence of an SRP, and its peroxygenase activity correlated with proper localization of RD20 to LD. Our data suggest that Arabidopsis SRPs play roles as positive factors for LD biogenesis to provide a proper localization of LD-localizing proteins in vegetative tissues.

Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses.

Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed.

Constitutive expression of CaPLA1 conferred enhanced growth and grain yield in transgenic rice plants.

Phospholipids are not only important components of cell membranes, but participate in diverse processes in higher plants. In this study, we generated Capsicum annuum phospholipiase A1 (CaPLA1) overexpressing transgenic rice (Oryza sativa L.) plants under the control of the maize ubiquitin promoter. The T4 CaPLA1-overexpressing rice plants (Ubi:CaPLA1) had a higher root:shoot mass ratio than the wild-type plants in the vegetative stage. Leaf epidermal cells from transgenic plants had more cells than wild-type plants. Genes that code for cyclin and lipid metabolic enzymes were up-regulated in the transgenic lines. When grown under typical paddy field conditions, the transgenic plants produced more tillers, longer panicles and more branches per panicle than the wild-type plants, all of which resulted in greater grain yield. Microarray analysis suggests that gene expressions that are related with cell proliferation, lipid metabolism, and redox state were widely altered in CaPLA1-overexpressing transgenic rice plants. Ubi:CaPLA1 plants had a reduced membrane peroxidation state, as determined by malondialdehyde and conjugated diene levels and higher peroxidase activity than wild-type rice plants. Furthermore, three isoprenoid synthetic genes encoding terpenoid synthase, hydroxysteroid dehydrogenase and 3-hydroxy-3-methyl-glutaryl-CoA reductase were up-regulated in CaPLA1-overexpressing plants. We suggest that constitutive expression of CaPLA1 conferred increased grain yield with enhanced growth in transgenic rice plants by alteration of gene activities related with cell proliferation, lipid metabolism, membrane peroxidation state and isoprenoid biosynthesis.

Expression, subcellular localization, and enzyme activity of a recombinant human extra-cellular superoxide dismutase in tobacco (Nicotiana benthamiana L.).

Human extracellular superoxide dismutase (hEC-SOD) is an enzyme that scavenges reactive oxygen species (ROS). Because of its antioxidant activity, hEC-SOD has been used as a therapeutic protein to treat skin disease and arthritis in mammalian systems. In this study, codon-optimized hEC-SOD was expressed in tobacco (Nicotiana benthamiana L.) via a plant-based transient protein expression system. Plant expression binary vectors containing full-length hEC-SOD (f-hEC-SOD) and modified hEC-SOD (m-hEC-SOD), in which the signal peptide and heparin-binding domain were deleted, were constructed for the cytosolic-, endoplasmic reticulum (ER)-, and chloroplast-localizations in tobacco leaf mesophyll cells. The results demonstrated that f-hEC-SOD was more efficiently expressed in the cytosolic fractions than in the ER or chloroplasts of tobacco cells. Our data further indicated that differently localized f-hEC-SOD and m-hEC-SOD displayed SOD enzyme activities, suggesting that the hEC-SODs expressed by plants may be functionally active. The f-hEC-SOD was expressed up to 3.8% of the total leaf soluble protein and the expression yield was calculated to be 313.7 µg f-hEC-SOD per g fresh weight of leaf. Overall, our results reveal that it was possible to express catalytically active hEC-SODs by means of a transient plant expression system in tobacco leaf cells.

Draft genome sequence of bacterium Bacillus proteolyticus strain IMGN4 from soil.

Here, we present the draft genome of Bacillus proteolyticus IMGN4, the gram-positive, soil-dwelling bacterium discovered in mountain Maemi, Republic of Korea in May 2019. The assembly resulted in 7 contigs, comprising a total of 6,063,502 base pairs and have 6,115 coding sequences.

Cooperation and functional diversification of two closely related galactolipase genes for jasmonate biosynthesis.

Jasmonic acid (JA) plays pivotal roles in diverse plant biological processes, including wound response. Chloroplast lipid hydrolysis is a critical step for JA biosynthesis, but the mechanism of this process remains elusive. We report here that DONGLE (DGL), a homolog of DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), encodes a chloroplast-targeted lipase with strong galactolipase and weak phospholipase A(1) activity. DGL is expressed in the leaves and has a specific role in maintaining basal JA content under normal conditions, and this expression regulates vegetative growth and is required for a rapid JA burst after wounding. During wounding, DGL and DAD1 have partially redundant functions for JA production, but they show different induction kinetics, indicating temporally separated roles: DGL plays a role in the early phase of JA production, and DAD1 plays a role in the late phase of JA production. Whereas DGL and DAD1 are necessary and sufficient for JA production, phospholipase D appears to modulate wound response by stimulating DGL and DAD1 expression.

Constitutive expression of CaRma1H1, a hot pepper ER-localized RING E3 ubiquitin ligase, increases tolerance to drought and salt stresses in transgenic tomato plants.

UNLABELLED: CaRma1H1, an endoplasmic reticulum (ER)-localized hot pepper really interesting new genes (RING) E3 Ub ligase, was previously reported to be a positive regulator of drought stress responses. To address the possibility that CaRma1H1 can be used to improve tolerance to abiotic stress in crop plants, CaRma1H1 was constitutively expressed in transgenic tomato (Solanum lycopersicum) plants. CaRma1H1-overexpressing tomato plants (35S:CaRma1H1) exhibited greatly enhanced tolerance to high-salinity treatments compared with wild-type plants. Leaf chlorophyll and proline contents in CaRma1H1 overexpressors were 4.3- to 8.5-fold and 1.2- to 1.5-fold higher, respectively, than in wild-type plants after 300 mM NaCl treatment. Transgenic cotyledons developed and their roots elongated in the presence of NaCl up to 200 mM. In addition, 35S:CaRma1H1 lines were markedly more tolerant to severe drought stress than were wild-type plants. Detached leaves of CaRma1H1 overexpressors preserved water more efficiently than did wild-type leaves during a rapid dehydration process. The ER chaperone genes LePDIL1, LeBIP1, and LeCNX1 were markedly up-regulated in 35S:CaRma1H1 tomatoes compared with wild-type plants. Therefore, overexpression of CaRma1H1 may enhance tomato plant ER responses to drought stress by effectively removing nonfunctional ubiquitinated proteins. Collectively, constitutive expression of CaRma1H1 in tomatoes conferrred strongly enhanced tolerance to salt- and water-stress. This raises the possibility that CaRma1H1 may be useful for developing abiotic stress-tolerant tomato plants. KEY MESSAGE: CaRma1H1 increases drought tolerance in transgenic tomato plants.

Harnessing epigenetic variability for crop improvement: current status and future prospects.

BACKGROUND: The epigenetic mechanisms play critical roles in a vast diversity of biological processes of plants, including development and response to environmental challenges. Particularly, DNA methylation is a stable epigenetic signature that supplements the genetics-based view of complex life phenomena. In crop breeding, the decrease in genetic diversity due to artificial selection of conventional breeding methods has been a long-standing concern. Therefore, the epigenetic diversity has been proposed as a new resource for future crop breeding, which will be hereinafter referred to as epibreeding. DISCUSSION: The induction of methylome changes has been performed in plants by several methods including chemical drugs treatment and tissue culture. Target-specific epigenetic engineering has been also attempted by exogenous RNAi mediated by virus-induced gene silencing and grafting. Importantly, the new and innovative techniques including the CRISPR-Cas9 system have recently been adopted in epigenetic engineering of plant genomes, facilitating the efforts for epibreeding. CONCLUSION: In this review, we introduce several examples of natural and induced epigenetic changes impacting on agronomic traits and discuss the methods for generating epigenomic diversity and site-specific epigenetic engineering.

Tracing Mobile DNAs: From Molecular to Population Scales.

Transposable elements (TEs, transposons) are mobile DNAs that are prevalent in most eukaryotic genomes. In plants, their mobility has vastly contributed to genetic diversity which is essential for adaptive changes and evolution of a species. Such mobile nature of transposon has been also actively exploited in plant science research by generating genetic mutants in non-model plant systems. On the other hand, transposon mobilization can bring about detrimental effects to host genomes and they are therefore mostly silenced by the epigenetic mechanisms. TEs have been studied as major silencing targets and acted a main feature in the remarkable growth of the plant epigenetics field. Despite the importance of transposon in plant biology and biotechnology, their mobilization and the underlying mechanisms are largely left unanswered. This is mainly because of the sequence repetitiveness of transposons, which makes their detection and analyses difficult and complicated. Recently, some attempts have been made to develop new experimental methods detecting active transposons and their mobilization behavior. These techniques reveal TE mobility in various levels, including the molecular, cellular, organismal and population scales. In this review, we will highlight the novel technical approaches in the study of mobile genetic elements and discuss how these techniques impacted on the advancement of transposon research and broadened our understanding of plant genome plasticity.

The Arabidopsis sn-1-specific mitochondrial acylhydrolase AtDLAH is positively correlated with seed viability.

Lipid-derived molecules produced by acylhydrolases play important roles in the regulation of diverse cellular functions in plants. In Arabidopsis, the DAD1-like phospholipase A1 family consists of 12 members, all of which possess a lipase 3 domain. In this study, the biochemical and cellular functions of AtDLAH, an Arabidopsis thaliana DAD1-like acylhydrolase, were examined. Bacterially expressed AtDLAH contained phospholipase A1 activity for catalysing the hydrolysis of phospholipids at the sn-1 position. However, AtDLAH displayed an even stronger preference for 1-lysophosphatidylcholine, 1-monodiacylglycerol, and phosphatidic acid, suggesting that AtDLAH is a sn-1-specific acylhydrolase. The AtDLAH gene was highly expressed in young seedlings, and its encoded protein was exclusively localized to the mitochondria. AtDLAH-overexpressing transgenic seeds (35S:AtDLAH) were markedly tolerant to accelerated-ageing treatment and thus had higher germination percentages than wild-type seeds. In contrast, the atdlah loss-of-function knockout mutant seeds were hypersusceptible to accelerated-ageing conditions. The 35S:AtDLAH seeds, as opposed to the atdlah seeds, exhibited a dark red staining pattern following tetrazolium treatment under both normal and accelerated-ageing conditions, suggesting that AtDLAH expression is positively correlated with seed viability. The enhanced viability of 35S:AtDLAH seeds was accompanied by more densely populated epidermal cells, lower levels of accumulated lipid hydroperoxides, and higher levels of polar lipids as compared with wild-type and atdlah mutant seeds. These results suggest that AtDLAH, a mitochondrial-localized sn-1-specific acylhydrolase, plays an important role in Arabidopsis seed viability.

Enrichment of Cytoplasmic RNA Granules from Arabidopsis Seedlings.

RNA granules (RGs) are membraneless intracellular compartments that play important roles in the post-transcriptional control of gene expression. Stress granules (SGs) are a type of RGs that form under environmental challenges and/or internal cellular stresses. Stress treatments lead to strong mRNAs translational inhibition and storage in SGs until the normal growth conditions are restored. Intriguingly, we recently showed that plant stress granules are associated with siRNA bodies, where the RDR6-mediated and transposon-derived siRNA biogenesis occurs ( Kim et al., 2021 ). This protocol provides a technical workflow for the enrichment of cytoplasmic RGs from Arabidopsis seedlings. We used the DNA methylation-deficient ddm1 mutant in our study, but the method can be applied to any other plant samples with strong RG formation. The resulting RG fractions can be further tested for either RNAs or proteins using RNA-seq and mass spectrometry-based proteomics.

High-Throughput Profiling of Extrachromosomal Linear DNAs of Long Terminal Repeat Retrotransposons by ALE-seq.

Extrachromosomal linear DNA (eclDNA) is the reverse-transcribed cDNA intermediate derived from long terminal repeat (LTR) transposable elements (TEs) (Cho et al., Nat Plants 5:26-33, 2018). Given that the eclDNAs are the final intermediate of LTR-TE life cycle prior to integration to the host chromosomes, their presence is considered a strong indication of active LTR retrotransposons (Cho et al., Nat Plants 5:26-33, 2018; Lanciano et al., PLoS Genet 13:e1006630, 2017). Here, we describe a method of amplification of LTR extrachromosomal DNA followed by sequencing (ALE-seq) which determines the 5' LTR sequences of eclDNAs. Briefly, ALE-seq consists of two steps of amplification, in vitro transcription of adaptor-ligated eclDNAs and subsequent reverse transcription to cDNAs primed at the conserved primer binding site (PBS) (Cho et al., Nat Plants 5:26-33, 2018). ALE-seq allows the high-throughput identification of novel LTR-TEs which are active in plants that could be potentially useful for crop biotechnology.