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1.
Plant Biotechnol J ; 2021 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-34459083

RESUMO

High humidity during harvest season often causes pre-harvest sprouting in barley (Hordeum vulgare). Prolonged grain dormancy prevents pre-harvest sprouting; however, extended dormancy can interfere with malt production and uniform germination upon sowing. In this study, we used Cas9-induced targeted mutagenesis to create single and double mutants in QTL FOR SEED DORMANCY 1 (Qsd1) and Qsd2 in the same genetic background. We performed germination assays in independent qsd1 and qsd2 single mutants, as well as in two double mutants, which revealed a strong repression of germination in the mutants. These results demonstrated that normal early grain germination requires both Qsd1 and Qsd2 function. However, germination of qsd1 was promoted by treatment with 3% hydrogen peroxide, supporting the notion that the mutants exhibit delayed germination. Likewise, exposure to cold temperatures largely alleviated the block of germination in the single and double mutants. Notably, qsd1 mutants partially suppress the long dormancy phenotype of qsd2, while qsd2 mutant grains failed to germinate in the light, but not in the dark. Consistent with the delay in germination, abscisic acid accumulated in all mutants relative to the wild type, but abscisic acid levels cannot maintain long-term dormancy and only delay germination. Elucidation of mutant allele interactions, such as those shown in this study, are important for fine-tuning traits that will lead to the design of grain dormancy through combinations of mutant alleles. Thus, these mutants will provide the necessary germplasm to study grain dormancy and germination in barley.

2.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34210100

RESUMO

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8'-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticalecan beincreased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Edição de Genes/métodos , Proteínas de Plantas/metabolismo , Triticale/metabolismo , Sistemas CRISPR-Cas , Sistema Enzimático do Citocromo P-450/genética , Genes Reporter , Mutação INDEL , Mutagênese Sítio-Dirigida , Proteínas de Plantas/genética , Transfecção , Triticale/genética
3.
Methods Mol Biol ; 2287: 199-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270031

RESUMO

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.


Assuntos
Endonucleases/metabolismo , Haploidia , Hordeum/crescimento & desenvolvimento , Hordeum/genética , Mutagênese Sítio-Dirigida/métodos , Melhoramento Vegetal/métodos , RNA Guia/genética , Sistemas CRISPR-Cas , Meios de Cultura , Endonucleases/genética , Edição de Genes , Engenharia Genética , Genoma de Planta , Homozigoto , Hordeum/embriologia , Plantas Geneticamente Modificadas , Pólen/genética , Pólen/crescimento & desenvolvimento
4.
Methods Mol Biol ; 2287: 215-226, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270032

RESUMO

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.


Assuntos
Hordeum/crescimento & desenvolvimento , Hordeum/genética , Melhoramento Vegetal/métodos , Técnicas de Cultura de Tecidos/métodos , Genótipo , Haploidia , Hordeum/embriologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Polinização
5.
Front Plant Sci ; 11: 543895, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33193477

RESUMO

Biotic stresses caused by microbial pathogens impair crop yield and quality if not restricted by expensive and often ecologically problematic pesticides. For a sustainable agriculture of tomorrow, breeding or engineering of pathogen-resistant crop varieties is therefore a major cornerstone. Maize is one of the four most important cereal crops in the world. The biotrophic fungal pathogen Ustilago maydis causes galls on all aerial parts of the maize plant. Biotrophic pathogens like U. maydis co-evolved with their host plant and depend during their life cycle on successful manipulation of the host's cellular machinery. Therefore, removing or altering plant susceptibility genes is an effective and usually durable way to obtain resistance in plants. Transcriptional time course experiments in U. maydis-infected maize revealed numerous maize genes being upregulated upon establishment of biotrophy. Among these genes is the maize LIPOXYGENASE 3 (LOX3) previously shown to be a susceptibility factor for other fungal genera as well. Aiming to engineer durable resistance in maize against U. maydis and possibly other pathogens, we took a Cas endonuclease technology approach to generate loss of function mutations in LOX3. lox3 maize mutant plants react with an enhanced PAMP-triggered ROS burst implicating an enhanced defense response. Based on visual assessment of disease symptoms and quantification of relative fungal biomass, homozygous lox3 mutant plants exposed to U. maydis show significantly decreased susceptibility. U. maydis infection assays using a transposon mutant lox3 maize line further substantiated that LOX3 is a susceptibility factor for this important maize pathogen.

6.
BMC Plant Biol ; 20(Suppl 1): 255, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33050877

RESUMO

BACKGROUND: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. RESULTS: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. CONCLUSIONS: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high ß-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Hordeum/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Grão Comestível/genética , Técnicas de Inativação de Genes , Marcação de Genes , Hordeum/anatomia & histologia , Fenótipo , RNA Guia/metabolismo
7.
Plant J ; 103(5): 1869-1884, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32530511

RESUMO

Cereal endosperm represents the most important source of the world's food; nevertheless, the molecular mechanisms underlying cell and tissue differentiation in cereal grains remain poorly understood. Endosperm cellularization commences at the maternal-filial intersection of grains and generates endosperm transfer cells (ETCs), a cell type with a prominent anatomy optimized for efficient nutrient transport. Barley HISTIDINE KINASE1 (HvHK1) was identified as a receptor component with spatially restricted expression in the syncytial endosperm where ETCs emerge. Here, we demonstrate its function in ETC fate acquisition using RNA interference-mediated downregulation of HvHK1. Repression of HvHK1 impairs cell specification in the central ETC region and the development of transfer cell morphology, and consecutively defects differentiation of adjacent endosperm tissues. Coinciding with reduced expression of HvHK1, disturbed cell plate formation and fusion were observed at the initiation of endosperm cellularization, revealing that HvHK1 triggers initial cytokinesis of ETCs. Cell-type-specific RNA sequencing confirmed loss of transfer cell identity, compromised cell wall biogenesis and reduced transport capacities in aberrant cells and elucidated two-component signaling and hormone pathways that are mediated by HvHK1. Gene regulatory network modeling was used to specify the direct targets of HvHK1; this predicted non-canonical auxin signaling elements as the main regulatory links governing cellularization of ETCs, potentially through interaction with type-B response regulators. This work provides clues to previously unknown molecular mechanisms directing ETC specification, a process with fundamental impact on grain yield in cereals.


Assuntos
Diferenciação Celular , Endosperma/crescimento & desenvolvimento , Histidina Quinase/metabolismo , Hordeum/metabolismo , Proteínas de Plantas/metabolismo , Divisão Celular , Polaridade Celular , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Histidina Quinase/fisiologia , Hordeum/enzimologia , Hordeum/crescimento & desenvolvimento , Proteínas de Plantas/fisiologia
9.
Plant J ; 102(3): 631-642, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31823436

RESUMO

Many plant genomes display high levels of repetitive sequences. The assembly of these complex genomes using short high-throughput sequence reads is still a challenging task. Underestimation or disregard of repeat complexity in these datasets can easily misguide downstream analysis. Detection of repetitive regions by k-mer counting methods has proved to be reliable. Easy-to-use applications utilizing k-mer counting are in high demand, especially in the domain of plants. We present Kmasker plants, a tool that uses k-mer count information as an assistant throughout the analytical workflow of genome data that is provided as a command-line and web-based solution. Beside its core competence to screen and mask repetitive sequences, we have integrated features that enable comparative studies between different cultivars or closely related species and methods that estimate target specificity of guide RNAs for application of site-directed mutagenesis using Cas9 endonuclease. In addition, we have set up a web service for Kmasker plants that maintains pre-computed indices for 10 of the economically most important cultivated plants. Source code for Kmasker plants has been made publically available at https://github.com/tschmutzer/kmasker. The web service is accessible at https://kmasker.ipk-gatersleben.de.


Assuntos
Genoma de Planta/genética , Algoritmos , Edição de Genes , Genômica , RNA Guia/genética , Análise de Sequência de DNA , Software
10.
Front Plant Sci ; 11: 613731, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33519872

RESUMO

Chloroplasts, the sites of photosynthesis in higher plants, have evolved several means to tolerate short episodes of drought stress through biosynthesis of diverse metabolites essential for plant function, but these become ineffective when the duration of the stress is prolonged. Cyanobacteria are the closest bacterial homologs of plastids with two photosystems to perform photosynthesis and to evolve oxygen as a byproduct. The presence of Flv genes encoding flavodiiron proteins has been shown to enhance stress tolerance in cyanobacteria. In an attempt to support the growth of plants exposed to drought, the Synechocystis genes Flv1 and Flv3 were expressed in barley with their products being targeted to the chloroplasts. The heterologous expression of both Flv1 and Flv3 accelerated days to heading, increased biomass, promoted the number of spikes and grains per plant, and improved the total grain weight per plant of transgenic lines exposed to drought. Improved growth correlated with enhanced availability of soluble sugars, a higher turnover of amino acids and the accumulation of lower levels of proline in the leaf. Flv1 and Flv3 maintained the energy status of the leaves in the stressed plants by converting sucrose to glucose and fructose, immediate precursors for energy production to support plant growth under drought. The results suggest that sugars and amino acids play a fundamental role in the maintenance of the energy status and metabolic activity to ensure growth and survival under stress conditions, that is, water limitation in this particular case. Engineering chloroplasts by Flv genes into the plant genome, therefore, has the potential to improve plant productivity wherever drought stress represents a significant production constraint.

11.
Nat Plants ; 5(11): 1129-1135, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712760

RESUMO

Global food security depends on cereal crops with durable disease resistance. Most cereals are colonized by rust fungi, which are pathogens of major significance for global agriculture1. Cereal rusts display a high degree of host specificity and one rust species or forma specialis generally colonizes only one cereal host2. Exploiting the non-host status and transferring non-host resistance genes between cereal crop species has been proposed as a strategy for durable rust resistance breeding. The molecular determinants that define the host status to rusts, however, are largely unknown. Here, we show that orthologous genes at the Rphq2 locus for quantitative leaf rust resistance from cultivated barley3 and Rph22 from wild bulbous barley4 affect the host status to leaf rusts. Both genes encode lectin receptor-like kinases. We transformed Rphq2 and Rph22 into an experimental barley line that has been bred for susceptibility to non-adapted leaf rusts, which allowed us to quantify resistance responses against various leaf rust species. Rphq2 conferred a much stronger resistance to the leaf rust of wild bulbous barley than to the leaf rust adapted to cultivated barley, while for Rph22 the reverse was observed. We hypothesize that adapted leaf rust species mitigate perception by cognate host receptors by lowering ligand recognition. Our results provide an example of orthologous genes that connect the quantitative host with non-host resistance to cereal rusts. Such genes provide a basis to exploit non-host resistance in molecular breeding.


Assuntos
Basidiomycota/fisiologia , Grão Comestível/enzimologia , Hordeum/enzimologia , Doenças das Plantas/imunologia , Proteínas Quinases/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Resistência à Doença/genética , Grão Comestível/microbiologia , Hordeum/microbiologia , Melhoramento Vegetal , Doenças das Plantas/microbiologia , Especificidade da Espécie
12.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146387

RESUMO

Domestication and breeding have created productive crops that are adapted to the climatic conditions of their growing regions. Initially, this process solely relied on the frequent occurrence of spontaneous mutations and the recombination of resultant gene variants. Later, treatments with ionizing radiation or mutagenic chemicals facilitated dramatically increased mutation rates, which remarkably extended the genetic diversity of crop plants. However, a major drawback of conventionally induced mutagenesis is that genetic alterations occur simultaneously across the whole genome and at very high numbers per individual plant. By contrast, the newly emerging Cas endonuclease technology allows for the induction of mutations at user-defined positions in the plant genome. In fundamental and breeding-oriented research, this opens up unprecedented opportunities for the elucidation of gene functions and the targeted improvement of plant performance. This review covers historical aspects of the development of customizable endonucleases, information on the mechanisms of targeted genome modification, as well as hitherto reported applications of Cas endonuclease technology in barley and wheat that are the agronomically most important members of the temperate cereals. Finally, current trends in the further development of this technology and some ensuing future opportunities for research and biotechnological application are presented.


Assuntos
Sistemas CRISPR-Cas , Engenharia Genética/métodos , Hordeum/genética , Melhoramento Vegetal/métodos , Triticum/genética
13.
Plant Cell ; 31(7): 1430-1445, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31023840

RESUMO

Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature from proplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genes impair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Such mutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplast biogenesis. Characterization of the green-white variegated albostrians mutant of barley (Hordeum vulgare) has greatly broadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification of the ALBOSTRIANS gene HvAST (also known as Hordeum vulgare CCT Motif Family gene 7, HvCMF7) by positional cloning as well as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes (TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvCMF7 in the original albostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation. HvCMF7 is a homolog of the Arabidopsis (Arabidopsis thaliana) CONSTANS, CO-like, and TOC1 (CCT) Motif transcription factor gene CHLOROPLAST IMPORT APPARATUS2, which was reported to be involved in the expression of nuclear genes essential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence for nuclear localization.


Assuntos
Cloroplastos/metabolismo , Genes de Plantas , Hordeum/genética , Folhas de Planta/fisiologia , Proteínas de Plantas/genética , Alelos , Sequência de Aminoácidos , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Cloroplastos/ultraestrutura , Mapeamento Cromossômico , Proteínas de Fluorescência Verde/metabolismo , Hordeum/ultraestrutura , Mutagênese Sítio-Dirigida , Mutação/genética , Folhas de Planta/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo
14.
New Phytol ; 223(2): 853-866, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30913300

RESUMO

The wheat Lr34res allele, coding for an ATP-binding cassette transporter, confers durable resistance against multiple fungal pathogens. The Lr34sus allele, differing from Lr34res by two critical nucleotide polymorphisms, is found in susceptible wheat cultivars. Lr34res is functionally transferrable as a transgene into all major cereals, including rice, barley, maize, and sorghum. Here, we used transcriptomics, physiology, genetics, and in vitro and in vivo transport assays to study the molecular function of Lr34. We report that Lr34res results in a constitutive induction of transcripts reminiscent of an abscisic acid (ABA)-regulated response in transgenic rice. Lr34-expressing rice was altered in biological processes that are controlled by this phytohormone, including dehydration tolerance, transpiration and seedling growth. In planta seedling and in vitro yeast accumulation assays revealed that both LR34res and LR34sus act as ABA transporters. However, whereas the LR34res protein was detected in planta the LR34sus version was not, suggesting a post-transcriptional regulatory mechanism. Our results identify ABA as a substrate of the LR34 ABC transporter. We conclude that LR34res-mediated ABA redistribution has a major effect on the transcriptional response and physiology of Lr34res-expressing plants and that ABA is a candidate molecule that contributes to Lr34res-mediated disease resistance.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Abscísico/metabolismo , Resistência à Doença/genética , Genes de Plantas , Triticum/genética , Regulação da Expressão Gênica de Plantas , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato
15.
Methods Mol Biol ; 1900: 195-215, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30460567

RESUMO

Genome engineering involves methods of genetic modification of cells at predefined genomic sites. Here, we used transcription activator-like effector nucleases (TALENs) for the site-directed mutagenesis in barley. Target gene-specific TALEN-encoding expression units were designed and delivered to totipotent cells of either cultivated embryogenic pollen or immature embryos. The analysis of resulting transgenic plants revealed that the described approach allows for the generation of site-specific, heritable mutations at reasonable efficiency.


Assuntos
Engenharia Genética/métodos , Genoma de Planta , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Agrobacterium/genética , Vetores Genéticos/metabolismo , Mutação/genética , Plantas Geneticamente Modificadas , Plasmídeos/genética , Pólen/genética
16.
J Exp Bot ; 70(7): 2143-2155, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30452688

RESUMO

To survive under water deficiency, plants alter gene expression patterns, make structural and physiological adjustments, and optimize the use of water. Rapid degradation and turnover of proteins is required for effective nutrient recycling. Here, we examined the transcriptional responses of the C1A cysteine protease family to drought in barley and found that four genes were up-regulated in stressed plants. Knock-down lines for the protease-encoding genes HvPap-1 and HvPap-19 showed unexpected changes in leaf cuticle thickness and stomatal pore area. The efficiency of photosystem II and the total amount of proteins were almost unaltered in stressed transgenic plants while both parameters decreased in stressed wild-type plants. Although the patterns of proteolytic activities in the knock-down lines did not change, the amino acid accumulation increased in response to drought, concomitant with a higher ABA content. Whilst jasmonic acid (JA) and JA-Ile concentrations increased in stressed leaves of the wild-type and the HvPap-1 knock-down lines, their levels were lower in the HvPap-19 knock-down lines, suggesting the involvement of a specific hormone interaction in the process. Our data indicate that the changes in leaf cuticle thickness and stomatal pore area had advantageous effects on leaf defense against fungal infection and mite feeding mediated by Magnaporthe oryzae and Tetranychus urticae, respectively.


Assuntos
Cisteína Proteases/genética , Secas , Regulação da Expressão Gênica de Plantas , Hordeum/fisiologia , Família Multigênica/genética , Proteínas de Plantas/genética , Cisteína Proteases/metabolismo , Hordeum/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Estresse Fisiológico , Regulação para Cima
17.
Theor Appl Genet ; 132(3): 593-605, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30569366

RESUMO

The ability to generate (doubled) haploid plants significantly accelerates the crop breeding process. Haploids have been induced mainly through the generation of plants from cultivated gametophic (haploid) cells and tissues, i.e., in vitro haploid technologies, or through the selective loss of a parental chromosome set upon inter- or intraspecific hybridization. Here, we focus our review on the mechanisms responsible for the in vivo formation of haploids in the context of inter- and intraspecific hybridization. The application of a modified CENH3 for uniparental genome elimination, the IG1 system used for paternal as well as the BBM-like and the patatin-like phospholipase essential for maternal haploidy induction are discussed in detail.


Assuntos
Haploidia , Melhoramento Vegetal/métodos , Centrômero/metabolismo , Produtos Agrícolas/genética , Hibridização Genética , Partenogênese
18.
J Integr Plant Biol ; 60(12): 1127-1153, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30387552

RESUMO

Since the discovery that nucleases of the bacterial CRISPR (clustered regularly interspaced palindromic repeat)-associated (Cas) system can be used as easily programmable tools for genome engineering, their application massively transformed different areas of plant biology. In this review, we assess the current state of their use for crop breeding to incorporate attractive new agronomical traits into specific cultivars of various crop plants. This can be achieved by the use of Cas9/12 nucleases for double-strand break induction, resulting in mutations by non-homologous recombination. Strategies for performing such experiments - from the design of guide RNA to the use of different transformation technologies - are evaluated. Furthermore, we sum up recent developments regarding the use of nuclease-deficient Cas9/12 proteins, as DNA-binding moieties for targeting different kinds of enzyme activities to specific sites within the genome. Progress in base deamination, transcriptional induction and transcriptional repression, as well as in imaging in plants, is also discussed. As different Cas9/12 enzymes are at hand, the simultaneous application of various enzyme activities, to multiple genomic sites, is now in reach to redirect plant metabolism in a multifunctional manner and pave the way for a new level of plant synthetic biology.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Plantas/genética , Biologia Sintética/métodos , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Endonucleases/genética , Endonucleases/metabolismo , Edição de Genes/métodos , Melhoramento Vegetal
19.
Genome Biol ; 19(1): 116, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-30111359

RESUMO

BACKGROUND: The large and highly repetitive genomes of the cultivated species Hordeum vulgare (barley), Triticum aestivum (wheat), and Secale cereale (rye) belonging to the Triticeae tribe of grasses appear to be particularly rich in gene-like sequences including partial duplicates. Most of them have been classified as putative pseudogenes. In this study we employ transient and stable gene silencing- and over-expression systems in barley to study the function of HvARM1 (for H. vulgare Armadillo 1), a partial gene duplicate of the U-box/armadillo-repeat E3 ligase HvPUB15 (for H. vulgare Plant U-Box 15). RESULTS: The partial ARM1 gene is derived from a gene-duplication event in a common ancestor of the Triticeae and contributes to quantitative host as well as nonhost resistance to the biotrophic powdery mildew fungus Blumeria graminis. In barley, allelic variants of HvARM1 but not of HvPUB15 are significantly associated with levels of powdery mildew infection. Both HvPUB15 and HvARM1 proteins interact in yeast and plant cells with the susceptibility-related, plastid-localized barley homologs of THF1 (for Thylakoid formation 1) and of ClpS1 (for Clp-protease adaptor S1) of Arabidopsis thaliana. A genome-wide scan for partial gene duplicates reveals further events in barley resulting in stress-regulated, potentially neo-functionalized, genes. CONCLUSION: The results suggest neo-functionalization of the partial gene copy HvARM1 increases resistance against powdery mildew infection. It further links plastid function with susceptibility to biotrophic pathogen attack. These findings shed new light on a novel mechanism to employ partial duplication of protein-protein interaction domains to facilitate the expansion of immune signaling networks.


Assuntos
Sequência Conservada/genética , Resistência à Doença/genética , Evolução Molecular , Duplicação Gênica , Interações Hospedeiro-Patógeno/genética , Poaceae/genética , Alelos , Sequência de Bases , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Marcadores Genéticos , Hordeum/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Característica Quantitativa Herdável
20.
Int J Mol Sci ; 19(3)2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29494488

RESUMO

Cystatins have been largely used for pest control against phytophagous species. However, cystatins have not been commonly overexpressed in its cognate plant species to test their pesticide capacity. Since the inhibitory role of barley HvCPI-6 cystatin against the phytophagous mite Tetranychus urticae has been previously demonstrated, the purpose of our study was to determine if barley transgenic lines overexpressing its own HvIcy6 gene were more resistant against this phytophagous infestation. Besides, a transcriptomic analysis was done to find differential expressed genes among wild-type and transformed barley plants. Barley plants overexpressing HvIcy6 cystatin gene remained less susceptible to T. urticae attack when compared to wild-type plants, with a significant lesser foliar damaged area and a lower presence of the mite. Transcriptomic analysis revealed a certain reprogramming of cellular metabolism and a lower expression of several genes related to photosynthetic activity. Therefore, although caution should be taken to discard potential deleterious pleiotropic effects, cystatins may be used as transgenes with impact on agricultural crops by conferring enhanced levels of resistance to phytophagous pests.


Assuntos
Resistência à Doença/genética , Expressão Gênica , Hordeum/genética , Hordeum/parasitologia , Interações Hospedeiro-Parasita/genética , Doenças das Plantas/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/genética , Perfilação da Expressão Gênica , Fenótipo , Folhas de Planta/genética , Folhas de Planta/parasitologia , Plantas Geneticamente Modificadas , Transcriptoma
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