G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type–specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.

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To accurately measure gene expression using quantitative reverse transcription PCR (qRT-PCR), reliable reference gene(s) are required for data normalization. Corchorus capsularis, an annual herbaceous fiber crop with predominant biodegradability and renewability, has not been investigated for the stability of reference genes with qRT-PCR. In this study, 11 candidate reference genes were selected and their expression levels were assessed using qRT-PCR. To account for the influence of experimental approach and tissue type, 22 different jute samples were selected from abiotic and biotic stress conditions as well as three different tissue types. The stability of the candidate reference genes was evaluated using geNorm, NormFinder, and BestKeeper programs, and the comprehensive rankings of gene stability were generated by aggregate analysis. For the biotic stress and NaCl stress subsets, ACT7 and RAN were suitable as stable reference genes for gene expression normalization. For the PEG stress subset, UBC, and DnaJ were sufficient for accurate normalization. For the tissues subset, four reference genes TUBβ, UBI, EF1α, and RAN were sufficient for accurate normalization. The selected genes were further validated by comparing expression profiles of WRKY15 in various samples, and two stable reference genes were recommended for accurate normalization of qRT-PCR data. Our results provide researchers with appropriate reference genes for qRT-PCR in C. capsularis, and will facilitate gene expression study under these conditions.

An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase–hexahistidine–2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.

Strigolactones positively regulate defense against infection by root-knot nematode in tomato through a process that is dependent on MYC2 integrating SL, ABA, and JA signaling

Brassinosteroids (BRs) regulate plant development and stress response. Although much has been learned about their roles in plant development, the mechanisms by which BRs regulate plant stress tolerance remain unclear. Chilling is a major stress that adversely affects plant growth. Here, we report that BR positively regulates chilling tolerance in tomato. BR partial deficiency aggravated chilling-induced oxidized protein accumulation, membrane lipid peroxidation, and decrease of maximum quantum efficiency of photosystem II (Fv/Fm). By contrast, overexpression of BR biosynthetic gene Dwarf or treatment with 24-epibrassinolide (EBR) attenuated chilling-induced oxidative damages and resulted in an increase of Fv/Fm. BR increased transcripts of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and GLUTAREDOXIN (GRX) genes, and BR-induced chilling tolerance was associated with an increase in the ratio of reduced/oxidized 2-cysteine peroxiredoxin (2-Cys Prx) and activation of antioxidant enzymes. However, RBOH1-RNAi plants failed to respond to EBR as regards to the induction of GRX genes, activation of antioxidant capacity, and attenuation of chilling-induced oxidative damages. Furthermore, silencing of GRXS12 and S14 compromised EBR-induced increases in the ratio of reduced/oxidized 2-Cys Prx and activities of antioxidant enzymes. Our study suggests that BR enhances chilling tolerance through a signalling cascade involving RBOH1, GRXs, and 2-Cys Prx in tomato.

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BackgroundGenetic mapping and quantitative trait locus (QTL) detection are powerful methodologies in plant improvement and breeding. White jute (Corchorus capsularis L.) is an important industrial raw material fiber crop because of its elite characteristics. However, construction of a high-density genetic map and identification of QTLs has been limited in white jute due to a lack of sufficient molecular markers. The specific locus amplified fragment sequencing (SLAF-seq) strategy combines locus-specific amplification and high-throughput sequencing to carry out de novo single nuclear polymorphism (SNP) discovery and large-scale genotyping. In this study, SLAF-seq was employed to obtain sufficient markers to construct a high-density genetic map for white jute. Moreover, with the development of abundant markers, genetic dissection of fiber yield traits such as plant height was also possible. Here, we present QTLs associated with plant height that were identified using our newly constructed genetic linkage groups.ResultsAn F8 population consisting of 100 lines was developed. In total, 69,446 high-quality SLAFs were detected of which 5,074 SLAFs were polymorphic; 913 polymorphic markers were used for the construction of a genetic map. The average coverage for each SLAF marker was 43-fold in the parents, and 9.8-fold in each F8 individual. A linkage map was constructed that contained 913 SLAFs on 11 linkage groups (LGs) covering 1621.4 cM with an average density of 1.61 cM per locus. Among the 11 LGs, LG1 was the largest with 210 markers, a length of 406.34 cM, and an average distance of 1.93 cM between adjacent markers. LG11 was the smallest with only 25 markers, a length of 29.66 cM, and an average distance of 1.19 cM between adjacent markers. ‘SNP_only’ markers accounted for 85.54% and were the predominant markers on the map. QTL mapping based on the F8 phenotypes detected 11 plant height QTLs including one major effect QTL across two cultivation locations, with each QTL accounting for 4.14–15.63% of the phenotypic variance.ConclusionsTo our knowledge, the linkage map constructed here is the densest one available to date for white jute. This analysis also identified the first QTL in white jute. The results will provide an important platform for gene/QTL mapping, sequence assembly, genome comparisons, and marker-assisted selection breeding for white jute.