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Belanger, S.

Publications and source records attributed to Belanger, S..

16 recordsLinked to original sources

The Annotated Blueprint: Integrated Functional Genomic Resources for a Model Tetraploid Wheat Triticum turgidum cv. Kronos

Triticum turgidum cv. Kronos is a tetraploid wheat cultivar that underpins one of the richest community platforms for functional genomics. Over the past decade, about 3,000 exome- and promoter-capture datasets, linked to mutagenized seed stocks, and transcriptomic and phenotypic resources have accumulated, yet the absence of a reference genome has constrained their impact. Here, we present a chromosome-scale reference genome of Kronos with high-confidence annotations, including manual curation of over 1,000 disease resistance (NLR) genes. This reference revealed previously hidden NLR diversity and clarified their genomic organization at chromosomal ends. Re-analysis of exome- and promoter-capture datasets enabled high-resolution mutation discovery in genes and regulatory regions that were previously inaccessible, uncovering the full standing variation present in Kronos mutant lines. We further re-curated transcriptomic and small RNA datasets, generating improved, genome-wide maps of microRNAs and phasiRNAs important for wheat development. Collectively, these resources elevate Kronos to reference quality and establish it as a versatile platform for functional and translational wheat research.

plant biology↗

Distinct classes of 21 and 24-nt phasiRNAs suggest diverse mechanisms of biogenesis and function in rice anther development

PhasiRNAs (phased small interfering RNAs) are a major class of plant small RNAs known to be key regulators in male reproductive development of maize (Zea mays) and rice (Oryza sativa), among other plants. Earlier research focused primarily on premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNAs, while new studies uncovered a premeiotic class of 24-nt phasiRNAs. The biogenesis and function of these phasiRNAs remain unclear. We conducted an integrative analysis combining small RNA (sRNA) sequencing and transcriptomic profiling of sRNA-associated genes across ten developmental stages of anther in Kitaake rice to map associations between sRNA-related genes and phasiRNA classes. We identified previously undescribed classes of postmeiotic 21-nucleotide (nt) and 24-nt phasiRNA-producing loci and characterized their unique accumulation patterns. Additionally, our findings reveal distinct nucleotide composition and register accumulation among the phasiRNA classes, suggesting the presence of diverse mechanisms of biogenesis and function. Our results provide new insights into the regulatory complexity of phasiRNAs, establishing a foundation for further functional studies and advancing our understanding of their roles in anther development and their underlying mechanisms. Plain Language SummarySmall RNAs play important roles in controlling how plants develop, but their function in plant reproduction is still not well understood. In rice, small RNAs called phasiRNAs help regulate how pollen develops, but past research has mostly focused on just two types: one found before meiosis and one found during meiosis. In this study, we analyzed small RNA activity across ten stages of rice anther development and found new phasiRNA-producing genes that are active after meiosis. We also discovered that different types of phasiRNAs have distinct patterns of accumulation and composition, suggesting that they have specialized roles in reproductive development. These findings reveal new layers of complexity in how small RNAs function and set the stage for future research to understand their exact roles in pollen formation. This work may help improve our understanding of plant fertility and contribute to advancements in crop breeding. Core ideasO_LIStage-specific accumulation patterns suggest more specialized functions of phasiRNAs. C_LIO_LIMeiotic 24-nt phasiRNAs are compositionally distinct from other 24-nt phasiRNAs. C_LIO_LINewly described phasiRNA clusters may differ in mechanism of biogenesis and function. C_LI

plant biology↗

Comparative transcriptome profiling identifies stage-specific phasiRNAs and co-expressed Argonaute genes in Bambusoideae and Pooideae species

PhasiRNAs (phased, small interfering RNAs) play a crucial role in supporting male fertility in grasses. Earlier work in maize (Zea mays) and rice (Oryza sativa) - and subsequently many other plant species - identified premeiotic 21-nt and meiotic 24-nt phasiRNAs. More recently, a group of premeiotic 24-nt phasiRNAs were discovered in the anthers of two Pooideae species, barley (Hordeum vulgare) and bread wheat (Triticum aestivum). The conservation of premeiotic 24-nt phasiRNAs and other classes of reproductive phasiRNAs across Pooideae species remains unclear. We conducted a comparative RNA profiling of three anther stages in six Pooideae species and one Bambusoideae species. We observed complex temporal accumulation patterns of 21-nt and 24-nt phasiRNAs in Pooideae and Bambusoideae grasses. In Bambusoideae, 21-nt phasiRNAs accumulated during meiosis, whereas 24-nt phasiRNAs were present in both premeiotic and postmeiotic stages. We identified premeiotic 24-nt phasiRNAs in all seven species examined. These phasiRNAs exhibit distinct biogenesis mechanisms and potential Argonaute effectors compared to meiotic 24-nt phasiRNAs. We show that specific Argonaute genes co-expressed with stage-specific phasiRNAs are conserved across Bambusoideae and Pooideae species. Our degradome analysis identified a set of conserved miRNA target genes across species, while 21-nt phasiRNAs targets were species-specific. Cleavage of few targets was observed for 24-nt phasiRNAs.

plant biology↗

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known. SIGNIFICANCEWe previously reported two classes of reproductive phasiRNAs in maize, the premeiotic 21-nt phasiRNAs and the meiotic 24-nt phasiRNAs. Here we report a third class of reproductive phasiRNAs - premeiotic 24-nt phasiRNAs - that are present in the Zea genus, including all five maize inbred lines and three teosinte species/subspecies that we examined, plus rice. We show that in the Zea genus the premeiotic 24-nt phasiRNAs are distinct from the meiotic 24-nt phasiRNAs in triggering mechanism, effector protein, and molecular function.

plant biology↗

Fluorescence hybridization chain reaction enables localization of multiple molecular classes combined with plant cell ultrastructure

BackgroundRecent developments in hybridization chain reaction (HCR) have enabled robust simultaneous localization of multiple mRNA transcripts using fluorescence in situ hybridization (FISH). Once multiple split initiator oligonucleotide probes bind their target mRNA, HCR uses DNA base-pairing of fluorophore-labeled hairpin sets to self-assemble into large polymers, amplifying the fluorescence signal and reducing non-specific background. Few studies have applied HCR in plants, despite its demonstrated utility in whole mount animal tissues and cell culture. Our aim was to optimize this technique for sectioned plant tissues embedded with paraffin and methacrylate resins, and to test its utility in combination with immunolocalization and subsequent correlation with cell ultrastructure using scanning electron microscopy. ResultsApplication of HCR to 10 {micro}m paraffin sections of 17-day-old Setaria viridis (green millet) inflorescences using confocal microscopy revealed that the transcripts of the transcription factor KNOTTED 1 (KN1) were localized to developing floret meristem and vascular tissue while SHATTERING 1 (SH1) and MYB26 transcripts were co-localized to the breakpoint below the floral structures (the abscission zone). We also used methacrylate de-embedment with 1.5 {micro}m and 0.5 {micro}m sections of 3-day-old Arabidopsis thaliana seedlings to show tissue specific CHLOROPHYLL BINDING FACTOR a/b (CAB1) mRNA highly expressed in photosynthetic tissues and ELONGATION FACTOR 1 ALPHA (EF1) highly expressed in meristematic tissues of the shoot apex. The housekeeping gene ACTIN7 (ACT7) mRNA was more uniformly distributed with reduced signals using lattice structured-illumination microscopy. HCR using 1.5 {micro}m methacrylate sections was followed by backscattered imaging and scanning electron microscopy thus demonstrating the feasibility of correlating fluorescent localization with ultrastructure. ConclusionHCR was successfully adapted for use with both paraffin and methacrylate de-embedment on diverse plant tissues in two model organisms, allowing for concurrent cellular and subcellular localization of multiple mRNAs, antibodies and other affinity probe classes. The mild hybridization conditions used in HCR made it highly amenable to observe immunofluorescence in the same section. De-embedded semi-thin methacrylate sections with HCR were compatible with correlative electron microscopy approaches. Our protocol provides numerous practical tips for successful HCR and affinity probe labeling in electron microscopy-compatible, sectioned plant material.

plant biology↗

Mixed Lineage Leukemia (Mll1) establishes global histone H3K4 trimethylation and stem like memory Cd8 T cell formation

CD8+ T cells with stem cell-like properties (TSCM) sustain adaptive immunity to intracellular pathogens and tumors. However, the developmental origins and chromatin regulatory factors (CRFs) that establish their differentiation are unclear. Using an RNA interference screen of all CRFs we discovered the histone methylase Mll1 was required during T cell receptor (TCR) stimulation for development of a TSCM precursor state and mature memory (TMEM) cells, but not short-lived or transitory effector cell-like states, in response to viral infections and tumors. Mll1 was essential for widespread de novo deposition of histone H3 lysine 4 trimethylation (H3K4me3) upon TCR stimulation, which accounted for 70% of all activation-induced sites in mature TMEM cells. Mll1 promoted both H3K4me3 deposition and reduced TCR-induced Pol II pausing at genes whose single-cell transcriptional dynamics explained trajectories into nascent TSCM precursor states during viral infection. Our results suggest Mll1-dependent control of Pol II elongation and H3K4me3 establishes and maintains differentiation of CD8+ TSCM cell states.

immunology↗

Plastid double-strand RNA transgenes trigger small RNA-based gene silencing of nuclear-encoded genes

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escape to the cytoplasm occurs. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment deficient plants. Furthermore, plastid-expressed double-stranded RNA (dsRNA) with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs broadly, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open new fields of study in plastid development, compartmentalization and small RNA biogenesis.

plant biology↗

IL-2 secreting T helper cells promote EF B cell maturation via intrinsic regulation of B cell mTOR/AKT/Blimp-1 axis.

SUMMARYB cells are fundamental players in the secretion of antibodies and the establishment of long-term memory-based immunity. Integration of signals from TLRs, BCR stimulation, and T helper cell-derived cytokines can all dictate B cell differentiation and their metabolic state. However, while important components of this interaction have been described, the precise signaling networks and mechanisms regulating B cell fate are not fully understood. Here, we elucidated the role of interleukin-2 (IL-2) in determining early B cell fate decisions and inducing plasma cell reprogramming. Using both in vitro culture systems and in vivo models of immunization, alongside CRISPR-based genome editing of antigen-specific T and B cells, we identify a role for T helper-secreted IL-2 in inducing high expression of Irf4 and Blimp-1 in activated cognate B cells, enhancing plasma cell differentiation. Induction of this cascade promotes their differentiation and drives metabolic reprogramming through the regulation of mTOR/AKT/Blimp-1 axis.

immunology↗

A Versatile Enhanced Freeze-Substitution Protocol for Volume Electron Microscopy

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including 2% osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% OsO4. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues, nematodes, yeast, and bacteria. Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.

plant biology↗

Genome-wide analysis of small RNA biogenesis proteins refine the evolution of Dicer-like and Argonaute gene families in flowering plants

Several protein families play a role in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-directed RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to Acorus americanus, the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained or further duplicated in sub-groups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17 and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins provide insights into the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways.

plant biology↗

The Streptochaeta genome and the evolution of the grasses

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size, capturing most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses ({rho}) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

evolutionary biology↗

The Evolutionary History of Small RNAs in the Solanaceae

The Solanaceae or "nightshade" family is an economically important group that harbors a remarkable amount of diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the familys small RNA genomic landscape, we downloaded over 255 publicly available small RNA datasets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major small RNA classes: (1) microRNAs (miRNAs), typically 20-22 nt RNAs generated from a hairpin precursor and functioning in gene silencing, and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs (hc-siRNAs) typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs (phasiRNAs) and trans-acting siRNAs (tasiRNAs) generated via miRNA-directed cleavage of a Pol II-derived RNA precursor. Our analyses described thousands of small RNA loci, including poorly-understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these small RNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar small RNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities. ONE-SENTENCE SUMMARYWe use over 255 publicly-available small RNA datasets to characterize the small RNA landscape for the Solanaceae family.

plant biology↗

Pre-meiotic, 21-nucleotide Reproductive PhasiRNAs Emerged in Seed Plants and Diversified in Flowering Plants

Plant small RNAs (sRNAs) are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. They have critical roles in most pathways involved in plant growth and reproductive development. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have not been described in eudicots. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we describe additional variants of 21-nt reproductive phasiRNAs, including those triggered by miR11308 in wild strawberry, a eudicot, and we validate the presence of this pathway in rose. We report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots, with novel biogenesis triggers in the basal eudicot columbine and the rosid flax. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports a singular finding in spruce, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

plant biology↗

Pre-meiotic, 24-nt reproductive phasiRNAs are abundant in anthers of wheat and barley but not rice and maize

Two classes of pre-meiotic (21-nt) and meiotic (24-nt) phasiRNAs and their patterns of accumulation have been described in maize and rice anthers. Their precise function remains unclear, but some studies have shown that they support male fertility. The important role of phasiRNAs in anthers underpins our current study to their characterization in wheat and barley anthers. In this study, we staged anthers at every 0.2 mm of development for one wheat and two barley varieties. We isolated pre-meiotic (0.2 mm, 0.4 mm and 0.6 mm), meiotic (0.8 mm, 1.0 mm and 1.4 mm) and post-meiotic (1.8 mm) anthers for which we then investigated accumulation patterns of RNAs, including reproductive phasiRNAs. We annotated a total 12,821 and 2,897 PHAS loci in the wheat and barley genomes, respectively. When comparing the total number of PHAS loci in genomes of maize, rice, barley and wheat, we characterized an expansion of reproductive PHAS loci in the genomes of Poaceae subfamilies from Panicoideae to Oryzoideae and to Poideae. In addition to the two classes of pre-meiotic (21-nt) and meiotic (24-nt) phasiRNAs, previously described in maize and rice anthers, we described a group of 24-nt phasiRNAs that accumulate in pre-meiotic anthers. The absence of pre-meiotic 24-nt phasiRNAs in maize and rice suggests a divergence in grass species of the Poideae subfamily. Additionally, we performed a co-expression gene analysis describing the regulation of phasiRNA biogenesis in wheat and barley anthers. We highlight AGO9 and AGO6 as candidate binding partners of pre-meiotic and meiotic 24-nt phasiRNAs, respectively. One sentence summaryIn wheat and barley anthers, 24-nt reproductive phasiRNAs are abundant in both meiotic and pre-meiotic stages.

plant biology↗

The commitment of barley microspores into embryogenesis involves miRNA-directed regulation of members of the SPL, GRF and HD-ZIPIII transcription factor families

Microspore embryogenesis is a model for developmental plasticity and cell fate decisions. To investigate the role of miRNAs in this development, we sequenced sRNAs and the degradome of barley microspores collected prior to (day 0) and after (days 2 and 5) the application of a stress treatment known to induce embryogenesis. Microspores isolated at these timepoints were uniform in both appearance and in their complements of sRNAs. We detected 68 miRNAs in microspores. The abundance of 51 of these miRNAs differed significantly during microspore development. One group of miRNAs was induced when the stress treatment was applied, prior to being repressed when microspores transitioned to embryogenesis. Another group of miRNAs were up-regulated in day-2 microspores and their abundance remained stable or increased in day-5 microspores, a timepoint at which the first clear indications of the transition towards embryogenesis were visible. Collectively, these miRNAs might play a role in the modulation of the stress response, the repression of gametic development, and/or the gain of embryogenic potential. A degradome analysis allowed us to validate the role of miRNAs in regulating 41 specific transcripts. We showed that the transition of microspores toward the embryogenesis pathway involves miRNA-directed regulation of members of the ARF, SPL, GRF and HD-ZIPIII transcription factor families. We noted that 41.5% of these targets were shared between day-2 and day-5 microspores while 26.8% were unique to day-5 microspores. The former set may act to disrupt transcripts involved in pollen development while the latter set may drive the commitment to embryogenesis.

plant biology↗

CD4+ follicular helper-like T cells are key players in anti-tumor immunity

To determine the nature of CD4+ T cells that provide help for generating robust anti-tumor CD8+ cytotoxic T cell (CTL) responses, we profiled the transcriptomes of patient-matched CD4+ and CD8+ T cells present in the tumor micro-environment (TME) and analyzed them jointly using integrated weighted gene correlation network analysis. We found the follicular helper T cell (TFH) program in CD4+ T cells was strongly associated with proliferation and tissue-residency in CD8+ CTLs. Single-cell analysis demonstrated the presence of TFH-like cells and features linked to cytotoxic function and their provision of CD8+ T cell help. Tumor-infiltrating TFH-like cells expressed PD-1 and were enriched in tumors following checkpoint blockade, suggesting that they may respond to anti-PD-1 therapy. Adoptive transfer or induction of TFH cells in mouse models resulted in augmented CD8+ CTL responses and impairment of tumor growth, indicating an important role of TFH-like CD4+ T cells in anti-tumor immunity.

immunology↗