SCR106 splicing factor modulates abiotic stress responses by maintaining RNA splicing in rice.

Plants employ sophisticated molecular machinery to fine-tune their responses to growth, developmental, and stress cues. Gene expression influences plant cellular responses through regulatory processes such as transcription and splicing. Pre-mRNA is alternatively spliced to increase the genome coding potential and further regulate expression. Serine/arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Several studies have reported SR protein genes in the rice genome, subdivided into six subfamilies based on their domain structures. Here, we identified a new splicing factor in rice with an RNA recognition motif (RRM) and SR-dipeptides, which is related to the SR proteins, subfamily SC. OsSCR106 regulates pre-mRNA splicing under abiotic stress conditions. It localizes to the nuclear speckles, a major site for pre-mRNA splicing in the cell. The loss-of-function scr106 mutant is hypersensitive to salt, abscisic acid, and low-temperature stress, and harbors a developmental abnormality indicated by the shorter length of the shoot and root. The hypersensitivity to stress phenotype was rescued by complementation using OsSCR106 fused behind its endogenous promoter. Global gene expression and genome-wide splicing analysis in wild-type and scr106 seedlings revealed that OsSCR106 regulates its targets, presumably through regulating the alternative 3'-splice site. Under salt stress conditions, we identified multiple splice isoforms regulated by OsSCR106. Collectively, our results suggest that OsSCR106 is an important splicing factor that plays a crucial role in accurate pre-mRNA splicing and regulates abiotic stress responses in plants.

Crop bioengineering via gene editing: reshaping the future of agriculture.

Genome-editing technologies have revolutionized research in plant biology, with major implications for agriculture and worldwide food security, particularly in the face of challenges such as climate change and increasing human populations. Among these technologies, clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR-associated protein [Cas] systems are now widely used for editing crop plant genomes. In this review, we provide an overview of CRISPR-Cas technology and its most significant applications for improving crop sustainability. We also review current and potential technological advances that will aid in the future breeding of crops to enhance food security worldwide. Finally, we discuss the obstacles and challenges that must be overcome to realize the maximum potential of genome-editing technologies for future crop and food production.

Pre-mRNA splicing repression triggers abiotic stress signaling in plants.

Alternative splicing (AS) of precursor RNAs enhances transcriptome plasticity and proteome diversity in response to diverse growth and stress cues. Recent work has shown that AS is pervasive across plant species, with more than 60% of intron-containing genes producing different isoforms. Mammalian cell-based assays have discovered various inhibitors of AS. Here, we show that the macrolide pladienolide B (PB) inhibits constitutive splicing and AS in plants. Also, our RNA sequencing (RNA-seq) data revealed that PB mimics abiotic stress signals including salt, drought and abscisic acid (ABA). PB activates the abiotic stress- and ABA-responsive reporters RD29A::LUC and MAPKKK18::uidA in Arabidopsis thaliana and mimics the effects of ABA on stomatal aperture. Genome-wide analysis of AS by RNA-seq revealed that PB perturbs the splicing machinery and leads to a striking increase in intron retention and a reduction in other forms of AS. Interestingly, PB treatment activates the ABA signaling pathway by inhibiting the splicing of clade A PP2C phosphatases while still maintaining to some extent the splicing of ABA-activated SnRK2 kinases. Taken together, our data establish PB as an inhibitor and modulator of splicing and a mimic of abiotic stress signals in plants. Thus, PB reveals the molecular underpinnings of the interplay between stress responses, ABA signaling and post-transcriptional regulation in plants.

Engineering plant architecture via CRISPR/Cas9-mediated alteration of strigolactone biosynthesis.

BACKGROUND: Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). RESULTS: We used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a reduced height, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography-mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. CONCLUSIONS: Taken together, our results show the potential and feasibility of the use of the CRISPR/Cas9 system for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

Herboxidiene triggers splicing repression and abiotic stress responses in plants.

BACKGROUND: Constitutive and alternative splicing of pre-mRNAs from multiexonic genes controls the diversity of the proteome; these precisely regulated processes also fine-tune responses to cues related to growth, development, and stresses. Small-molecule inhibitors that perturb splicing provide invaluable tools for use as chemical probes to uncover the molecular underpinnings of splicing regulation and as potential anticancer compounds. RESULTS: Here, we show that herboxidiene (GEX1A) inhibits both constitutive and alternative splicing. Moreover, GEX1A activates genome-wide transcriptional patterns involved in abiotic stress responses in plants. GEX1A treatment -activated ABA-inducible promoters, and led to stomatal closure. Interestingly, GEX1A and pladienolide B (PB) elicited similar cellular changes, including alterations in the patterns of transcription and splicing, suggesting that these compounds might target the same spliceosome complex in plant cells. CONCLUSIONS: Our study establishes GEX1A as a potent splicing inhibitor in plants that can be used to probe the assembly, dynamics, and molecular functions of the spliceosome and to study the interplay between splicing stress and abiotic stresses, as well as having potential biotechnological applications.

Expression analysis of Arabidopsis XH/XS-domain proteins indicates overlapping and distinct functions for members of this gene family.

RNA-directed DNA methylation (RdDM) is essential for de novo DNA methylation in higher plants, and recent reports established novel elements of this silencing pathway in the model organism Arabidopsis thaliana. Involved in de novo DNA methylation 2 (IDN2) and the closely related factor of DNA methylation (FDM) are members of a plant-specific family of dsRNA-binding proteins characterized by conserved XH/XS domains and implicated in the regulation of RdDM at chromatin targets. Genetic analyses have suggested redundant as well as non-overlapping activities for different members of the gene family. However, detailed insights into the function of XH/XS-domain proteins are still elusive. By the generation and analysis of higher-order mutant combinations affected in IDN2 and further members of the gene family, we have provided additional evidence for their redundant activity. Distinct roles for members of the XH/XS-domain gene family were indicated by differences in their expression and subcellular localization. Fluorescent protein-tagged FDM genes were expressed either in nuclei or in the cytoplasm, suggestive of activities of XH/XS-domain proteins in association with chromatin as well as outside the nuclear compartment. In addition, we observed altered location of a functional FDM1-VENUS reporter from the nucleus into the cytoplasm under conditions when availability of further FDM proteins was limited. This is suggestive of a mechanism by which redistribution of XH/XS-domain proteins could compensate for the loss of closely related proteins.

Putative Arabidopsis transcriptional adaptor protein (PROPORZ1) is required to modulate histone acetylation in response to auxin.

Plant development is highly adaptable and controlled by a combination of various regulatory circuits that integrate internal and environmental cues. The phytohormone auxin mediates such growth responses, acting as a dynamic signal in the control of morphogenesis via coordinating the interplay between cell cycle progression and cell differentiation. Mutants in the chromatin-remodeling component PROPORZ1 (PRZ1; also known as AtADA2b) are impaired in auxin effects on morphogenesis, suggestive of an involvement of PRZ1-dependent control of chromatin architecture in the determination of hormone responses. Here we demonstrate that PRZ1 is required for accurate histone acetylation at auxin-controlled loci. Specifically, PRZ1 is involved in the modulation of histone modifications and corresponding adjustments in gene expression of Arabidopsis KIP RELATED PROTEIN (KRP) CDK inhibitor genes in response to auxin. Deregulated KRP expression in KRP silencer lines phenocopies prz1 hyperproliferative growth phenotypes, whereas in a KRP overexpression background some mutant phenotypes are suppressed. Collectively, our findings support a model in which translation of positional signals into developmental cues involves adjustments in chromatin modifications that orchestrate auxin effects on cell proliferation.

Multi-omics resources for targeted agronomic improvement of pigmented rice.

Pigmented rice (Oryza sativa L.) is a rich source of nutrients, but pigmented lines typically have long life cycles and limited productivity. Here we generated genome assemblies of 5 pigmented rice varieties and evaluated the genetic variation among 51 pigmented rice varieties by resequencing an additional 46 varieties. Phylogenetic analyses divided the pigmented varieties into four varietal groups: Geng-japonica, Xian-indica, circum-Aus and circum-Basmati. Metabolomics and ionomics profiling revealed that black rice varieties are rich in aromatic secondary metabolites. We established a regeneration and transformation system and used CRISPR-Cas9 to knock out three flowering time repressors (Hd2, Hd4 and Hd5) in the black Indonesian rice Cempo Ireng, resulting in an early maturing variety with shorter stature. Our study thus provides a multi-omics resource for understanding and improving Asian pigmented rice.

Synthetic evolution of herbicide resistance using a T7 RNAP-based random DNA base editor.

Synthetic directed evolution via localized sequence diversification and the simultaneous application of selection pressure is a promising method for producing new, beneficial alleles that affect traits of interest in diverse species; however, this technique has rarely been applied in plants. Here, we designed, built, and tested a chimeric fusion of T7 RNA Polymerase (RNAP) and deaminase to enable the localized sequence diversification of a target sequence of interest. We tested our T7 RNAP-DNA base editor in <i>Nicotiana benthamiana</i> transient assays to target a transgene expressing <i>GFP</i> under the control of the T7 promoter and observed C-to-T conversions. We then targeted the T7 promoter-driven <i>acetolactate synthase</i> sequence that had been stably integrated in the rice genome and generated C-to-T and G-to-A transitions. We used herbicide treatment as selection pressure for the evolution of the <i>acetolactate synthase</i> sequence, resulting in the enrichment of herbicide-responsive residues. We then validated these herbicide-responsive regions in the transgenic rice plants. Thus, our system could be used for the continuous synthetic evolution of gene functions to produce variants with improved herbicide resistance.

High-efficiency retron-mediated single-stranded DNA production in plants.

Retrons are a class of retroelements that produce multicopy single-stranded DNA (ssDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the overproduction of ssDNA, genome engineering and directed evolution in bacteria, yeast and mammalian cells. Retron-mediated ssDNA production in plants could unlock their potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Here, we demonstrated retron-mediated overproduction of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated clustered regularly interspaced short palindromic repeats-retron-coupled ssDNA overproduction and targeted HDR in N. benthamiana. Overall, we present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications. Graphical Abstract.

The Rice Serine/Arginine Splicing Factor RS33 Regulates Pre-mRNA Splicing during Abiotic Stress Responses.

Abiotic stresses profoundly affect plant growth and development and limit crop productivity. Pre-mRNA splicing is a major form of gene regulation that helps plants cope with various stresses. Serine/arginine (SR)-rich splicing factors play a key role in pre-mRNA splicing to regulate different biological processes under stress conditions. Alternative splicing (AS) of SR transcripts and other transcripts of stress-responsive genes generates multiple splice isoforms that contribute to protein diversity, modulate gene expression, and affect plant stress tolerance. Here, we investigated the function of the plant-specific SR protein RS33 in regulating pre-mRNA splicing and abiotic stress responses in rice. The loss-of-function mutant rs33 showed increased sensitivity to salt and low-temperature stresses. Genome-wide analyses of gene expression and splicing in wild-type and rs33 seedlings subjected to these stresses identified multiple splice isoforms of stress-responsive genes whose AS are regulated by RS33. The number of RS33-regulated genes was much higher under low-temperature stress than under salt stress. Our results suggest that the plant-specific splicing factor RS33 plays a crucial role during plant responses to abiotic stresses.

Overlapping roles of spliceosomal components SF3B1 and PHF5A in rice splicing regulation.

The SF3B complex, a multiprotein component of the U2 snRNP of the spliceosome, plays a crucial role in recognizing branch point sequence and facilitates spliceosome assembly and activation. Several chemicals that bind SF3B1 and PHF5A subunits of the SF3B complex inhibit splicing. We recently generated a splicing inhibitor-resistant SF3B1 mutant named SF3B1 GEX1A RESISTANT 4 (SGR4) using CRISPR-mediated directed evolution, whereas splicing inhibitor-resistant mutant of PHF5A (Overexpression-PHF5A GEX1A Resistance, OGR) was generated by expressing an engineered version PHF5A-Y36C. Global analysis of splicing in wild type and these two mutants revealed the role of SF3B1 and PHF5A in splicing regulation. This analysis uncovered a set of genes whose intron retention is regulated by both proteins. Further analysis of these retained introns revealed that they are shorter, have a higher GC content, and contain shorter and weaker polypyrimidine tracts. Furthermore, splicing inhibition increased seedlings sensitivity to salt stress, consistent with emerging roles of splicing regulation in stress responses. In summary, we uncovered the functions of two members of the plant branch point recognition complex. The novel strategies described here should be broadly applicable in elucidating functions of splicing regulators, especially in studying the functions of redundant paralogs in plants.

CRISPR-Based Directed Evolution for Crop Improvement.

Directed evolution involves generating diverse sequence variants of a gene of interest to produce a desirable trait under selective pressure. CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) systems can be programmed to target any genomic locus and perform targeted directed evolution. Here, we discuss the opportunities and challenges of this emerging platform for targeted crop improvement.

Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice.

Precise genome editing by systems such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) requires high-efficiency homology-directed repair (HDR). Different technologies have been developed to improve HDR but with limited success. Here, we generated a fusion between the Cas9 endonuclease and the Agrobacterium VirD2 relaxase (Cas9-VirD2). This chimeric protein combines the functions of Cas9, which produces targeted and specific DNA double-strand breaks (DSBs), and the VirD2 relaxase, which brings the repair template in close proximity to the DSBs, to facilitate HDR. We successfully employed our Cas9-VirD2 system for precise ACETOLACTATE SYNTHASE (OsALS) allele modification to generate herbicide-resistant rice (Oryza sativa) plants, CAROTENOID CLEAVAGE DIOXYGENASE-7 (OsCCD7) to engineer plant architecture, and generate in-frame fusions with the HA epitope at HISTONE DEACETYLASE (OsHDT) locus. The Cas9-VirD2 system expands our ability to improve agriculturally important traits in crops and opens new possibilities for precision genome engineering across diverse eukaryotic species.

Multiplex CRISPR Mutagenesis of the Serine/Arginine-Rich (SR) Gene Family in Rice.

Plant growth responds to various environmental and developmental cues via signaling cascades that influence gene expression at the level of transcription and pre-mRNA splicing. Alternative splicing of pre-mRNA increases the coding potential of the genome from multiexon genes and regulates gene expression through multiple mechanisms. Serine/arginine-rich (SR) proteins, a conserved family of splicing factors, are the key players of alternative splicing and regulate pre-mRNA splicing under stress conditions. The rice (Oryza sativa) genome encodes 22 SR proteins categorized into six subfamilies. Three of the subfamilies are plant-specific with no mammalian orthologues, and the functions of these SR proteins are not well known. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a genome engineering tool that cleaves the target DNA at specific locations directed by a guide RNA (gRNA). Recent advances in CRISPR/Cas9-mediated plant genome engineering make it possible to generate single and multiple functional knockout mutants in diverse plant species. In this study, we targeted each rice SR locus and produced single knockouts. To overcome the functional redundancy within each subfamily of SR genes, we utilized a polycistronic tRNA-gRNA multiplex targeting system and targeted all loci of each subfamily. Sanger sequencing results indicated that most of the targeted loci had knockout mutations. This study provides useful resource materials for understanding the molecular role of SR proteins in plant development and biotic and abiotic stress responses.

Serine/Arginine-rich protein family of splicing regulators: New approaches to study splice isoform functions.

Serine/arginine-rich (SR) proteins are conserved RNA-binding proteins that play major roles in RNA metabolism. They function as molecular adaptors, facilitate spliceosome assembly and modulate constitutive and alternative splicing of pre-mRNAs. Pre-mRNAs encoding SR proteins and many other proteins involved in stress responses are extensively alternatively spliced in response to diverse stresses. Hence, it is proposed that stress-induced changes in splice isoforms contribute to the adaptation of plants to stress responses. However, functions of most SR genes and their splice isoforms in stress responses are not known. Lack of easy and robust tools hindered the progress in this area. Emerging technologies such as CRISPR/Cas9 will facilitate studies of SR function by enabling the generation of single and multiple knock-out mutants of SR subfamily members. Moreover, CRISPR/Cas13 allows targeted manipulation of splice isoforms from SR and other genes in a constitutive or tissue-specific manner to evaluate functions of individual splice variants. Identification of the in vivo targets of SR proteins and their splice variants using the recently developed TRIBE (Targets of RNA-binding proteins Identified By Editing) and other methods will help unravel their mode of action and splicing regulatory elements under various conditions. These new approaches are expected to provide significant new insights into the roles of SRs and splice isoforms in plants adaptation to diverse stresses.

Large Language Model Performance on Practice Epilepsy Board Examinations.

This diagnostic study examines whether large language models are able to pass practice licensing examinations for epilepsy.

CRISPR directed evolution of the spliceosome for resistance to splicing inhibitors.

Increasing genetic diversity via directed evolution holds great promise to accelerate trait development and crop improvement. We developed a CRISPR/Cas-based directed evolution platform in plants to evolve the rice (Oryza sativa) SF3B1 spliceosomal protein for resistance to splicing inhibitors. SF3B1 mutant variants, termed SF3B1-GEX1A-Resistant (SGR), confer variable levels of resistance to splicing inhibitors. Studies of the structural basis of the splicing inhibitor binding to SGRs corroborate the resistance phenotype. This directed evolution platform can be used to interrogate and evolve the molecular functions of key biomolecules and to engineer crop traits for improved performance and adaptation under climate change conditions.