Transposable elements (TEs) are ubiquitous components of plant genomes that profoundly influence plant diversity, adaptation, and genome structure. Transposition of TEs is primarily suppressed by distinct DNA methylation systems. However, the distribution of DNA methylation at the level of individual TEs in plants remains poorly understood. Here, we address this question by generating per-base cytosine methylation maps of individual long terminal repeat retrotransposons (LTR-RTEs) for the large sunflower (Helianthus annuus L.) and the small Arabidopsis thaliana genomes. A. thaliana was selected as the model species, for which genome-wide DNA methylation profiles have been extensively characterized in prior studies. Our analysis revealed significant heterogeneity in methylation patterns both between and within individual LTR-RTE lineages. We also found that the sunflower genes harboring intact or fragmented LTR-RTE insertions exhibit altered DNA methylation and expression profiles, with intact LTR-RTE insertions enriched in stress-response and regulatory pathways. Our interspecies comparison of DNA methylation patterns indicates that methylation patterns are intrinsic features of LTR-RTE lineages, conserved across diverse plant species but influenced by factors such as insertion age, element length, and proximity to genes. Furthermore, we identified epigenetically distinct clusters of Tork and Athila sunflower elements corresponding to separate phylogenetic clades, suggesting a link between epigenetic regulation and the genetic diversity of plant LTR-RTEs.

Transposable elements (TEs) are major drivers of plant genome plasticity, but the immediate molecular consequences of new TE insertions remain poorly understood. In this study, we generated a wild-type Arabidopsis thaliana population with novel insertions of ONSEN retrotransposon to investigate early epigenomic and transcriptomic changes using whole-genome and cDNA nanopore sequencing. We found that novel ONSEN insertions were distributed non-randomly, with a strong preference for genic regions, particularly in chromatin enriched for H2A.Z, H3K27me3, and H3K4me2. Most full-length ONSEN insertions within genes were rapidly recognized and spliced out as new introns (intronization), thereby mitigating potential deleterious effects on transcript isoforms. In some cases, ONSEN insertions provided alternative transcription start or termination sites, generating novel transcript isoforms. Genome-wide methylation analysis revealed that new ONSEN copies were efficiently and precisely targeted by DNA methylation. Independently on the location of the original ONSEN element, the euchromatic and heterochromatic insertions display distinct methylation signatures, reflecting the action of different epigenetic pathways. In conclusion, our results demonstrate that DNA methylation and alternative splicing are effective control mechanisms safeguarding the plant genome and transcriptome integrity after retrotransposition burst.

Mobile elements, particularly long terminal repeat retrotransposons (LTR-RTEs), are abundant and dynamic components of plant genomes. Although viral infections are known to transcriptionally activate retrotransposons, it remains unclear whether such virus-induced activation leads to their mobilization. To address this question, we examined LTR-RTE activation in Arabidopsis thaliana, Brassica napus, and Nicotiana benthamiana following infection with the RNA viruses Tobacco rattle virus (TRV), Potato virus X (PVX), and Tobacco ringspot virus (TRSV). Nanopore cDNA sequencing revealed virus-specific transcriptional responses, with PVX uniquely triggering a strong transcriptional burst of diverse LTR-RTE families in N. benthamiana. To test the role of viral suppressors of RNA silencing (VSRs) in this process, we analyzed extrachromosomal circular DNA (eccDNA) from plants infected with TRV expressing the VSR P19. This analysis identified eccDNA derived from Ty3/Gypsy Galadriel elements, demonstrating that viral infection can promote not only retrotransposon transcription but also eccDNA production, which may indicate the ability of LTR-RTEs to transpose. These findings clearly illustrate that plant-virus interactions can induce not only changes in gene transcription, but also the activation of multiple retrotransposons, highlighting a potential evolutionary interface linking antiviral defense and transposon regulation.

Long Terminal Repeat (LTR) retrotransposons (LTR-RTEs) comprise up to 90% of some plant genomes and drive genome diversification through their amplification. Novel insertions arise during the final stages of the LTR-RTE life cycle, which depends on both LTR-RTE-encoded proteins and host cellular factors. The LTR-RTE elements require host transcriptional machinery for RNA production, followed by nuclear processing/export, translation, virus-like particle assembly, reverse transcription, and genomic integration. This review addresses the following question: What host proteins promote LTR-RTE transposition in plants? Our analysis of recent literature on host factors and cellular compartments implicated in the retrotransposition cycle reveals the extensive integration of LTR-RTEs into host processes. Nonetheless, the precise mechanisms remain poorly resolved, especially in plants with their rich repertoire of LTR-RTEs. We propose integrating plant mobilomics with transposition reporters, genome editing, synthetic biology, and interactomics to elucidate plant-specific mechanisms.

The rapid identification of allele variants in target genes is crucial for accelerating marker-assisted selection (MAS) in plant breeding. Although current high-throughput genotyping methods are efficient in detecting known polymorphisms, they are limited when multiple variant sites are scattered along the gene. This study presents a target amplicon sequencing approach using Oxford Nanopore Technologies (ONT-TAS) to rapidly sequence full-length genes and identify allele variants in sunflower and wheat collections. This procedure combines multiplex PCR and a rapid sequencing kit, significantly reducing the time and cost compared to previous methods. The efficiency of the approach was demonstrated by sequencing four genes (Ahasl1, Ahasl2, Ahasl3, and FAD2) in 40 sunflower genotypes and three genes (Ppo, Wx, and Lox) in 30 wheat genotypes. The ONT-TAS revealed a complete picture of SNPs and InDels distributed over the individual alleles, enabling rapid (4.5 h for PCR and sequencing) characterization of the genetic diversity of the target genes in the germplasm collections. The results showed a significant diversity of the Ahasl1/Ahasl3 and Wx-A/Lox-B genes in the sunflower and wheat collections, respectively. This method offers a high-throughput, cost-effective (USD 3.4 per gene) solution for genotyping and identifying novel allele variants in plant breeding programs.

LTR retrotransposons are widespread genomic elements that significantly impact genome structure and function. In Arabidopsis thaliana, the EVD LTR retrotransposon encodes a GAG protein essential for retrotransposon particle assembly. Here, we present a comprehensive analysis of the structural features, intracellular localization, and transcriptomic effects of the EVD GAG (evdGAG) protein. Using AlphaFold3, we identified canonical capsid (CA-NTD and CA-CTD) and nucleocapsid (NC) domains, with predicted disordered regions likely facilitating oligomerization. Transient expression of GFP-tagged evdGAG in protoplasts of A. thaliana and distant plant species (Nicotiana benthamiana and Helianthus annuus) revealed the formation of multiple large cytoplasmic aggregates resembling retrosomes, often localized near the nucleus. Stable overexpression of evdGAG in wild-type and ddm1 mutant backgrounds induced significant transcriptomic changes, including up-regulation of stress response and defense-related genes and downregulation of photosynthesis and chloroplast-associated pathways. Importantly, genes linked to stress granule formation were also up-regulated, suggesting a role for evdGAG in modulating cellular stress responses. Our findings provide novel insights into the cellular and molecular properties of plant retrotransposon GAG proteins and their influence on host gene expression.

Transposable elements (TEs) are a major force in the evolution of plant genomes. Differences in the transposition activities and landscapes of TEs can vary substantially, even in closely related species. Interspecific hybridization, a widely employed technique in tomato breeding, results in the creation of novel combinations of TEs from distinct species. The implications of this process for TE transposition activity have not been studied in modern cultivars. In this study, we used nanopore sequencing of extrachromosomal circular DNA (eccDNA) and identified two highly active Ty1/Copia LTR retrotransposon families of tomato (Solanum lycopersicum), called Salsa and Ketchup. Elements of these families produce thousands of eccDNAs under controlled conditions and epigenetic stress. EccDNA sequence analysis revealed that the major parts of eccDNA produced by Ketchup and Salsa exhibited low similarity to the S. lycopersicum genomic sequence. To trace the origin of these TEs, whole-genome nanopore sequencing and de novo genome assembly were performed. We found that these TEs occurred in a tomato breeding line via interspecific introgression from S. peruvianum. Our findings collectively show that interspecific introgressions can contribute to both genetic and phenotypic diversity not only by introducing novel genetic variants, but also by importing active transposable elements from other species.

Genetic variation in high molecular weight glutenin (HMW-GS) genes is tightly linked with the breadmaking quality of wheat. Hundreds of different alleles have been identified in HMW-GS genes worldwide. Such huge variability makes it difficult to distinguish them using conventional genotyping methods (for example, SDS-PAGE, SNP detection, etc.). Here, we exploited the nanopore amplicon sequencing technique (Amplicon-Seq) to uncover genetic variants distributed along the full-length sequence of six HMW-GSs, including the promoter and protein-coding regions. We analyzed 23 wheat accessions for allelic variants of HMW-GSs using the Amplicon-Seq and SDS-PAGE methods. We obtained sufficient (>50×) target gene coverage by ONT reads in just one hour. Using the obtained data, we identified numerous single nucleotide polymorphisms and InDels in the protein coding and promoter regions. Moreover, Amplicon-Seq allowed for the identification of new alleles (Glu-A1x1-T) of the Glu-1Ax gene that could not be recognized by SDS-PAGE. Collectively, our results showed that Amplicon-Seq is a rapid, multiplexed, and efficient method for high-throughput genotyping of full-length genes in large and complex genomes. This opens new avenues for the assessment of target gene variation to select novel alleles and create unique combinations of desirable traits in plant breeding programs.

Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62–91

Одним из перспективных методов для создания таких модификаций является вирус-опосредованный сайленсинг генов (VIGS), который может быть применён для подавления генов, связанных с метилированием ДНК и сайленсингом LTRретротранспозонов (LTR-RT) [3].

Plant genomes possess numerous transposable element (TE) insertions that have occurred during evolution. Most TEs are silenced or diverged; therefore, they lose their ability to encode proteins and are transposed in the genome. Knowledge of active plant TEs and TE-encoded proteins essential for transposition and evasion of plant cell transposon silencing mechanisms remains limited. This study investigated active long terminal repeat (LTR) retrotransposons (RTEs) in sunflowers (Helianthus annuus), revealing heterogeneous and phylogenetically distinct RTEs triggered by epigenetic changes and heat stress. Many of these RTEs belong to three distinct groups within the Tekay clade, showing significant variations in chromosomal insertion distribution. Through protein analysis of these active RTEs, it was found that Athila RTEs and Tekay group 2 elements possess additional open reading frames (aORFs). The aORF-encoded proteins feature a transposase domain, a transmembrane domain, and nuclear localization signals. The aORF proteins of the Tekay subgroup exhibited remarkable conservation among over 500 Tekay members, suggesting their functional importance in RTE mobility. The predicted 3D structure of the sunflower Tekay aORF protein showed significant homology with Tekay proteins in rice, maize, and sorghum. Additionally, the structural features of aORF proteins resemble those of plant DRBM-containing proteins, suggesting their potential role in RNA-silencing modulation. These findings offer insights into the diversity and activity of sunflower RTEs, emphasizing the conservation and structural characteristics of aORF-encoded proteins.

CRISPR-Cas системы – прокариотический инструмент для защиты от фаговых инфекций. Cas-9 белок обладает эндонуклеазной активностью и в комплексе с гидовой молекулой способен распознавать чужеродные геномные последовтельности и разрезать их, тем самым предотвращая распространение инфекции. Эта система показала свою эффективность и в эукариотических организмах, став перспективным инструментом для геномного редактирования.

AbstractTransposable elements (TEs) are a major force in the evolution of plant genomes. Differences in the transposition activities and landscapes of TEs can vary substantially, even in closely related species. Interspecific hybridization, a widely employed technique in tomato breeding, results in the creation of novel combinations of TEs from distinct species. The implications of this process for TE transposition activity have not been studied in modern cultivars. In this study, we used nanopore sequencing of extrachromosomal circular DNA (eccDNA) and identified two highly active Ty1/CopiaLTR retrotransposon families of tomato (Solanum lycopersicum), calledSalsaandKetchup. Elements of these families produce thousands of eccDNAs under controlled conditions and epigenetic stress. EccDNA sequence analysis revealed that the major parts of eccDNA produced byKetchupandSalsaexhibited low similarity to theS. lycopersicumgenomic sequence. To trace the origin of these TEs, whole-genome nanopore sequencing and de novo genome assembly were performed. We found that these TEs occurred in a tomato breeding line via interspecific introgression fromS. peruvianum. Our findings collectively show that interspecific introgressions can contribute to both genetic and phenotypic diversity not only by introducing novel genetic variants, but also by importing active transposable elements from other species.

Transposable element insertions (TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large, complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that reflect mobilome dynamics are difficult to track using short reads. To address these challenges, we combined Cas9-targeted Nanopore sequencing (CANS) with the novel pipeline NanoCasTE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADÉ ( EVD ) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40x sequence coverage. Analysis of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1 genome uncovered the crucial role of DNA methylation in shaping EVD insertion preference. We also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSEN s. Finally, we detected hypomethylation of novel somatic insertions for two ONSEN s. CANS and NanoCasTE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.

Мобильные элементы обнаружены почти во всех организмах и часто составляют основную часть генома. Они (МЭ) представляют собой фрагменты ДНК способные перемещаться по геному, и интегрироваться в новые места, приводя к увеличению генетического разнообразия и получению новых признаков, включающих вариации окраски плодовых и бобовых, а также приобретение устойчивости к биотическим и абиотическим факторам. Эти признаки могут стать ценными материалом для селекции сельскохозяйственных растений.

Мягкая пшеница (Triticum aestivum L.) – важнейшая продовольственная и кормовая сельскохозяйственная культура в мире. По данным ФАО в 2021 году пшеница возделывалась на площади 220,8 млн га, являясь лидером по этому показателю, а валовый сбор составил 770,9 млн т при средней урожайности в 3,5 т/га (http://faostat.fao.org/). Высокая востребованность этой культуры связана с тем, что её зерно является основным сырьём для хлебопекарной промышленности. При этом ценность используемого для хлебопечения зерна зависит в первую очередь от качества и количества содержащегося в нем белка, и в особенности клейковины (Савельев, 2015).

Genetic diversity is a key factor for plant breeding. The birth of novel genic and genomic variants is also crucial for plant adaptation in nature. Therefore, the genomes of almost all living organisms possess natural mutagenic mechanisms. Transposable elements (TEs) are a major mutagenic force driving genetic diversity in wild plants and modern crops. The relatively rare TE transposition activity during the thousand-year crop domestication process has led to the phenotypic diversity of many cultivated species. The utilization of TE mutagenesis by artificial and transient acceleration of their activity in a controlled mode is an attractive foundation for a novel type of mutagenesis called TE-mediated biological mutagenesis. Here, I focus on TEs as mutagenic sources for plant breeding and discuss existing and emerging transgene-free approaches for TE activation in plants. Furthermore, I also review the non-randomness of TE insertions in a plant genome and the molecular and epigenetic factors involved in shaping TE insertion preferences. Additionally, I discuss the molecular mechanisms that prevent TE transpositions in germline plant cells (e.g., meiocytes, pollen, egg and embryo cells, and shoot apical meristem), thereby reducing the chances of TE insertion inheritance. Knowledge of these mechanisms can expand the TE activation toolbox using novel gene targeting approaches. Finally, the challenges and future perspectives of plant populations with induced novel TE insertions (iTE plant collections) are discussed.

Геном большинства сельскохозяйственных растений в значительной мере представлен мобильными элементами (МЭ, транспозоны), способными к перемещению и пролиферации внутри генома, что также может приводить к появлению генов нового функционального значения. Мобилом растений в большей степени представлен LTR-ретротранспозонами, активность которых сыграла важную роль в адаптации и эволюции растений (Lisch, 2013).

Достижения в технологии секвенирования следующего поколения привели к аккумулированию большого объема геномных данных, открыв новые перспективы для поиска функциональных генов. Для установления и изучения функций генов могут быть использованы методы обратной генетики, например с помощью сайленсинга их экспрессии. Вирус-индуцированнный сайленсинг генов (англ. Virus-Induced Gene Silencing, VIGS) – это метод, основанный на пост-транскрипционном подавлении генов (англ. - Post-Transcriptional Gene Silencing, PTGS), реализуемый с применением естественного механизма РНК-интерференции, используемого растениями для борьбы с вирусными инфекциями.