Biology subjects

Meyers, B. C.

Publications and source records attributed to Meyers, B. C..

51 records · Page 3Linked to original sources

bioRxiv2020

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↗

bioRxiv2020

Visualizing cassava bacterial blight at the molecular level using CRISPR-mediated homology-directed repair

Research on a few model, plant-pathogen systems has benefitted from years of tool and resource development. This is not the case for the vast majority of economically and nutritionally important plants, creating a crop improvement bottleneck. Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam), is an important disease in all regions where cassava (Manihot esculenta Crantz) is grown. Here we describe development of a tool for molecular-level visualization of CBB dynamics in vivo. Using CRISPR-mediated homology-directed repair (HDR), we generated plants containing scarless insertion of a GFP reporter at the CBB susceptibility (S) gene MeSWEET10a. Activation of MeSWEET10a-GFP by Xam was subsequently visualized at transcriptional and translational levels. Development of this tool was facilitated by a time-saving, adaptable strategy for identifying successful products of HDR, currently a limiting factor in plant research. This strategy has the potential to enable such research in other systems, improving the practicality of HDR-based experimentation.

plant biology↗

bioRxiv2020

Reproductive phasiRNA loci and DICER-LIKE5, but not microRNA loci, diversified in monocotyledonous plants

In monocots other than maize and rice, the repertoire and diversity of microRNAs (miRNAs) and the populations of phased, secondary, small interfering RNAs (phasiRNAs) are poorly characterized. To remedy this, we sequenced small RNAs from vegetative and dissected inflorescence tissue in 28 phylogenetically diverse monocots and from several early-diverging angiosperm lineages, as well as publicly available data from 10 additional monocot species. We annotated miRNAs, siRNAs and phasiRNAs across the monocot phylogeny, identifying miRNAs apparently lost or gained in the grasses relative to other monocot families, as well as a number of tRNA fragments misannotated as miRNAs. Using our miRNA database cleaned of these misannotations, we identified conservation at the 8th, 9th, 19th and 3 end positions that we hypothesize are signatures of selection for processing, targeting, or Argonaute sorting. We show that 21-nt reproductive phasiRNAs are far more numerous in grass genomes than other monocots. Based on sequenced monocot genomes and transcriptomes, DICER-LIKE5 (DCL5), important to 24-nt phasiRNA biogenesis, likely originated via gene duplication before the diversification of the grasses. This curated database of phylogenetically diverse monocot miRNAs, siRNAs, and phasiRNAs represents a large collection of data that should facilitate continued exploration of small RNA diversification in flowering plants.

plant biology↗

bioRxiv2020

A new Spirodela polyrhiza genome and proteome reveal a conserved chromosomal structure with high abundances of proteins favoring energy production

Duckweeds are a monophyletic group of rapidly reproducing aquatic monocots in the Lemnaceae family. Spirodela polyrhiza, the Greater Duckweed, has the largest body plan yet the smallest genome size in the family (1C = 150 Mb). Given their clonal, exponentially fast reproduction, a key question is whether genome structure is conserved across the species in the absence of meiotic recombination. We generated a highly contiguous, chromosome-scale assembly of Spirodela polyrhiza line Sp7498 using Oxford Nanopore plus Hi-C scaffolding (Sp7498_HiC) which is highly syntenic with a related line (Sp9509). Both the Sp7498_HiC and Sp9509 genome assemblies reveal large chromosomal misorientations in a recent PacBio assembly of Sp7498, highlighting the necessity of orthogonal long-range scaffolding techniques like Hi-C and BioNano optical mapping. Shotgun proteomics of Sp7498 verified the expression of [~]2,250 proteins and revealed a high abundance of proteins involved in photosynthesis and carbohydrate metabolism among other functions. In addition, a strong increase in chloroplast proteins was observed that correlated to chloroplast density. This Sp7498_HiC genome was generated cheaply and quickly with a single Oxford Nanopore MinION flow cell and one Hi-C library in a classroom setting. Combining these data with a mass spectrometry-generated proteome illustrates the utility of duckweed as a model for genomics- and proteomics-based education.

plant biology↗

bioRxiv2019

Abundant expression of maternal siRNAs is a conserved feature of seed development

Small RNAs are abundant in plant reproductive tissues, especially 24-nt short interfering (si)RNAs. Most 24-nt siRNAs are dependent on RNA Pol IV and RDR2, and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=128 SRC="FIGDIR/small/866806v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@68de55org.highwire.dtl.DTLVardef@dca8eaorg.highwire.dtl.DTLVardef@d5d489org.highwire.dtl.DTLVardef@74824a_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2019

sRNAanno --- a database repository of uniformly-annotated small RNAs in plants

Small RNAs (sRNAs) are essential regulatory molecules, including three mayor classes in plants, microRNAs (miRNAs), phased small interfering RNAs (phased siRNAs or phasiRNAs), and heterochromatic siRNAs (hc-siRNAs). Except miRNAs, the other two classes are not well-annotated and collected in public databases for most sequenced plant genomes. We performed comprehensive sRNA annotation for 138 plant species, which have fully sequenced genomes and public next-generation-sequencing (NGS) sRNA data available. The results are available via an online repository called sRNAanno (www.plantsRNAs.org). Compared to plant miRNAs deposited in miRBase, we obtained much more miRNAs, which are more complete and reliable because of consistent and high-stringent criteria used in our miRNA annotation. sRNAanno also provides free access to genomic information for >16,000 PHAS loci and >21,000,000 hc-siRNA loci annotated from these 138 plants. On the basis of Integrative Genomics Viewer (IGV), we developed a visualization tool for browsing NGS sRNA data (IGV-sRNA), which have been integrated a series of new functions compatible to specific sRNA features. To make sRNA annotation an easy task, sRNAanno also provides free service of sRNA annotation to the community. In summary, sRNAanno and IGV-sRNA are great resources to facilitate the genomic and genetic research of plant small RNAs.

genomics↗

bioRxiv2019

Quantitative, super-resolution localization of small RNAs with sRNA-PAINT

Small RNAs are non-coding RNAs that play important roles in the lives of both animals and plants. They are 21- to 24-nt in length and around 10 nanometers (nm) in size. Their small size and high diversity have made it challenging to develop detection methods that have sufficient resolution and specificity to multiplex and quantify. We created a method, sRNA-PAINT, for the detection of small RNAs with 20 nm resolution by combining the super-resolution method, DNA-based points accumulation in nanoscale topography (DNA-PAINT), and the specificity of locked nucleic acid (LNA) probes for the in situ detection of multiple small RNAs. The method relies on designing probes to target small RNAs that combine DNA oligonucleotides (oligos) for PAINT with LNA-containing oligos for hybridization; therefore, we developed an online tool called "Vetting & Analysis of RNA for in situ Hybridization probes" (VARNISH) for probe design. Our method utilizes advances in DNA-PAINT methodologies, including qPAINT for quantification, and Exchange-PAINT for multiplexing. We demonstrated these capabilities of sRNA-PAINT by detecting and quantifying small RNAs in different cell layers of early developmental stage maize anthers that are important for male sexual reproduction.

cell biology↗

bioRxiv2019

Variation and inheritance of small RNAs in maize inbreds and F1 hybrids

Small RNAs (sRNAs) regulate gene expression, play important roles in epigenetic pathways, and have been hypothesised to contribute to hybrid vigor in plants. Prior investigations have provided valuable insights into associations between sRNAs and heterosis, often using a single hybrid genotype or tissue. However, our understanding of the role of sRNAs and their potential value to plant breeding are limited by an incomplete picture of sRNA variation between diverse genotypes and development stages. Here, we provide a deep exploration of sRNA variation and inheritance among a panel of 108 maize samples spanning five tissues from eight inbred parents and 12 hybrid genotypes, covering a spectrum of heterotic groups, genetic variation, and levels of heterosis for various traits. We document substantial developmental and genotypic influences on sRNA expression, with varying patterns for 21-nt, 22-nt and 24-nt sRNAs. We provide a detailed view of the distribution of sRNAs in the maize genome, revealing a complex make-up that also shows developmental plasticity, particularly for 22-nt sRNAs. sRNAs exhibited substantially more variation between inbreds as compared to observed variation for gene expression. In hybrids, we identify locus-specific examples of non-additive inheritance, mostly characterised as partial or complete dominance, but rarely outside the parental range. However, the global abundance of 21-nt, 22-nt and 24-nt sRNAs varies very little between inbreds and hybrids, suggesting that hybridization affects sRNA expression principally at specific loci rather than on a global scale. This study provides a valuable resource for understanding the potential role of sRNAs in hybrid vigor.\n\nOne-sentence summaryCharacterizing the roles of development and genotype in driving expression variation of different small RNA populations in maize inbreds and their F1 hybrids.

genomics↗

bioRxiv2018

Sex chromosome evolution via two genes

The origin of sex chromosomes has been hypothesized to involve the linkage of factors with antagonistic effects on male and female function. Garden asparagus (Asparagus officinalis L.) is an ideal species to test this hypothesis, as the X and Y chromosomes are cytologically homomorphic and recently evolved from an ancestral autosome pair in association with a shift from hermaphroditism to dioecy. Mutagenesis screens paired with singlemolecule fluorescence in situ hybridization (smFISH) directly implicate Y-specific genes that respectively suppress female organ development and are necessary for male gametophyte development. Comparison of contiguous X and Y chromosome shows that loss of recombination between the genes suppressing female function (SUPPRESSOR OF FEMALE FUNCTION, SOFF) and promoting male function (TAPETAL DEVELOPMENT AND FUNCTION 1, aspTDF1) is due to hemizygosity. We also experimentally demonstrate the function of aspTDF1. These finding provide direct evidence that sex chromosomes can evolve from autosomes via two sex determination genes: a dominant suppressor of femaleness and a promoter of maleness.\n\nOne Sentence SummarySex chromosomes can evolve from autosomes by mutations in two sex determination genes.

genetics↗

bioRxiv2018

Developmental genetics of corolla tube formation: role of the tasiRNA-ARF pathway

More than 80,000 angiosperm species produce flowers with petals fused into a corolla tube. As an important element of the tremendous diversity of flower morphology, the corolla tube plays a critical role in many specialized interactions between plants and animal pollinators (e.g., beeflies, hawkmoths, hummingbirds, nectar bats), which in turn drives rapid plant speciation. Despite its clear significance in plant reproduction and evolution, the corolla tube remains one of the least understood plant structures from a developmental genetics perspective. Through mutant analyses and transgenic experiments, here we show that the tasiRNA-ARF pathway is required for corolla tube formation in the monkeyflower species Mimulus lewisii. Loss-of-function mutations in the M. lewisii orthologs of ARGONAUTE7 and SUPPRESSOR OF GENE SILENCING 3 cause a dramatic decrease in abundance of TAS3-derived small RNAs and a moderate up-regulation of AUXIN RESPONSE FACTOR 3 (ARF3) and ARF4, which lead to inhibition of lateral expansion of the bases of petal primordia and complete arrest of the upward growth of the inter-primordial regions, resulting in unfused corollas. By using an auxin reporter construct, we discovered that auxin distribution is continuous along the petal primordium base and the inter-primordial region during the critical stage of corolla tube formation in the wild-type, and that this auxin distribution is much weaker and more restricted in the mutant. Together, these results suggest a new conceptual model highlighting the central role of auxin directed synchronized growth of the petal primordium base and the inter-primordial region in corolla tube formation.

plant biology↗

bioRxiv2018

Reproductive phasiRNAs in grasses are compositionally distinct from other classes of small RNAs

Summary and keywordsO_LILittle is known about the characteristics and function of reproductive phased, secondary, small interfering RNAs (phasiRNAs) in the Poaceae, despite the availability of significant genomic resources, experimental data, and a growing number of computational tools. We utilized machine-learning methods to identify sequence-based and structural features that distinguish phasiRNAs in rice and maize from other small RNAs (sRNAs).\nC_LIO_LIWe developed Random Forest classifiers that can distinguish reproductive phasiRNAs from other sRNAs in complex sets of sequencing data, utilizing sequence-based (k-mers) and features describing position-specific sequence biases.\nC_LIO_LIThe classification performance attained is >80% in accuracy, sensitivity, specificity, and positive predicted value. Feature selection identified important features in both ends of phasiRNAs. We demonstrated that phasiRNAs have strand specificity and position-specific nucleotide biases potentially influencing AGO sorting; we also predicted targets to infer functions of phasiRNAs, and computationally-assessed their sequence characteristics relative to other sRNAs.\nC_LIO_LIOur results demonstrate that machine-learning methods effectively identify phasiRNAs despite the lack of characteristic features typically present in precursor loci of other small RNAs, such as sequence conservation or structural motifs. The 5-end features we identified provide insights into AGO-phasiRNA interactions; we describe a hypothetical model of competition for AGO loading between phasiRNAs of different nucleotide compositions.\nC_LI

plant biology↗

bioRxiv2018

The basis of accumulation differences in plant 21-nt reproductive phasiRNAs, and their cis-directed activity

O_LIPost-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield non-coding RNAs that are processed into 21- and 24-nt phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275.\nC_LIO_LIWe characterized these \"reproductive phasiRNAs\" from rice panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions.\nC_LIO_LIWe demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice (Oryza sativa) and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production.\nC_LIO_LIWe characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived \"top\" strand phasiRNAs are consistently higher abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.\nC_LI

plant biology↗

bioRxiv2017

Revisiting criteria for plant miRNA annotation in the era of big data

MicroRNAs (miRNAs) are ~21 nucleotide-long regulatory RNAs that arise from endonucleolytic processing of hairpin precursors. Many function as essential post-transcriptional regulators of target mRNAs and long non-coding RNAs. Alongside miRNAs, plants also produce large numbers of short interfering RNAs (siRNAs), which are distinguished from miRNAs primarily by their biogenesis (typically processed from long double-stranded RNA instead of single-stranded hairpins) and functions (typically via roles in transcriptional regulation instead of post-transcriptional regulation). Next-generation DNA sequencing methods have yielded extensive datasets of plant small RNAs, resulting in many miRNA annotations, occasionally inaccurately curated. The sheer number of endogenous siRNAs compared to miRNAs has been a major factor in the erroneous annotation of siRNAs as miRNAs. Here, we provide updated criteria for the confident annotation of plant miRNAs, suitable for the era of \"big data\" from DNA sequencing. The updated criteria emphasize replication, the minimization of false positives, and they require next-generation sequencing of small RNAs. We argue that improved annotation systems are needed for miRNAs and all other classes of plant small RNAs. Finally, to illustrate the complexities of miRNA and siRNA annotation, we review the evolution and functions of miRNAs and siRNAs in plants.

plant biology↗

bioRxiv2017

CRISPR/Cas9-mediated resistance to cauliflower mosaic virus

Viral diseases are a leading cause of worldwide yield losses in crop production. Breeding of resistance genes (R gene) into elite crop cultivars has been the standard and most cost-effective practice. However, R gene-mediated resistance is limited by the available R genes within genetic resources and in many cases, by strain specificity. Therefore, it is important to generate new and broad-spectrum antiviral strategies. The CRISPR-Cas9 (clustered regularly interspaced palindromic repeat, CRISPR-associated) editing system has been employed to confer resistance to human viruses and several plant single-stranded DNA geminiviruses, pointing out the possible application of the CRISPR-Cas9 system for virus control. Here we demonstrate that strong viral resistance to cauliflower mosaic virus (CaMV), a pararetrovirus with a double-stranded DNA genome, can be achieved through Cas9-mediated multiplex targeting of the viral coat protein sequence. We further show that small interfering RNAs (siRNA) are produced and mostly map to the 3' end of guide RNAs (gRNA), although very low levels of siRNAs map to the spacer region as well. However, these siRNAs are not responsible for the inhibited CaMV infection because there is no resistance if Cas9 is not present. We have also observed edited viruses in systematically infected leaves in some transgenic plants, with short deletions or insertions consistent with Cas9-induced DNA breaks at the gRNA target sites in coat protein coding sequence. These edited coat proteins, in most cases, led to earlier translation stop and thus, non-functional coat proteins. We also recovered wild-type CP sequence in these infected transgenic plants, suggesting these edited viral genomes were packaged by wild-type coat proteins. Our data demonstrate that the CRISPR-Cas9 system can be used for virus control against plant pararetroviruses with further modifications.

plant biology↗

bioRxiv2016

The emergence, evolution, and diversification of the miR390-TAS3-ARF pathway in land plants

In plants, miR390 directs the production of tasiRNAs from TRANS-ACTING SIRNA 3 (TAS3) transcripts to regulate AUXIN RESPONSIVE FACTOR (ARF) genes, transcription factors critical for auxin signaling; these tasiRNAs are known as tasiARFs. This pathway is highly conserved, with the TAS3 as the only one noncoding gene present almost ubiquitously in land plants. To understand the evolution of this miR390-TAS3-ARF pathway, we characterized homologs of these three genes from thousands of plant species, from bryophytes to angiosperms. Both miR390 and TAS3 are present and functional in liverworts, confirming their ancestral role to regulate ARFs in land plants. We found the lower-stem region of MIR390 genes, critical for accurate DCL1 (DICER-LIKE 1) processing, is conserved in sequence in seed plants. We propose a model for the transition of functional tasiRNA sequences in TAS3 genes occurred at the emergence of vascular plants, in which the two miR390 target sites of TAS3 genes showed distinct pairing patterns in different plant lineages. Based on the cleavability of miR390 target sites and the distance between target site and tasiARF we inferred a potential bidirectional processing mechanism exists for some TAS3 genes. We also demonstrated a tight mutual selection between tasiARF and its target genes, and characterized unusual aspects and diversity of regulatory components of this pathway. Taken together, these data illuminate the evolutionary path of the miR390-TAS3-ARF pathway in land plants, and demonstrate the significant variation that occurs in the production of phasiRNAs in plants, even in the functionally important and archetypal miR390-TAS3-ARF regulatory circuit.

evolutionary biology↗