Identifying genes associated with abiotic stress tolerance suitable for CRISPR/Cas9 editing in upland rice cultivars adapted to acid soils.

Five genes of large phenotypic effect known to confer abiotic stress tolerance in rice were selected to characterize allelic variation in commercial Colombian tropical japonica upland rice cultivars adapted to drought-prone acid soil environments (cv. Llanura11 and Porvenir12). Allelic variants of the genes ART1, DRO1, SUB1A, PSTOL1, and SPDT were characterized by PCR and/or Sanger sequencing in the two upland cultivars and compared with the Nipponbare and other reference genomes. Two genes were identified as possible targets for gene editing: SUB1A (Submergence 1A), to improve tolerance to flooding, and SPDT (SULTR3;4) (SULTR-like Phosphorus Distribution Transporter), to improve phosphorus utilization efficiency and grain quality. Based on technical and regulatory considerations, SPDT was targeted for editing. The two upland cultivars were shown to carry the SPDT wild-type (nondesirable) allele based on sequencing, RNA expression, and phenotypic evaluations under hydroponic and greenhouse conditions. A gene deletion was designed using the CRISPR/Cas9 system, and specialized reagents were developed for SPDT editing, including vectors targeting the gene and a protoplast transfection transient assay. The desired edits were confirmed in protoplasts and serve as the basis for ongoing plant transformation experiments aiming to improve the P-use efficiency of upland rice grown in acidic soils.

Spelling Changes and Fluorescent Tagging With Prime Editing Vectors for Plants.

Prime editing is an adaptation of the CRISPR-Cas system that uses a Cas9(H840A)-reverse transcriptase fusion and a guide RNA amended with template and primer binding site sequences to achieve RNA-templated conversion of the target DNA, allowing specified substitutions, insertions, and deletions. In the first report of prime editing in plants, a variety of edits in rice and wheat were described, including insertions up to 15 bp. Several studies in rice quickly followed, but none reported a larger insertion. Here, we report easy-to-use vectors for prime editing in dicots as well as monocots, their validation in Nicotiana benthamiana, rice, and Arabidopsis, and an insertion of 66 bp that enabled split-GFP fluorescent tagging.

Global Analysis of RNA-Dependent RNA Polymerase-Dependent Small RNAs Reveals New Substrates and Functions for These Proteins and SGS3 in Arabidopsis.

RNA silencing pathways control eukaryotic gene expression transcriptionally or posttranscriptionally in a sequence-specific manner. In RNA silencing, the production of double-stranded RNA (dsRNA) gives rise to various classes of 20-24 nucleotide (nt) small RNAs (smRNAs). In Arabidopsis thaliana, smRNAs are often derived from long dsRNA molecules synthesized by one of the six genomically encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding cofactors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their cofactors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDRγ clade proteins (RDR3-5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, RDR2, and RDR6) as well as the RDR2 cofactor RNA-directed DNA methylation 12 (RDM12) and the RDR6 cofactor suppressor of gene silencing 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general cofactor for the RDR family of proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses, including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in rdr6 and sgs3 mutant plants. Correspondingly, plants that lack these proteins (rdr6 and sgs3 mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have a higher survival rate under salt treatment in comparison to wild-type plants. In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses.

Messenger RNA 5' NAD+ Capping Is a Dynamic Regulatory Epitranscriptome Mark That Is Required for Proper Response to Abscisic Acid in Arabidopsis.

Although eukaryotic messenger RNAs (mRNAs) normally possess a 5' end N7-methyl guanosine (m7G) cap, a non-canonical 5' nicotinamide adenine dinucleotide (NAD+) cap can tag certain transcripts for degradation mediated by the NAD+ decapping enzyme DXO1. Despite this importance, whether NAD+ capping dynamically responds to specific stimuli to regulate eukaryotic transcriptomes remains unknown. Here, we reveal a link between NAD+ capping and tissue- and hormone response-specific mRNA stability. In the absence of DXO1 function, transcripts displaying a high proportion of NAD+ capping are instead processed into RNA-dependent RNA polymerase 6-dependent small RNAs, resulting in their continued turnover likely to free the NAD+ molecules. Additionally, the NAD+-capped transcriptome is significantly remodeled in response to the essential plant hormone abscisic acid in a mechanism that is primarily independent of DXO1. Overall, our findings reveal a previously uncharacterized and essential role of NAD+ capping in dynamically regulating transcript stability during specific physiological responses.

Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana.

Vegetative phase change is regulated by a decrease in the abundance of the miRNAs, miR156 and miR157, and the resulting increase in the expression of their targets, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. To determine how miR156/miR157 specify the quantitative and qualitative changes in leaf morphology that occur during vegetative phase change, we measured their abundance in successive leaves and characterized the phenotype of mutations in different MIR156 and MIR157 genes. miR156/miR157 decline rapidly between leaf 1&2 and leaf 3 and decrease more slowly after this point. The amount of miR156/miR157 in leaves 1&2 greatly exceeds the threshold required to specify their identity. Subsequent leaves have relatively low levels of miR156/miR157 and are sensitive to small changes in their abundance. In these later-formed leaves, the amount of miR156/miR157 is close to the threshold required to specify juvenile vs. adult identity; a relatively small decrease in the abundance of miR156/157 in these leaves produces a disproportionately large increase in SPL proteins and a significant change in leaf morphology. miR157 is more abundant than miR156 but has a smaller effect on shoot morphology and SPL gene expression than miR156. This may be attributable to the inefficiency with which miR157 is loaded onto AGO1, as well as to the presence of an extra nucleotide at the 5' end of miR157 that is mis-paired in the miR157:SPL13 duplex. miR156 represses different targets by different mechanisms: it regulates SPL9 by a combination of transcript cleavage and translational repression and regulates SPL13 primarily by translational repression. Our results offer a molecular explanation for the changes in leaf morphology that occur during shoot development in Arabidopsis and provide new insights into the mechanism by which miR156 and miR157 regulate gene expression.

Genome-Wide Mapping of Uncapped and Cleaved Transcripts Reveals a Role for the Nuclear mRNA Cap-Binding Complex in Cotranslational RNA Decay in Arabidopsis.

RNA turnover is necessary for controlling proper mRNA levels posttranscriptionally. In general, RNA degradation is via exoribonucleases that degrade RNA either from the 5' end to the 3' end, such as XRN4, or in the opposite direction by the multisubunit exosome complex. Here, we use genome-wide mapping of uncapped and cleaved transcripts to reveal the global landscape of cotranslational mRNA decay in the Arabidopsis thaliana transcriptome. We found that this process leaves a clear three nucleotide periodicity in open reading frames. This pattern of cotranslational degradation is especially evident near the ends of open reading frames, where we observe accumulation of cleavage events focused 16 to 17 nucleotides upstream of the stop codon because of ribosomal pausing during translation termination. Following treatment of Arabidopsis plants with the translation inhibitor cycloheximide, cleavage events accumulate 13 to 14 nucleotides upstream of the start codon where initiating ribosomes have been stalled with these sequences in their P site. Further analysis in xrn4 mutant plants indicates that cotranslational RNA decay is XRN4 dependent. Additionally, studies in plants lacking CAP BINDING PROTEIN80/ABA HYPERSENSITIVE1, the largest subunit of the nuclear mRNA cap binding complex, reveal a role for this protein in cotranslational decay. In total, our results demonstrate the global prevalence and features of cotranslational RNA decay in a plant transcriptome.

Protein Interaction Profile Sequencing (PIP-seq) in Plants.

RNA secondary structure and RNA-protein interactions are necessary for maintaining biological functionality and regulatory mechanisms within eukaryotic transcriptomes. Determining the structural characteristics and protein-bound sites of RNA molecules has therefore become a major research objective and requires the development of global methods for probing intra- and intermolecular RNA interaction sites. Sequencing RNAs treated with single-strand- and double-strand-specific ribonucleases in the absence of proteins allows the inference of RNA secondary structure. These samples can be compared to samples treated with nucleases in the presence of interacting proteins to identify protein-bound sequences. Thus, these four libraries reveal a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites in a single experiment for any plant species of interest. © 2016 by John Wiley & Sons, Inc.

The Arabidopsis Mediator CDK8 module genes CCT (MED12) and GCT (MED13) are global regulators of developmental phase transitions.

Temporal coordination of developmental programs is necessary for normal ontogeny, but the mechanism by which this is accomplished is still poorly understood. We have previously shown that two components of the Mediator CDK8 module encoded by CENTER CITY (CCT; Arabidopsis MED12) and GRAND CENTRAL (GCT; Arabidopsis MED13) are required for timing of pattern formation during embryogenesis. A morphological, molecular and genomic analysis of the post-embryonic phenotype of gct and cct mutants demonstrated that these genes also promote at least three subsequent developmental transitions: germination, vegetative phase change, and flowering. Genetic and molecular analyses indicate that GCT and CCT operate in parallel to gibberellic acid, a phytohormone known to regulate these same three transitions. We demonstrate that the delay in vegetative phase change in gct and cct is largely due to overexpression of miR156, and that the delay in flowering is due in part to upregulation of FLC. Thus, GCT and CCT coordinate vegetative and floral transitions by repressing the repressors miR156 and FLC. Our results suggest that MED12 and MED13 act as global regulators of developmental timing by fine-tuning the expression of temporal regulatory genes.

Global Regulation of Embryonic Patterning in Arabidopsis by MicroRNAs.

The development of the embryo in Arabidopsis (Arabidopsis thaliana) involves a carefully controlled set of cell divisions and cell fate decisions that lead to a mature embryo containing shoot and root meristems and all basic tissue types. Over the last 20 years, a number of transcriptional regulators of embryonic patterning have been described, but little is known about the role of posttranscriptional regulators such as microRNAs (miRNAs). Previous work has centered on the study of null or very weak alleles of miRNA biosynthetic genes, but these mutants either arrest early in embryogenesis or have wild-type-looking embryos. Here, we significantly extend those analyses by characterizing embryos mutant for a strong hypomorphic allele of DICER-LIKE1 (dcl1-15). Our data demonstrate that miRNAs are required for the patterning of most regions of the embryo, with the exception of the protoderm. In mutant embryos with the most severe morphological defects, the majority of tissue identities are lost. Different levels of miRNAs appear to be required to specify cell fates in various regions of the embryo. The suspensor needs the lowest levels, followed by the root apical meristem and hypocotyl, cotyledons, and shoot apical meristem. Furthermore, we show that erecta acts as a suppressor of dcl1-15, a novel role for this signaling pathway in embryos. Our results also indicate that the regulation of the messenger RNA levels of miRNA targets involves not just the action of miRNAs but has a significant transcriptional component as well.

Improved genome-wide mapping of uncapped and cleaved transcripts in eukaryotes--GMUCT 2.0.

The advent of high-throughput sequencing has led to an explosion of studies into the diversity, expression, processing, and lifespan of RNAs. Recently, three different high-throughput sequencing-based methods have been developed to specifically study RNAs that are in the process of being degraded. All three methods-genome-wide mapping of uncapped and cleaved transcripts (GMUCT), parallel analysis of RNA ends (PARE), and degradome sequencing-take advantage of the fact that Illumina sequencing libraries use T4 RNA ligase 1 to ligate an adapter to the 5' end of RNAs that have a free 5'-monophosphate. This condition for T4 RNA ligase 1 substrates means that mature mRNAs are not substrates of the enzyme because they have a 5'-cap moiety. As a result, these sequencing libraries are specifically made up of clones of decapped or degrading mRNAs resulting from 5'-to-3' or nonsense-mediated decay (NMD) and the 3' fragment of cleaved microRNA (miRNA) and small interfering RNA (siRNA) target RNAs. Here, we present a massively streamlined protocol for GMUCT that takes 2-3days, can be initiated with as little as 5µg of starting total RNA, and involves only one gel size-selection step. We show that the resulting datasets are similar to those produced using the previous GMUCT and PARE protocols. In total, our results suggest that this method will be the preferable approach for future studies of RNA degradation intermediates and small RNA-mediated cleavage in eukaryotic transcriptomes.

Regulatory impact of RNA secondary structure across the Arabidopsis transcriptome.

The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, and function. However, the global influence of this feature on plant gene expression is still largely unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of rRNA-depleted (RNA sequencing), small RNA, and ribosome-bound RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis thaliana. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing evidence that secondary structure is necessary for these RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We further reveal that RNA folding is significantly anticorrelated with overall transcript abundance, which is often due to the increased propensity of highly structured mRNAs to be degraded and/or processed into small RNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. These findings provide a global assessment of RNA folding and its significant regulatory effects in a plant transcriptome.

Is there a role for trihelix transcription factors in embryo maturation?

The development of the angiosperm seed includes the accumulation of storage products, the loss of most of its water and the establishment of dormancy. While much is known about the pathways that initiate maturation during mid-embryogenesis or repress it after germination, only recently has it been shown that other mechanisms repress the program during early embryogenesis.Two recent reports have shown that microRNAs are critical regulators of maturation in Arabidopsis early embryogenesis. Two closely related trihelix transcription factors, ASIL1 and ASIL2, were identified as probable partially redundant repressors of early maturation downstream of the microRNA-synthesizing enzyme DICER-LIKE1. An overlap between the genes upregulated in asil1-1 and dcl1-15 mutants support this conclusion. ASIL2 orthologs are found across seed plants, indicating that their role in maturation might be conserved. ASIL1 arose from the ancestral ASIL2 clade by a gene duplication event in the Brassicaceae, although it is not clear whether its function has diverged.

Experimental design, preprocessing, normalization and differential expression analysis of small RNA sequencing experiments.

Prior to the advent of new, deep sequencing methods, small RNA (sRNA) discovery was dependent on Sanger sequencing, which was time-consuming and limited knowledge to only the most abundant sRNA. The innovation of large-scale, next-generation sequencing has exponentially increased knowledge of the biology, diversity and abundance of sRNA populations. In this review, we discuss issues involved in the design of sRNA sequencing experiments, including choosing a sequencing platform, inherent biases that affect sRNA measurements and replication. We outline the steps involved in preprocessing sRNA sequencing data and review both the principles behind and the current options for normalization. Finally, we discuss differential expression analysis in the absence and presence of biological replicates. While our focus is on sRNA sequencing experiments, many of the principles discussed are applicable to the sequencing of other RNA populations.

MicroRNAs regulate the timing of embryo maturation in Arabidopsis.

The seed is a key evolutionary adaptation of land plants that facilitates dispersal and allows for germination when the environmental conditions are adequate. Mature seeds are dormant and desiccated, with accumulated storage products that are to be used by the seedling after germination. These properties are imposed on the developing embryo by a maturation program, which operates during the later part of embryogenesis. A number of "master regulators" (the "LEC genes") required for the induction of the maturation program have been described, but it is not known what prevents this program from being expressed during early embryogenesis. Here, we report that Arabidopsis (Arabidopsis thaliana) embryos mutant for strong alleles of DICER-LIKE1, the enzyme responsible for the biosynthesis of microRNAs (miRNAs), mature earlier than their wild-type counterparts. This heterochronic phenotype indicates that miRNAs are key regulators of the timing of the maturation program. We demonstrate that miRNAs operate in part by repressing the master regulators LEAFY COTYLEDON2 and FUSCA3 and identify the trihelix transcription factors ARABIDOPSIS 6B-INTERACTING PROTEIN1-LIKE1 (ASIL1) and ASIL2 and the histone deacetylase HDA6/SIL1 as components that act downstream of miRNAs to repress the maturation program early in embryogenesis. Both ASIL1 and HDA6/SIL1 are known to act to prevent the expression of embryonic maturation genes after germination, but to our knowledge, this is the first time they have been shown to have a role during embryogenesis. Our data point to a common negative regulatory module of maturation during early embryogenesis and seedling development.

The effect of the floral repressor FLC on the timing and progression of vegetative phase change in Arabidopsis.

Plants undergo two major post-embryonic developmental transitions--the juvenile-to-adult vegetative transition (vegetative phase change) and the adult-to-reproductive transition (flowering). In woody plants, these transitions can be separated by years, but in herbaceous species they are often very close together, making it difficult to differentiate the effects of vegetative phase change and floral induction on vegetative development. To distinguish between these factors, we have compared the vegetative morphology of plants highly expressing the floral repressor FLC (FRI;FLC) with plants mutant for this gene (FRI;flc-3) under both photoinductive (long day, LD and night interruption, NI) and non-photoinductive (short day, SD) conditions. We show that the onset of abaxial trichome production is insensitive to floral induction, but the distribution and overall number of abaxial trichomes, as well as several other leaf traits associated with vegetative change, are strongly influenced by flowering. Most of the major differences in leaf morphology between FRI;FLC and FRI;flc-3 plants grown in LD can be attributed to the early flowering phenotype of FRI;flc-3, because these differences are not apparent in plants grown in SD. These include differences in leaf size, hydathode number and the distribution of abaxial trichomes along the length of the leaf. Leaf shape and the total number of abaxial trichomes are affected by FLC independently of its effect on flowering. Our results demonstrate that the onset and the progression of vegetative phase change are regulated by different combinations of endogenous and environmental factors, and reveal a role for FLC in vegetative development.

The Functions of RNA-Dependent RNA Polymerases in Arabidopsis.

One recently identified mechanism that regulates mRNA abundance is RNA silencing, and pioneering work in Arabidopsis thaliana and other genetic model organisms helped define this process. RNA silencing pathways are triggered by either self-complementary fold-back structures or the production of double-stranded RNA (dsRNA) that gives rise to small RNAs (smRNAs) known as microRNAs (miRNAs) or small-interfering RNAs (siRNAs). These smRNAs direct sequence-specific regulation of various gene transcripts, repetitive sequences, viruses, and mobile elements via RNA cleavage, translational inhibition, or transcriptional silencing through DNA methylation and heterochromatin formation. Early genetic screens in Arabidopsis were instrumental in uncovering numerous proteins required for these important regulatory pathways. Among the factors identified by these studies were RNA-dependent RNA polymerases (RDRs), which are proteins that synthesize siRNA-producing dsRNA molecules using a single-stranded RNA (ssRNA) molecule as a template. Recently, a growing body of evidence has implicated RDR-dependent RNA silencing in many different aspects of plant biology ranging from reproductive development to pathogen resistance. Here, we focus on the specific functions of the six Arabidopsis RDRs in RNA silencing, their ssRNA substrates and resulting RDR-dependent smRNAs, and the numerous biological functions of these proteins in plant development and stress responses.