A tripartite rheostat controls self-regulated host plant resistance to insects.

Plants deploy receptor-like kinases and nucleotide-binding leucine-rich repeat receptors to confer host plant resistance (HPR) to herbivores1. These gene-for-gene interactions between insects and their hosts have been proposed for more than 50 years2. However, the molecular and cellular mechanisms that underlie HPR have been elusive, as the identity and sensing mechanisms of insect avirulence effectors have remained unknown. Here we identify an insect salivary protein perceived by a plant immune receptor. The BPH14-interacting salivary protein (BISP) from the brown planthopper (Nilaparvata lugens Stål) is secreted into rice (Oryza sativa) during feeding. In susceptible plants, BISP targets O. satvia RLCK185 (OsRLCK185; hereafter Os is used to denote O. satvia-related proteins or genes) to suppress basal defences. In resistant plants, the nucleotide-binding leucine-rich repeat receptor BPH14 directly binds BISP to activate HPR. Constitutive activation of Bph14-mediated immunity is detrimental to plant growth and productivity. The fine-tuning of Bph14-mediated HPR is achieved through direct binding of BISP and BPH14 to the selective autophagy cargo receptor OsNBR1, which delivers BISP to OsATG8 for degradation. Autophagy therefore controls BISP levels. In Bph14 plants, autophagy restores cellular homeostasis by downregulating HPR when feeding by brown planthoppers ceases. We identify an insect saliva protein sensed by a plant immune receptor and discover a three-way interaction system that offers opportunities for developing high-yield, insect-resistant crops.

Cell type-specific attenuation of brassinosteroid signaling precedes stomatal asymmetric cell division.

In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

LeafNet: a tool for segmenting and quantifying stomata and pavement cells.

Stomata play important roles in gas and water exchange in leaves. The morphological features of stomata and pavement cells are highly plastic and are regulated during development. However, it is very laborious and time-consuming to collect accurate quantitative data from the leaf surface by manual phenotyping. Here, we introduce LeafNet, a tool that automatically localizes stomata, segments pavement cells (to prepare them for quantification), and reports multiple morphological parameters for a variety of leaf epidermal images, especially bright-field microscopy images. LeafNet employs a hierarchical strategy to identify stomata using a deep convolutional network and then segments pavement cells on stomata-masked images using a region merging method. LeafNet achieved promising performance on test images for quantifying different phenotypes of individual stomata and pavement cells compared with six currently available tools, including StomataCounter, Cellpose, PlantSeg, and PaCeQuant. LeafNet shows great flexibility, and we improved its ability to analyze bright-field images from a broad range of species as well as confocal images using transfer learning. Large-scale images of leaves can be efficiently processed in batch mode and interactively inspected with a graphic user interface or a web server (https://leafnet.whu.edu.cn/). The functionalities of LeafNet could easily be extended and will enhance the efficiency and productivity of leaf phenotyping for many plant biologists.

Shaping brassinosteroid signaling through scaffold proteins.

Cellular responses to internal and external stimuli are orchestrated by intricate intracellular signaling pathways. To ensure an efficient and specific information flow, cells employ scaffold proteins as critical signaling organizers. With the ability to bind multiple signaling molecules, scaffold proteins can sequester signaling components within specific subcellular domains or modulate the efficiency of signal transduction. Scaffolds can also tune the output of signaling pathways by serving as regulatory targets. This review focuses on scaffold proteins associated with the plant GLYCOGEN SYNTHASE KINASE3-like kinase, BRASSINOSTEROID-INSENSITIVE2 (BIN2) that serve as a key negative regulator of brassinosteroid (BR) signaling. Here we summarize the current understanding of how scaffold proteins actively shape BR signaling outputs and crosstalk in plant cells via interactions with BIN2.

The pentatricopeptide repeat protein DG1 promotes the transition to bilateral symmetry during Arabidopsis embryogenesis through GUN1-mediated plastid signals.

During Arabidopsis embryogenesis, the transition of the embryo's symmetry from radial to bilateral between the globular and heart stage is a crucial event, involving the formation of cotyledon primordia and concurrently the establishment of a shoot apical meristem (SAM). However, a coherent framework of how this transition is achieved remains to be elucidated. In this study, we investigated the function of DELAYED GREENING 1 (DG1) in Arabidopsis embryogenesis using a newly identified dg1-3 mutant. The absence of chloroplast-localized DG1 in the mutants led to embryos being arrested at the globular or heart stage, accompanied by an expansion of WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) expression. This finding pinpoints the essential role of DG1 in regulating the transition to bilateral symmetry. Furthermore, we showed that this regulation of DG1 may not depend on its role in plastid RNA editing. Nevertheless, we demonstrated that the DG1 function in establishing bilateral symmetry is genetically mediated by GENOMES UNCOUPLED 1 (GUN1), which represses the transition process in dg1-3 embryos. Collectively, our results reveal that DG1 functionally antagonizes GUN1 to promote the transition of the Arabidopsis embryo's symmetry from radial to bilateral and highlight the role of plastid signals in regulating pattern formation during plant embryogenesis.

GhCASPL1 regulates secondary cell wall thickening in cotton fibers by stabilizing the cellulose synthase complex on the plasma membrane.

Cotton (Gossypium hirsutum) fibers are elongated single cells that rapidly accumulate cellulose during secondary cell wall (SCW) thickening, which requires cellulose synthase complex (CSC) activity. Here, we describe the CSC-interacting factor CASPARIAN STRIP MEMBRANE DOMAIN-LIKE1 (GhCASPL1), which contributes to SCW thickening by influencing CSC stability on the plasma membrane. GhCASPL1 is preferentially expressed in fiber cells during SCW biosynthesis and encodes a MARVEL domain protein. The ghcaspl1 ghcaspl2 mutant exhibited reduced plant height and produced mature fibers with fewer natural twists, lower tensile strength, and a thinner SCW compared to the wild type. Similarly, the Arabidopsis (Arabidopsis thaliana) caspl1 caspl2 double mutant showed a lower cellulose content and thinner cell walls in the stem vasculature than the wild type but normal plant morphology. Introducing the cotton gene GhCASPL1 successfully restored the reduced cellulose content of the Arabidopsis caspl1 caspl2 mutant. Detergent treatments, ultracentrifugation assays, and enzymatic assays showed that the CSC in the ghcaspl1 ghcaspl2 double mutant showed reduced membrane binding and decreased enzyme activity compared to the wild type. GhCASPL1 binds strongly to phosphatidic acid (PA), which is present in much higher amounts in thickening fiber cells compared to ovules and leaves. Mutating the PA-binding site in GhCASPL1 resulted in the loss of its colocalization with GhCesA8, and it failed to localize to the plasma membrane. PA may alter membrane structure to facilitate protein-protein interactions, suggesting that GhCASPL1 and PA collaboratively stabilize the CSC. Our findings shed light on CASPL functions and the molecular machinery behind SCW biosynthesis in cotton fibers.

Probiotics-Fermented Grifola frondosa Total Active Components: Better Antioxidation and Microflora Regulation for Alleviating Alcoholic Liver Damage in Mice.

Alcoholic liver damage is caused by long-term drinking, and it further develops into alcoholic liver diseases. In this study, we prepared a probiotic fermentation product of Grifola frondosa total active components (PFGF) by fermentation with Lactobacillus acidophilus, Lactobacillus rhamnosus, and Pediococcus acidilactici. After fermentation, the total sugar and protein content in the PFGF significantly decreased, while the lactic acid level and antioxidant activity of the PFGF increased. Afterward, we investigated the alleviating effect of PFGF on alcoholic liver injury in alcohol-fed mice. The results showed that the PFGF intervention reduced the necrosis of the liver cells, attenuated the inflammation of the liver and intestines, restored the liver function, increased the antioxidant factors of the liver, and maintained the cecum tissue barrier. Additionally, the results of the 16S rRNA sequencing analysis indicated that the PFGF intervention increased the relative abundance of beneficial bacteria, such as Lactobacillus, Ruminococcaceae, Parabacteroids, Parasutterella, and Alistipes, to attenuate intestinal inflammation. These results demonstrate that PFGF can potentially alleviate alcoholic liver damage by restoring the intestinal barrier and regulating the intestinal microflora.

MaGenDB: a functional genomics hub for Malvaceae plants.

Malvaceae is a family of flowering plants containing many economically important plant species including cotton, cacao and durian. Recently, the genomes of several Malvaceae species have been decoded, and many omics data were generated for individual species. However, no integrative database of multiple species, enabling users to jointly compare and analyse relevant data, is available for Malvaceae. Thus, we developed a user-friendly database named MaGenDB (http://magen.whu.edu.cn) as a functional genomics hub for the plant community. We collected the genomes of 13 Malvaceae species, and comprehensively annotated genes from different perspectives including functional RNA/protein element, gene ontology, KEGG orthology, and gene family. We processed 374 sets of diverse omics data with the ENCODE pipelines and integrated them into a customised genome browser, and designed multiple dynamic charts to present gene/RNA/protein-level knowledge such as dynamic expression profiles and functional elements. We also implemented a smart search system for efficiently mining genes. In addition, we constructed a functional comparison system to help comparative analysis between genes on multiple features in one species or across closely related species. This database and associated tools will allow users to quickly retrieve large-scale functional information for biological discovery.

PROTEIN S-ACYL TRANSFERASE 13/16 modulate disease resistance by S-acylation of the nucleotide binding, leucine-rich repeat protein R5L1 in Arabidopsis.

Nucleotide binding, leucine-rich repeat (NB-LRR) proteins are critical for disease resistance in plants, while we do not know whether S-acylation of these proteins plays a role during bacterial infection. We identified 30 Arabidopsis mutants with mutations in NB-LRR encoding genes from the Nottingham Arabidopsis Stock Center and characterized their contribution to the plant immune response after inoculation with Pseudomonas syringae pv tomato DC3000 (Pst DC3000). Of the five mutants that were hyper-susceptible to the pathogen, three (R5L1, R5L2 and RPS5) proteins contain the conserved S-acylation site in the N-terminal coiled-coil (CC) domain. In wild-type (WT) Arabidopsis plants, R5L1 was transcriptionally activated upon pathogen infection, and R5L1 overexpression lines had enhanced resistance. Independent experiments indicated that R5L1 localized at the plasma membrane (PM) via S-acylation of its N-terminal CC domain, which was mediated by PROTEIN S-ACYL TRANSFERASE 13/16 (PAT13, PAT16). Modification of the S-acylation site reduced its affinity for binding the PM, with a consequent significant reduction in bacterial resistance. PM localization of R5L1 was significantly reduced in pat13 and pat16 mutants, similar to what was found for WT plants treated with 2-bromopalmitate, an S-acylation-blocking agent. Transgenic plants expressing R5L1 in the pat13 pat16 double mutant showed no enhanced disease resistance. Overexpression of R5L1 in WT Arabidopsis resulted in substantial accumulation of reactive oxygen species after inoculation with Pst DC3000; this effect was not observed with a mutant R5L1 carrying a mutated S-acylation site. Our data suggest that PAT13- and PAT16-mediated S-acylation of R5L1 is crucial for its membrane localization to activate the plant defense response.

The bioequivalence of a single dose of a generic solifenacin succinate tablet or Vesicare in fasting or postprandial healthy Chinese adults.

OBJECTIVE: The present study evaluated the bioequivalence of the test preparation (5 mg generic solifenacin succinate tablet, produced by Jiangsu Deyuan Pharmaceutical Co., Ltd.) or the reference preparation (5 mg solifenacin succinate tablets with the trade name Vesicare, produced by Astellas Pharma Europe B.V.) in either fasting or postprandial states in healthy Chinese subjects. MATERIALS AND METHODS: The present study was designed as an open-label, randomized, single-center, single-dose, dual-cycle, dual-crossover, fasting/postprandial study. 56 healthy Chinese subjects (28 each in the fasting and postprandial groups) were enrolled. WinNolin software (version 7.0 or above) was used to calculate the pharmacokinetic parameters. RESULTS: In the fasting and postprandial group, pharmacokinetic analysis and bioequivalence analysis were carried out based on the blood concentration-time data. The 90% confidence interval of the geometric mean ratio of Cmax, AUC0-t, and AUC0-∞ of the test preparation and the reference preparation were both within the acceptance range of 80.00 - 125.00%. CONCLUSION: A single administration of a 5-mg tablet of either the test preparation, generic solifenacin succinate, or the reference preparation, solifenacin succinate with the brand-name Vesicare, was safe and bioequivalent in healthy Chinese subjects in either fasting or postprandial states according to the criteria of bioequivalence of the State Drug Administration of China.

Two-Step Functional Innovation of the Stem-Cell Factors WUS/WOX5 during Plant Evolution.

WUS and WOX5, which are expressed, respectively, in the organizing center (OC) and the quiescent center (QC), are essential for shoot/root apical stem-cell maintenance in flowering plants. However, little is known about how these stem-cell factors evolved their functions in flowering plants. Here, we show that the WUS/WOX5 proteins acquired two distinct capabilities by a two-step functional innovation process in the course of plant evolution. The first-step is the apical stem-cell maintenance activity of WUS/WOX5, which originated in the common ancestor of ferns and seed plants, as evidenced by the interspecies complementation experiments, showing that ectopic expression of fern Ceratopteris richardii WUS-like (CrWUL) surrounding OC/QC, or exclusive OC-/QC-expressed gymnosperms/angiosperms WUS/WOX5 in Arabidopsis wus-1 and wox5-1 mutants, could rescue their phenotypes. The second-step is the intercellular mobility that emerged in the common ancestor of seed plants after divergence from the ferns. Evidence for this includes confocal imaging of GFP fusion proteins, showing that WUS/WOX5 from seed plants, rather than from the fern CrWUL, can migrate into cells adjacent to the OC/QC. Evolutionary analysis showed that the WUS-like gene was duplicated into two copies prior to the divergence of gymnosperms/angiosperms. Then the two gene copies (WUS and WOX5) have undergone similar levels of purifying selection, which is consistent with their conserved functions in angiosperm shoot/root stem-cell maintenance and floral organ formation. Our results highlight the critical roles and the essential prerequisites that the two-step functional innovation of these genes performs and represents in the origin of flowering plants.

Genome-scale analysis of the cotton KCS gene family revealed a binary mode of action for gibberellin A regulated fiber growth.

Production of ß-ketoacyl-CoA, which is catalyzed by 3-ketoacyl-CoA synthase (KCS), is the first step in very long chain fatty acid (VLCFA) biosynthesis. Here we identified 58 KCS genes from Gossypium hirsutum, 31 from G. arboreum and 33 from G. raimondii by searching the assembled cotton genomes. The gene family was divided into the plant-specific FAE1-type and the more general ELO-type. KCS transcripts were widely expressed and 32 of them showed distinct subgenome-specific expressions in one or more cotton tissues/organs studied. Six GhKCS genes rescued the lethality of elo2Δelo3Δ yeast double mutant, indicating that this gene family possesses diversified functions. Most KCS genes with GA-responsive elements (GAREs) in the promoters were significantly upregulated by gibberellin A3 (GA). Exogenous GA3 not only promoted fiber length, but also increased the thickness of cell walls significantly. GAREs present also in the promoters of several cellulose synthase (CesA) genes required for cell wall biosynthesis and they were all induced significantly by GA3 . Because GA treatment resulted in longer cotton fibers with thicker cell walls and higher dry weight per unit cell length, we suggest that it may regulate fiber elongation upstream of the VLCFA-ethylene pathway and also in the downstream steps towards cell wall synthesis.

Vascular calcification in chronic kidney disease is induced by bone morphogenetic protein-2 via a mechanism involving the Wnt/ß-catenin pathway.

BACKGROUND: Vascular calcification (VC), in which vascular smooth muscle cells (VSMCs) undergo a phenotypic transformation into osteoblast-like cells, is one of the emergent risk factors for the accelerated atherosclerosis process characteristic of chronic kidney disease (CKD). Phosphate is an important regulator of VC. METHODS: The expression of different smooth muscle cell or osteogenesis markers in response to high concentrations of phosphate or exogenous bone morphogenetic protein 2 (BMP-2) was examined by qRT-PCR and western blotting in rat VSMCs. Osteocalcin secretion was measured by radioimmunoassay. Differentiation and calcification of VSMCs were examined by alkaline phosphatase (ALP) activity assay and Alizarin staining. Short hairpin RNA-mediated silencing of ß-catenin was performed to examine the involvement of Wnt/ß-catenin signaling in VSMC calcification and osteoblastic differentiation induced by high phosphate or BMP-2. Apoptosis was determined by TUNEL assay and immunofluorescence imaging. RESULTS: BMP-2 serum levels were significantly higher in CKD patients than in controls. High phosphate concentrations and BMP-2 induced VSMC apoptosis and upregulated the expression of ß-catenin, Msx2, Runx2 and the phosphate cotransporter Pit1, whereas a BMP-2 neutralization antibody reversed these effects. Knockdown of ß-catenin abolished the effect of high phosphate and BMP-2 on VSMC apoptosis and calcification. CONCLUSIONS: BMP-2 plays a crucial role in calcium deposition in VSMCs and VC in CKD patients via a mechanism involving the Wnt/ß-catenin pathway.

A Genome-Wide Alternative Splicing Analysis of Gossypium arboreum and Gossypium raimondii During Fiber Development.

Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism that contributes to proteome complexity and versatility in different plant species. However, detailed AS exploration in diploid cotton during fiber development has not been reported. In this study, we comparatively analyzed G. arboreum and G. raimondii AS events during fiber development using transcriptome data and identified 9690 and 7617 AS events that were distributed in 6483 and 4859 genes, respectively. G. arboreum had more AS genes and AS events than G. raimondii, and most AS genes were distributed at both ends of all 13 chromosomes in both diploid cotton species. Four major AS types, including IR, SE, A3SS, and A5SS, were all experimentally validated through RT-PCR assays. G. arboreum and G. raimondii had only 1888 AS genes in common, accounting for one-third and one-half of the total number of AS genes, respectively. Furthermore, we found a lysine-specific demethylase coding gene with a different AS mechanism in G. arboreum and G. raimondii, in which AS isoforms lacked part of a key conserved domain. Our findings may provide new directions for the discovery of functional genes involved in cotton species differentiation.

Upgraded durian genome reveals the role of chromosome reshuffling during ancestral karyotype evolution, lignin biosynthesis regulation, and stress tolerance.

Durian (Durio zibethinus) is a tropical fruit that has a unique flavor and aroma. It occupies a significant phylogenetic position within the Malvaceae family. Extant core-eudicot plants are reported to share seven ancestral karyotypes that have undergone reshuffling, resulting in an abundant genomic diversity. However, the ancestral karyotypes of the Malvaceae family, as well as the evolution trajectory leading to the 28 chromosomes in durian, remain poorly understood. Here, we report the high-quality assembly of the durian genome with comprehensive comparative genomic analyses. By analyzing the collinear blocks between cacao and durian, we inferred 11 Malvaceae ancestral karyotypes. These blocks were present in a single-copy form in cacao and mainly in triplicates in durian, possibly resulting from a recent whole genome triplication (WGT) event that led to hexaploidization of the durian genome around 20 (17-24) million years ago. A large proportion of the duplicated genes in durian, such as those involved in the lignin biosynthesis module for phenylpropane biosynthesis, are derived directly from whole genome duplication, which makes it an important force in reshaping its genomic architecture. Transcriptome studies have revealed that genes involved in feruloyl-CoA formations were highly preferentially expressed in fruit peels, indicating that the thorns produced on durian fruit may comprise guaiacyl and syringyl lignins. Among all the analyzed transcription factors (TFs), members of the heat shock factor family (HSF) were the most significantly upregulated under heat stress. All subfamilies of genes encoding heat shock proteins (HSPs) in the durian genome appear to have undergone expansion. The potential interactions between HSF Dzi05.397 and HSPs were examined and experimentally verified. Our study provides a high-quality durian genome and reveals the reshuffling mechanism of ancestral Malvaceae chromosomes to produce the durian genome. We also provide insights into the mechanism underlying lignin biosynthesis and heat stress tolerance.

GhATL68b regulates cotton fiber cell development by ubiquitinating the enzyme required for ß-oxidation of polyunsaturated fatty acids.

E3 ligases are key enzymes required for protein degradation. Here, we identified a C3H2C3 RING domain-containing E3 ubiquitin ligase gene named GhATL68b. It is preferentially and highly expressed in developing cotton fiber cells and shows greater conservation in plants than in animals or archaea. The four orthologous copies of this gene in various diploid cottons and eight in the allotetraploid G. hirsutum were found to have originated from a single common ancestor that can be traced back to Chlamydomonas reinhardtii at about 992 million years ago. Structural variations in the GhATL68b promoter regions of G. hirsutum, G. herbaceum, G. arboreum, and G. raimondii are correlated with significantly different methylation patterns. Homozygous CRISPR-Cas9 knockout cotton lines exhibit significant reductions in fiber quality traits, including upper-half mean length, elongation at break, uniformity, and mature fiber weight. In vitro ubiquitination and cell-free protein degradation assays revealed that GhATL68b modulates the homeostasis of 2,4-dienoyl-CoA reductase, a rate-limiting enzyme for the ß-oxidation of polyunsaturated fatty acids (PUFAs), via the ubiquitin proteasome pathway. Fiber cells harvested from these knockout mutants contain significantly lower levels of PUFAs important for production of glycerophospholipids and regulation of plasma membrane fluidity. The fiber growth defects of the mutant can be fully rescued by the addition of linolenic acid (C18:3), the most abundant type of PUFA, to the ovule culture medium. This experimentally characterized C3H2C3 type E3 ubiquitin ligase involved in regulating fiber cell elongation may provide us with a new genetic target for improved cotton lint production.

Arabidopsis PDLP7 modulated plasmodesmata function is related to BG10-dependent glucosidase activity required for callose degradation.

The microdomains of plasmodesmata, specialized cell-wall channels responsible for communications between neighboring cells, are composed of various plasmodesmata-located proteins (PDLPs) and lipids. Here, we found that, among all PDLP or homologous proteins in Arabidopsis thaliana genome, PDLP5 and PDLP7 possessed a C-terminal sphingolipid-binding motif, with the latter being the only member that was significantly upregulated upon turnip mosaic virus and cucumber mosaic virus infections. pdlp7 mutant plants exhibited significantly reduced callose deposition, larger plasmodesmata diameters, and faster viral transmission. These plants exhibited increased glucosidase activity but no change in callose synthase activity. PDLP7 interacted specifically with glucan endo-1,3-ß-glucosidase 10 (BG10). Consistently, higher levels of callose deposition and slower virus transmission in bg10 mutants were observed. The interaction between PDLP7 and BG10 was found to depend on the presence of the Gnk2-homologous 1 (GnK2-1) domain at the N terminus of PDLP7 with Asp-35, Cys-42, Gln-44, and Leu-116 being essential. In vitro supplementation of callose was able to change the conformation of the GnK2-1 domain. Our data suggest that the GnK2-1 domain of PDLP7, in conjunction with callose and BG10, plays a key role in plasmodesmata opening and closure, which is necessary for intercellular movement of various molecules.

Actively Expressed Intergenic Genes Generated by Transposable Element Insertions in Gossypium hirsutum Cotton.

The genomes and annotated genes of allotetraploid cotton Gossypium hirsutum have been extensively studied in recent years. However, the expression, regulation, and evolution of intergenic genes (ITGs) have not been completely deciphered. In this study, we identified a novel set of actively expressed ITGs in G. hirsutum cotton, through transcriptome profiling based on deep sequencing data, as well as chromatin immunoprecipitation, followed by sequencing (ChIP-seq) of histone modifications and how the ITGs evolved. Totals of 17,567 and 8249 ITGs were identified in G. hirsutum and Gossypium arboreum, respectively. The expression of ITGs in G. hirsutum was significantly higher than that in G. arboreum. Moreover, longer exons were observed in G. hirsutum ITGs. Notably, 42.3% of the ITGs from G. hirsutum were generated by the long terminal repeat (LTR) insertions, while their proportion in genic genes was 19.9%. The H3K27ac and H3K4me3 modification proportions and intensities of ITGs were equivalent to genic genes. The H3K4me1 modifications were lower in ITGs. Additionally, evolution analyses revealed that the ITGs from G. hirsutum were mainly produced around 6.6 and 1.6 million years ago (Mya), later than the pegged time for genic genes, which is 7.0 Mya. The characterization of ITGs helps to elucidate the evolution of cotton genomes and shed more light on their biological functions in the transcriptional regulation of eukaryotic genes, along with the roles of histone modifications in speciation and diversification.

A telomere-to-telomere cotton genome assembly reveals centromere evolution and a Mutator transposon-linked module regulating embryo development.

Assembly of complete genomes can reveal functional genetic elements missing from draft sequences. Here we present the near-complete telomere-to-telomere and contiguous genome of the cotton species Gossypium raimondii. Our assembly identified gaps and misoriented or misassembled regions in previous assemblies and produced 13 centromeres, with 25 chromosomal ends having telomeres. In contrast to satellite-rich Arabidopsis and rice centromeres, cotton centromeres lack phased CENH3 nucleosome positioning patterns and probably evolved by invasion from long terminal repeat retrotransposons. In-depth expression profiling of transposable elements revealed a previously unannotated DNA transposon (MuTC01) that interacts with miR2947 to produce trans-acting small interfering RNAs (siRNAs), one of which targets the newly evolved LEC2 (LEC2b) to produce phased siRNAs. Systematic genome editing experiments revealed that this tripartite module, miR2947-MuTC01-LEC2b, controls the morphogenesis of complex folded embryos characteristic of Gossypium and its close relatives in the cotton tribe. Our study reveals a trans-acting siRNA-based tripartite regulatory pathway for embryo development in higher plants.