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In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing. AGO1 associates to the rough endoplasmic reticulum to conduct miRNA-mediated translational repression, mRNA cleavage, and biogenesis of phased siRNAs. Here, we show that a 37°C heat stress (HS) promotes AGO1 protein accumulation in cytosolic condensates where it colocalizes with components of siRNA bodies and of stress granules. AGO1 contains a prion-like domain in its poorly characterized N-terminal Poly-Q domain, which is sufficient to undergo phase separation independently of the presence of SGS3. HS only moderately affects the small RNA repertoire, the loading of AGO1 by miRNAs, and the signatures of target cleavage, suggesting that its localization in condensates protects AGO1 rather than promoting or impairing its activity in reprogramming gene expression during stress. Collectively, our work sheds new light on the impact of high temperature on a main effector of RNA silencing in plants.

Hybrid breeding for increased vigour has been used for over a century to boost agricultural outputs without requiring higher inputs. While this approach has led to some of the most substantial gains in crop productivity, breeding barriers have fundamentally limited soybean (Glycine max) from reaping the benefits of hybrid vigour. Soybean flowers self-pollinate prior to opening and thus are not readily amenable to outcrossing. In this study, we demonstrate that the barnase/barstar male sterility/rescue system can be used in soybean to produce hybrid seeds. By expressing the cytotoxic ribonuclease, barnase, under a tapetum-specific promoter in soybean anthers, we are able to completely block pollen maturation, creating male sterile plants. We show that fertility can be rescued in the F1 generation of these barnase-expressing lines when they are crossed with pollen from plants that express the barnase inhibitor, barstar. Importantly, we found that the successful rescue of male fertility is dependent on the relative dosage of barnase and barstar. When barnase and barstar were expressed under the same tapetum-specific promoter, the F1 offspring remained male sterile. When we expressed barstar under a relatively stronger promoter than barnase, we were able to achieve a successful rescue of male fertility in the F1 generation. This work demonstrates the successful implementation of a biotechnology approach to produce fertile hybrid offspring in soybean.

Small RNAs, including microRNAs (miRNAs), phased secondary small interfering RNAs (phasiRNA), and heterochromatic small interfering RNAs (hc-siRNA) are an essential component of gene regulation. To establish a broad potato small RNA atlas, we constructed an expression atlas of leaves, flowers, roots, and tubers of Desiree and Eva, which are commercially important potato (Solanum tuberosum) cultivars. All small RNAs identified were observed to be conserved between both cultivars, supporting the hypothesis that small RNAs have a low evolutionary rate and are mostly conserved between lineages. However, we also found that a few miRNAs showed differential accumulation between the two potato cultivars, and that hc-siRNAs have a tissue specific expression. We further identified dozens of reproductive and non-reproductive phasiRNAs originating from coding and noncoding regions that appeared to exhibit tissue-specific expression. Together, this study provides an extensive small RNA profiling of different potato tissues that might be used as a resource for future investigations.

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

Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs.

Aegilops tauschii is the donor of the D subgenome of hexaploid wheat and an important genetic resource. The reference-quality genome sequence Aet v4.0 for Ae. tauschii acc. AL8/78 was therefore an important milestone for wheat biology and breeding. Further advances in sequencing acc. AL8/78 and release of the Aet v5.0 sequence assembly are reported here. Two new optical maps were constructed and used in the revision of pseudomolecules. Gaps were closed with Pacific Biosciences long-read contigs, decreasing the gap number by 38,899. Transposable elements and protein-coding genes were reannotated. The number of annotated high-confidence genes was reduced from 39,635 in Aet v4.0 to 32,885 in Aet v5.0. A total of 2245 biologically important genes, including those affecting plant phenology, grain quality, and tolerance of abiotic stresses in wheat, was manually annotated and disease-resistance genes were annotated by a dedicated pipeline. Disease-resistance genes encoding nucleotide-binding site domains, receptor-like protein kinases, and receptor-like proteins were preferentially located in distal chromosome regions, whereas those encoding transmembrane coiled-coil proteins were dispersed more evenly along the chromosomes. Discovery, annotation, and expression analyses of microRNA (miRNA) precursors, mature miRNAs, and phasiRNAs are reported, including miRNA target genes. Other small RNAs, such as hc-siRNAs and tRFs, were characterized. These advances enhance the utility of the Ae. tauschii genome sequence for wheat genetics, biotechnology, and breeding.

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 toward 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.

Maize somatic embryogenesis (SE) requires the induction of embryogenic callus and establishment of proliferation before plant regeneration. The molecular mechanisms underlying callus embryogenic potential are not well understood. Here we explored the role of small RNAs (sRNAs) and the accumulation of their target transcripts in maize SE at the dedifferentiation step using VS-535 zygotic embryos collected at distinct developmental stages and displaying contrasting in vitro embryogenic potential and morphology. MicroRNAs (miRNAs), trans-acting siRNAs (tasiRNAs), heterochromatic siRNAs (hc-siRNAs) populations and their RNA targets were analyzed by high-throughput sequencing. Abundances of specific miRNAs, tasiRNAs and targets were validated by qRT-PCR. Unique accumulation patterns were found for immature embryo at 15 Days After Pollination (DAP) and for the callus induction from this explant, as compared to 23 DAP and mature embryos. miR156, miR164, miR166, tasiARFs and the 24 nt hc-siRNAs displayed the most strikingly different patterns between explants and during dedifferentiation. According to their role in auxin responses and developmental cues, we conclude that sRNA-target regulation operating within the 15 DAP immature embryo explant provides key molecular hints as to why this stage is relevant for callus induction with successful proliferation and plant regeneration.

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA-FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co-localization of miR2275 and a 24-nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi-photon fluorescence excitation microscopy can be used to separate the target sRNA-FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA-FISH signals can be imaged using super-resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super-resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step-by-step sRNA-FISH protocol for studying sRNAs at the cellular and even subcellular level.

Phased, secondary siRNAs (phasiRNAs) represent a class of small RNAs in plants generated via distinct biogenesis pathways, predominantly dependent on the activity of 22-nt miRNAs. Most 22-nt miRNAs are processed by DCL1 from miRNA precursors containing an asymmetric bulge, yielding a 22/21-nt miRNA/miRNA* duplex. Here we show that miR1510, a soybean miRNA capable of triggering phasiRNA production from numerous nucleotide-binding leucine-rich repeat (NB-LRRs), previously described as 21 nt in its mature form, primarily accumulates as a 22-nt isoform via monouridylation. We demonstrate that, in Arabidopsis, this uridylation is performed by HESO1. Biochemical experiments showed that the 3' terminus of miR1510 is only partially 2'-O-methylated because of the terminal mispairing in the miR1510/miR1510* duplex that inhibits HEN1 activity in soybean. miR1510 emerged in the Phaseoleae ∼41-42 million years ago with a conserved precursor structure yielding a 22-nt monouridylated form, yet a variant in mung bean is processed directly in a 22-nt mature form. This analysis of miR1510 yields two observations: (i) plants can utilize postprocessing modification to generate abundant 22-nt miRNA isoforms to more efficiently regulate target mRNA abundances; and (ii) comparative analysis demonstrates an example of selective optimization of precursor processing of a young plant miRNA.

SUMMARY: Noncoding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. We combined these data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs, and tRFs. Our differential and co‐expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs, and phasiRNAs preferentially accumulate in a stage‐specific manner. Members of the miR482/miR2118 superfamily and their 21‐nucleotide phasiRNAs originating from resistance genes show a robust co‐expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family stage‐specific manner, related to modulated hormonal responses and transcription factor expression. Significance Statement: Small RNAs in coffee floral development: unveiling stage‐specific accumulation patterns and regulatory networks. [ABSTRACT FROM AUTHOR]

Previously, we have shown that apoplastic wash fluid (AWF) purified from Arabidopsis leaves contains small RNAs (sRNAs). To investigate whether these sRNAs are encapsulated inside extracellular vesicles (EVs), we treated EVs isolated from Arabidopsis leaves with the protease trypsin and RNase A, which should degrade RNAs located outside EVs but not those located inside. These analyses revealed that apoplastic RNAs are mostly located outside and are associated with proteins. Further analyses of these extracellular RNAs (exRNAs) revealed that they include both sRNAs and long noncoding RNAs (lncRNAs), including circular RNAs (circRNAs). We also found that exRNAs are highly enriched in the posttranscriptional modification N6-methyladenine (m6A). Consistent with this, we identified a putative m6A-binding protein in AWF, GLYCINE-RICH RNA-BINDING PROTEIN 7 (GRP7), as well as the sRNA-binding protein ARGONAUTE2 (AGO2). These two proteins coimmunoprecipitated with lncRNAs, including circRNAs. Mutation of GRP7 or AGO2 caused changes in both the sRNA and lncRNA content of AWF, suggesting that these proteins contribute to the secretion and/or stabilization of exRNAs. We propose that exRNAs located outside of EVs mediate host-induced gene silencing, rather than RNA located inside EVs. [ABSTRACT FROM AUTHOR]

Background and Aims MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression via sequence-specific cleavage or translational repression of target transcripts. They are transcribed as long single-stranded RNA precursors with unique stem–loop structures that are processed by a DICER-Like (DCL) ribonuclease, typically DCL1, to produce mature miRNAs. Although a plethora of miRNAs have been found to be regulated by pathogen infection in plants, the biological function of most miRNAs remains largely unknown. Here, the contribution of OsDCL1 to rice immunity was investigated. Methods Activation-tagged Osdcl1a (Osdcl1a-Ac) rice mutants were examined for resistance to pathogen infection. mRNA and small RNA deep sequencing, quantitative real-time PCR (RT-qPCR) and stem–loop reverse tanscripion–PCR (RT–PCR) were used to examine DCL1a -mediated alterations in the rice transcriptome. Rice diterpene phytoalexins were quantified by liquid chromatography–tandem mass spectrometry (LC-MSMS). Accumulation of O2·– was determined by nitroblue tetrazolium (NBT) staining. Key Results dcl1a-Ac mutants exhibit enhanced susceptibility to infection by fungal pathogens which was associated with a weaker induction of defence gene expression. Comparison of the mRNA and miRNA transcriptomes of dcl1a-Ac and wild-type plants revealed misregulation of genes involved in detoxification of reactive oxygen species. Consequently, dcl1a-Ac plants accumulated O2·– in their leaves and were more sensitive to methyl viologen-induced oxidative stress. Furthermore, dcl1a-Ac plants showed downregulation of diterpenoid phytoalexin biosynthetic genes, these genes also being weakly induced during pathogen infection. Upon pathogen challenge, dcl1a-Ac plants failed to accumulate major diterpenoid phytoalexins. OsDCL1a activation resulted in marked alterations in the rice miRNAome, including both upregulation and downregulation of miRNAs. Conclusions OsDCL1a activation enhances susceptibility to infection by fungal pathogens in rice. Activation of OsDCL1a represses the pathogen-inducible host defence response and negatively regulates diterpenoid phytoalexin production. These findings provide a basis to understand the molecular mechanisms through which OsDCL1a mediates rice immunity. [ABSTRACT FROM AUTHOR]