RESUMEN
Cuscuta species (dodders) are agriculturally destructive, parasitic angiosperms. These parasitic plants use haustoria as physiological bridges to extract nutrients and water from hosts. Cuscuta campestris has a broad host range and wide geographical distribution. While some wild tomato relatives are resistant, cultivated tomatoes are generally susceptible to C. campestris infestations. However, some specific Heinz tomato (Solanum lycopersicum) hybrid cultivars exhibit resistance to dodders in the field, but their defense mechanism was previously unknown. Here, we discovered that the stem cortex in these resistant lines responds with local lignification upon C. campestris attachment, preventing parasite entry into the host. Lignin Induction Factor 1 (LIF1, an AP2-like transcription factor), SlMYB55, and Cuscuta R-gene for Lignin-based Resistance 1, a CC-NBS-LRR (CuRLR1) are identified as factors that confer host resistance by regulating lignification. SlWRKY16 is upregulated upon C. campestris infestation and potentially negatively regulates LIF1 function. Intriguingly, CuRLR1 may play a role in signaling or function as an intracellular receptor for receiving Cuscuta signals or effectors, thereby regulating lignification-based resistance. In summary, these four regulators control the lignin-based resistance response in specific Heinz tomato cultivars, preventing C. campestris from parasitizing resistant tomatoes. This discovery provides a foundation for investigating multilayer resistance against Cuscuta species and has potential for application in other essential crops attacked by parasitic plants.
Asunto(s)
Cuscuta , Solanum lycopersicum , Solanum , Cuscuta/fisiología , Especificidad del Huésped , Lignina , Solanum lycopersicum/genéticaRESUMEN
Plants produce specialized metabolites to protect themselves from biotic enemies. Members of the Solanaceae family accumulate phenylpropanoid-polyamine conjugates (PPCs) in response to attackers while also maintaining a chemical barrier of steroidal glycoalkaloids (SGAs). Across the plant kingdom, biosynthesis of such defense compounds is promoted by jasmonate signaling in which clade IIIe basic helix-loop-helix (bHLH) transcription factors play a central role. By characterizing hairy root mutants obtained through Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (CRISPR-Cas9) genome editing, we show that the tomato clade IIIe bHLH transcription factors, MYC1 and MYC2, redundantly control jasmonate-inducible PPC and SGA production, and are also essential for constitutive SGA biosynthesis. Double myc1 myc2 loss-of-function tomato hairy roots displayed suppressed constitutive expression of SGA biosynthesis genes, and severely reduced levels of the main tomato SGAs α-tomatine and dehydrotomatine. In contrast, basal expression of genes involved in PPC biosynthesis was not affected. CRISPR-Cas9(VQR) genome editing of a specific cis-regulatory element, targeted by MYC1/2, in the promoter of a SGA precursor biosynthesis gene led to decreased constitutive expression of this gene, but did not affect its jasmonate inducibility. Our results demonstrate that clade IIIe bHLH transcriptional regulators have evolved under the control of distinct regulatory cues to specifically steer constitutive and stress-inducible specialized metabolism.
Asunto(s)
Solanum lycopersicum , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Ciclopentanos/metabolismo , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Oxilipinas/metabolismo , Poliaminas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Creating true-breeding lines is a critical step in plant breeding. Novel, completely homozygous true-breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere-specific histone 3 variant (CENH3), including chimeric proteins, expression of non-native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild-type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS-inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9-mediated in-frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild-type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non-transgenic approaches to the generation of haploid inducers.
RESUMEN
Spatiotemporal regulation of transcription is fine-tuned at multiple levels, including chromatin compaction. Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which is the hallmark of a repressive chromatin state. Multiple PRC2 complexes have been reported in Arabidopsis thaliana to control the expression of genes involved in developmental transitions and maintenance of organ identity. Here, we show that PRC2 member genes display complex spatiotemporal gene expression patterns and function in root meristem and vascular cell proliferation and specification. Furthermore, PRC2 gene expression patterns correspond with vascular and nonvascular tissue-specific H3K27me3-marked genes. This tissue-specific repression via H3K27me3 regulates the balance between cell proliferation and differentiation. Using enhanced yeast one-hybrid analysis, upstream regulators of the PRC2 member genes are identified, and genetic analysis demonstrates that transcriptional regulation of some PRC2 genes plays an important role in determining PRC2 spatiotemporal activity within a developing organ.
Asunto(s)
Arabidopsis/metabolismo , Complejo Represivo Polycomb 2/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Complejo Represivo Polycomb 2/genética , Regiones Promotoras Genéticas/genéticaRESUMEN
Natural cis-antisense siRNAs (cis-nat-siRNAs) are a recently characterized class of small regulatory RNAs that are widespread in eukaryotes. Despite their abundance, the importance of their regulatory activity is largely unknown. The only functional role for eukaryotic cis-nat-siRNAs that has been described to date is in environmental stress responses in plants. Here we demonstrate that cis-nat-siRNA-based regulation plays key roles in Arabidopsis reproductive function, as it facilitates gametophyte formation and double fertilization, a developmental process of enormous agricultural value. We show that male gametophytic kokopelli (kpl) mutants display frequent single-fertilization events, and that KPL and a inversely transcribed gene, ARIADNE14 (ARI14), which encodes a putative ubiquitin E3 ligase, generate a sperm-specific nat-siRNA pair. In the absence of KPL, ARI14 RNA levels in sperm are increased and fertilization is impaired. Furthermore, ARI14 transcripts accumulate in several siRNA biogenesis pathway mutants, and overexpression of ARI14 in sperm phenocopies the reduced seed set of the kokopelli mutants. These results extend the regulatory capacity of cis-nat-siRNAs to development by identifying a role for cis-nat-siRNAs in controlling sperm function during double fertilization.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Fertilización/genética , Regulación de la Expresión Génica de las Plantas , ARN sin Sentido/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas de Arabidopsis/genética , Perfilación de la Expresión Génica , Mutación/genética , Óvulo Vegetal/crecimiento & desarrollo , Fenotipo , Polen/genética , ARN Interferente Pequeño/biosíntesisRESUMEN
Genetic markers are essential when developing or working with genetically variable populations. Indel Group in Genomes (IGG) markers are primer pairs that amplify single-locus sequences that differ in size for two or more alleles. They are attractive for their ease of use for rapid genotyping and their codominant nature. Here, we describe a heuristic algorithm that uses a k-mer-based approach to search two or more genome sequences to locate polymorphic regions suitable for designing candidate IGG marker primers. As input to the IGG pipeline software, the user provides genome sequences and the desired amplicon sizes and size differences. Primer sequences flanking polymorphic insertions/deletions are produced as output. IGG marker files for three sets of genomes, Solanum lycopersicum/Solanum pennellii, Arabidopsis (Arabidopsis thaliana) Columbia-0/Landsberg erecta-0 accessions, and S. lycopersicum/S. pennellii/Solanum tuberosum (three-way polymorphic) are included.
Asunto(s)
Marcadores Genéticos/genética , Genoma de Planta/genética , Mutación INDEL , Polimorfismo de Nucleótido Simple , Alelos , Arabidopsis/genética , Secuencia de Bases , Mapeo Cromosómico , Cromosomas de las Plantas/genética , Biología Computacional/métodos , Genotipo , Solanum lycopersicum/genética , Solanum/genética , Especificidad de la EspecieRESUMEN
Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.
Asunto(s)
Agrobacterium/fisiología , Regulación de la Expresión Génica de las Plantas , Modelos Biológicos , Raíces de Plantas/fisiología , Solanum lycopersicum/fisiología , Secuencia de Bases , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN de Plantas , Solanum lycopersicum/genética , Datos de Secuencia Molecular , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido NucleicoRESUMEN
While the Arabidopsis (Arabidopsis thaliana) root has been elegantly characterized with respect to specification of cell identity, its development is missing a number of cellular features present in other species. We have characterized the root development of a wild and a domesticated tomato species, Solanum pennellii and Solanum lycopersicum 'M82.' We found extensive differences between these species for root morphology and cellular development including root length, a novel gravity set point angle, differences in cortical cell layer patterning, stem cell niche structure, and radial cell division. Using an introgression line population between these two species, we identified numerous loci that regulate these distinct aspects of development. Specifically we comprehensively identified loci that regulate (1) root length by distinct mechanisms including regulation of cell production within the meristem and the balance between cell division and expansion, (2) the gravity set point angle, and (3) radial cell division or expansion either in specific cell types or generally across multiple cell types. Our findings provide a novel perspective on the regulation of root growth and development between species. These loci have exciting implications with respect to regulation of drought resistance or salinity tolerance and regulation of root development in a family that has undergone domestication.
Asunto(s)
Raíces de Plantas/citología , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/genética , Sitios de Carácter Cuantitativo , Solanum lycopersicum/citología , Solanum lycopersicum/genética , División Celular/genética , Variación Genética , Gravitación , Meristema/genética , Raíces de Plantas/fisiologíaRESUMEN
GEX1 is a plasma membrane protein that is conserved among plant species, and has previously been shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immediately after fertilization and in the pollen vegetative cell. We functionally characterize GEX1 in Arabidopsis thaliana, and show that it is a versatile protein that performs functions during male and female gametophyte development, and during early embryogenesis. gex1-1/+ plants, which synthesize a truncated GEX1 mRNA encoding a protein lacking the predicted cytoplasmic domain, but still targeted to the plasma membrane, had embryos that arrested before the pre-globular stage. gex1-3/+ plants, carrying a null GEX1 allele, had defects during male and female gametophyte development, and during early embryogenesis. Using an antisense GEX1 transgenic line we demonstrate that the predicted GEX1 extracellular domain is sufficient and necessary for GEX1 function during the development of both gametophytes. The predicted cytoplasmic domain is necessary for correct early embryogenesis and mediates homodimer formation at the plasma membrane. We propose that dimerization of GEX1 in the zygote might be an upstream step in a signaling cascade regulating early embryogenesis.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/genética , Gametogénesis en la Planta , Células Germinativas de las Plantas/crecimiento & desarrollo , Arabidopsis/embriología , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Membrana Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Mutación , Fenotipo , Plantas Modificadas Genéticamente/embriología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Multimerización de ProteínaRESUMEN
Strigolactones (SLs) are a class of plant hormones that regulate numerous processes of growth and development. SL perception and signal activation involves interaction between F-box E3 ubiquitin ligase D3/MAX2 and DWARF14 (D14) α/ß-hydrolase in a SL-dependent manner and targeting of D53/SMXL6/7/8 transcriptional repressors (SMXLs) for proteasome-mediated degradation. D3/MAX2 has been shown to exist in multiple conformational states in which the C-terminal helix (CTH) undergoes a closed-to-open dynamics and regulates D14 binding and SL perception. Despite the multiple modes of D3-D14 interactions found in vitro, the residues that regulate the conformational switch of D3/MAX2 CTH in targeting D53/SMXLs and the subsequent effect on SL signalling remain unclear. Here we elucidate the functional dynamics of ASK1-D3/MAX2 in SL signalling by leveraging conformational switch mutants in vitro and in plants. We report the crystal structure of a dislodged CTH of the ASK1-D3 mutant and demonstrate that disruptions in CTH plasticity via either CRISPR-Cas9 genome editing or expression of point mutation mutants result in impairment of SL signalling. We show that the conformational switch in ASK1-D3/MAX2 CTH directly regulates ubiquitin-mediated protein degradation. A dislodged conformation involved in D53/SMXLs SL-dependent recruitment and ubiquitination and an engaged conformation are required for the release of polyubiquitinated D53/SMXLs and subsequently D14 for proteasomal degradation. Finally, we uncovered an organic acid metabolite that can directly trigger the D3/MAX2 CTH conformational switch. Our findings unravel a new regulatory function of a SKP1-CUL1-F-box ubiquitin ligase in plant signalling.
Asunto(s)
Proteínas de Arabidopsis , Oryza , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Compuestos Heterocíclicos con 3 Anillos , Lactonas , Oryza/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina/metabolismoRESUMEN
Elicitor recognition plays a key role in the reaction of plants to pathogens and the induction of plant defense responses. Furthermore, plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. Ethylene-inducing xylanase (Eix) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). The Eix receptors (LeEix1 and LeEix2) belong to a superclade of leucine-rich repeat receptor-like proteins (RLP) with a signal for receptor-mediated endocytosis, which was shown to be essential for proper induction of defense responses. Both receptors are able to bind Eix, while only LeEix2 mediates defense responses. Here we demonstrate that LeEix1 heterodimerizes with LeEix2 upon application of the Eix elicitor. We show that LeEix1 attenuates Eix-induced internalization and signaling of the LeEix2 receptor. Furthermore, we demonstrate, using yeast two-hybrid and in planta bimolecular fluorescence complementation assays, that the brassinosteroid co-receptor, BAK1, binds LeEix1 but not LeEix2. In BAK1-silenced plants, LeEix1 was no longer able to attenuate plant responses to Eix, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires BAK1.
Asunto(s)
Endo-1,4-beta Xilanasas/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinasas/metabolismo , Solanum lycopersicum/metabolismo , Endocitosis , Endosomas/metabolismo , Etilenos/metabolismo , Etilenos/farmacología , Proteínas Fúngicas/metabolismo , Hongos/metabolismo , Hongos/fisiología , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Huésped-Patógeno , Inmunidad Innata , Proteínas Repetidas Ricas en Leucina , Solanum lycopersicum/genética , Solanum lycopersicum/microbiología , Microscopía Confocal , Enfermedades de las Plantas/microbiología , Reguladores del Crecimiento de las Plantas/metabolismo , Reguladores del Crecimiento de las Plantas/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/genética , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nicotiana/genética , Nicotiana/microbiología , Técnicas del Sistema de Dos HíbridosRESUMEN
Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.
Asunto(s)
Badnavirus/genética , Sistemas CRISPR-Cas , Edición Génica/métodos , Genoma de Planta , Genoma Viral , Musa/genética , Fitomejoramiento/métodos , Badnavirus/patogenicidad , Secuencia de Bases , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Quimera/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Musa/virología , Mutación , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , Plantas Modificadas Genéticamente , Plásmidos/química , Plásmidos/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Alineación de Secuencia , Estrés FisiológicoRESUMEN
Broomrapes (Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 (CCD8). Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P. aegyptiaca. Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato (Solanum lycopersicum L.) plants. Several CCD8Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks.
Asunto(s)
Dioxigenasas/genética , Resistencia a la Enfermedad/genética , Orobanche , Proteínas de Plantas/genética , Malezas , Solanum lycopersicum/parasitología , Sistemas CRISPR-Cas/genética , Carotenoides/metabolismo , Dioxigenasas/metabolismo , Exones/genética , Regulación de la Expresión Génica de las Plantas , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Lactonas/metabolismo , Solanum lycopersicum/enzimología , Solanum lycopersicum/genética , Mutagénesis , Fitomejoramiento , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.
Asunto(s)
Arabidopsis/genética , Edición Génica/métodos , Genómica/métodos , Mutagénesis , ARN Guía de Kinetoplastida/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sistemas CRISPR-Cas , Reparación del ADN por Unión de Extremidades , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ARN Guía de Kinetoplastida/metabolismo , ADN Polimerasa thetaRESUMEN
CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature.
Asunto(s)
Sistemas CRISPR-Cas/genética , Edición Génica , Patrón de Herencia/genética , Lactuca/genética , Proteínas de Plantas/genética , Alelos , Técnicas de Inactivación de Genes , Marcadores Genéticos , Células Germinativas/metabolismo , Germinación/genética , Calor , Mutación/genética , Fenotipo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , ARN Guía de Kinetoplastida/genética , Análisis de Secuencia de ADN , Transformación GenéticaRESUMEN
Regulation of plant root angle is critical for obtaining nutrients and water and is an important trait for plant breeding. A plant's final, long-term root angle is the net result of a complex series of decisions made by a root tip in response to changes in nutrient availability, impediments, the gravity vector and other stimuli. When a root tip is displaced from the gravity vector, the short-term process of gravitropism results in rapid reorientation of the root toward the vertical. Here, we explore both short- and long-term regulation of root growth angle, using natural variation in tomato to identify shared and separate genetic features of the two responses. Mapping of expression quantitative trait loci mapping and leveraging natural variation between and within species including Arabidopsis suggest a role for PURPLE ACID PHOSPHATASE 27 and CELL DIVISION CYCLE 73 in determining root angle.
Asunto(s)
Fosfatasa Ácida , Proteínas de Arabidopsis , Arabidopsis , Glicoproteínas , Gravitropismo/fisiología , Raíces de Plantas , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrolloRESUMEN
This corrects the article DOI: 10.1038/ncomms15235.
RESUMEN
Most living organisms developed systems to efficiently time environmental changes. The plant-clock acts in coordination with external signals to generate output responses determining seasonal growth and flowering time. Here, we show that two Arabidopsis thaliana transcription factors, FAR1 RELATED SEQUENCE 7 (FRS7) and FRS12, act as negative regulators of these processes. These proteins accumulate particularly in short-day conditions and interact to form a complex. Loss-of-function of FRS7 and FRS12 results in early flowering plants with overly elongated hypocotyls mainly in short days. We demonstrate by molecular analysis that FRS7 and FRS12 affect these developmental processes in part by binding to the promoters and repressing the expression of GIGANTEA and PHYTOCHROME INTERACTING FACTOR 4 as well as several of their downstream signalling targets. Our data reveal a molecular machinery that controls the photoperiodic regulation of flowering and growth and offer insight into how plants adapt to seasonal changes.
Asunto(s)
Aldehído Oxidorreductasas/genética , Arabidopsis/genética , Flores/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Aldehído Oxidorreductasas/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Ritmo Circadiano/fisiología , Flores/crecimiento & desarrollo , Flores/metabolismo , Hipocótilo/genética , Hipocótilo/crecimiento & desarrollo , Hipocótilo/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Luz , Fotoperiodo , Transducción de Señal , Transcripción GenéticaRESUMEN
Transcriptional regulation plays a major role in defining cell identity. Analysis of cell type-resolution expression profiling datasets is moving beyond cataloging gene expression patterns to reveal novel biological insights. Recently developed expression maps of the shoot apical meristem and gametophytes can be used as tools to help define novel cell types and pathways. Already these maps have revealed cell type-specific epigenetic regulatory mechanisms that play important roles in development. Further examples are provided that demonstrate how cell type-specific expression profiling can also be used to uncover genes and pathways in development and response to stress that would be nearly impossible to identify using traditional genetics.
Asunto(s)
Células Vegetales/fisiología , Plantas/genética , Plantas/metabolismo , Perfilación de la Expresión GénicaRESUMEN
Double fertilization in flowering plants occurs when the two sperm cells, carried by the pollen tube, are released in a synergid cell of the embryo sac and then fertilize the egg and the central cell. Proteins on the surfaces of the sperm, egg, central, and synergid cells might be important for guidance and recognition/fusion of the gametes. Here, we present functional analyses of Arabidopsis GEX3, which encodes a plasma membrane-localized protein that has homologs in other plants. GEX3 is expressed in both the vegetative and sperm cells of the male gametophyte and in the egg cell of the female gametophyte. Transgenic lines in which GEX3 was down-regulated or overexpressed, using the Arabidopsis GEX2 promoter, had reduced seed set. Reciprocal crosses and imaging after pollination with a reporter line showed that, in both cases, the defect causing reduced seed set occurred in the female. In the antisense lines, micropylar pollen tube guidance failed. In the overexpression lines, fertilization of mutant ovules was mostly blocked because pollen tube guidance failed, although, occasionally, non-viable embryos were formed. We conclude that properly regulated expression of GEX3 in the egg cell of Arabidopsis is essential for pollen tube guidance.