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1.
Hortic Res ; 11(7): uhae130, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38974188

RESUMO

Potato is the third most important food crop, but cultivation is challenged by numerous diseases and adverse abiotic conditions. To combat diseases, frequent fungicide application is common. Knocking out susceptibility genes by genome editing could be a durable option to increase resistance. DMR6 has been described as a susceptibility gene in several crops, based on data that indicates increased resistance upon interruption of the gene function. In potato, Stdmr6-1 mutants have been described to have increased resistance against the late blight pathogen Phytophthora infestans in controlled conditions. Here, we present field evaluations of CRISPR/Cas9 mutants, in a location with a complex population of P. infestans, during four consecutive years that indicate increased resistance to late blight without any trade-off in terms of yield penalty or tuber quality. Furthermore, studies of potato tubers from the field trials indicated increased resistance to common scab, and the mutant lines exhibit increased resistance to early blight pathogen Alternaria solani in controlled conditions. Early blight and common scab are problematic targets in potato resistance breeding, as resistance genes are very scarce. The described broad-spectrum resistance of Stdmr6-1 mutants may further extend to some abiotic stress conditions. In controlled experiments of either drought simulation or salinity, Stdmr6-1 mutant plants are less affected than the background cultivar. Together, these results demonstrate the prospect of the Stdmr6-1 mutants as a useful tool in future sustainable potato cultivation without any apparent trade-offs.

2.
Nat Commun ; 15(1): 5224, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890293

RESUMO

Continued climate change impose multiple stressors on crops, including pathogens, salt, and drought, severely impacting agricultural productivity. Innovative solutions are necessary to develop resilient crops. Here, using quantitative potato proteomics, we identify Parakletos, a thylakoid protein that contributes to disease susceptibility. We show that knockout or silencing of Parakletos enhances resistance to oomycete, fungi, bacteria, salt, and drought, whereas its overexpression reduces resistance. In response to biotic stimuli, Parakletos-overexpressing plants exhibit reduced amplitude of reactive oxygen species and Ca2+ signalling, and silencing Parakletos does the opposite. Parakletos homologues have been identified in all major crops. Consecutive years of field trials demonstrate that Parakletos deletion enhances resistance to Phytophthora infestans and increases yield. These findings demark a susceptibility gene, which can be exploited to enhance crop resilience towards abiotic and biotic stresses in a low-input agriculture.


Assuntos
Doenças das Plantas , Proteínas de Plantas , Solanum tuberosum , Estresse Fisiológico , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Solanum tuberosum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Espécies Reativas de Oxigênio/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Secas , Phytophthora infestans , Plantas Geneticamente Modificadas , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Deleção de Genes , Proteômica
3.
Plant Physiol Biochem ; 199: 107713, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37126903

RESUMO

Cassava (Manihot esculenta Crantz) is a predominant food security crop in several developing countries. Its storage roots, rich in carbohydrate, are deficient in essential micronutrients, including provitamin A carotenoids. Increasing carotenoid content in cassava storage roots is important to reduce the incidence of vitamin A deficiency, a public health problem in sub-Saharan Africa. However, cassava improvement advances slowly, mainly due to limited information on the molecular factors influencing ß-carotene accumulation in cassava. To address this problem, we performed comparative transcriptomic and untargeted metabolic analyses of roots and leaves of eleven African cassava landraces ranging from white to deep yellow colour, to uncover regulators of carotenoid biosynthesis and accumulation with conserved function in yellow cassava roots. Sequence analysis confirmed the presence of a mutation, known to influence ß-carotene content, in PSY transcripts of deep yellow but not of pale yellow genotypes. We identified genes and metabolites with expression and accumulation levels significantly associated with ß-carotene content. Particularly an increased activity of the abscisic acid catabolism pathway together with a reduced amount of L-carnitine, may be related to the carotenoid pathway flux, higher in yellow than in white storage roots. In fact, NCED_3.1 was specifically expressed at a lower level in all yellow genotypes suggesting that it could be a potential target for increasing carotenoid accumulation in cassava. These results expand the knowledge on metabolite compositions and molecular mechanisms influencing carotenoid biosynthesis and accumulation in cassava and provide novel information for biotechnological applications and genetic improvement of cassava with high nutritional values.


Assuntos
Manihot , beta Caroteno , beta Caroteno/análise , Vitamina A/análise , Vitamina A/metabolismo , Vitaminas/análise , Vitaminas/metabolismo , Manihot/genética , Manihot/metabolismo , Transcriptoma/genética , Carotenoides/metabolismo , Verduras , Metaboloma
4.
BMC Plant Biol ; 23(1): 130, 2023 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-36882678

RESUMO

BACKGROUND: Early blight, caused by the necrotrophic fungal pathogen Alternaria solani, is an economically important disease affecting the tuber yield worldwide. The disease is mainly controlled by chemical plant protection agents. However, over-using these chemicals can lead to the evolution of resistant A. solani strains and is environmentally hazardous. Identifying genetic disease resistance factors is crucial for the sustainable management of early blight but little effort has been diverted in this direction. Therefore, we carried out transcriptome sequencing of the A. solani interaction with different potato cultivars with varying levels of early blight resistance to identify key host genes and pathways in a cultivar-specific manner. RESULTS: In this study, we have captured transcriptomes from three different potato cultivars with varying susceptibility to A. solani,  namely Magnum Bonum, Désirée, and Kuras, at 18 and 36 h post-infection. We identified many differentially expressed genes (DEGs) between these cultivars, and the number of DEGs increased with susceptibility and infection time. There were 649 transcripts commonly expressed between the potato cultivars and time points, of which 627 and 22 were up- and down-regulated, respectively. Interestingly, overall the up-regulated DEGs were twice in number as compared to down-regulated ones in all the potato cultivars and time points, except Kuras at 36 h post-inoculation. In general, transcription factor families WRKY, ERF, bHLH, MYB, and C2H2 were highly enriched DEGs, of which a significant number were up-regulated. The majority of the key transcripts involved in the jasmonic acid and ethylene biosynthesis pathways were highly up-regulated. Many transcripts involved in the mevalonate (MVA) pathway, isoprenyl-PP, and terpene biosynthesis were also up-regulated across the potato cultivars and time points. Compared to Magnum Bonum and Désirée, multiple components of the photosynthesis machinery, starch biosynthesis and degradation pathway were down-regulated in the most susceptible potato cultivar, Kuras. CONCLUSIONS: Transcriptome sequencing identified many differentially expressed genes and pathways, thereby contributing to the improved understanding of the interaction between the potato host and A. solani. The transcription factors identified are attractive targets for genetic modification to improve potato resistance against early blight. The results provide important insights into the molecular events at the early stages of disease development, help to shorten the knowledge gap, and support potato breeding programs for improved early blight disease resistance.


Assuntos
Solanum tuberosum , Solanum tuberosum/genética , Resistência à Doença/genética , Melhoramento Vegetal , Perfilação da Expressão Gênica
5.
GM Crops Food ; 13(1): 290-298, 2022 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36263889

RESUMO

Late blight, caused by Phytophthora infestans, is the most devastating disease in potato production. Here, we show full late blight resistance in a location with a genetically diverse pathogen population with the use of GM potato stacked with three resistance (R) genes over three seasons. In addition, using this field trials, we demonstrate that in-the-field intervention among consumers led to change for more favorable attitude generally toward GM crops.


Assuntos
Phytophthora infestans , Solanum tuberosum , Solanum tuberosum/genética , Doenças das Plantas/genética , Plantas Geneticamente Modificadas/genética , Phytophthora infestans/genética , Atitude
7.
Methods Mol Biol ; 2354: 111-122, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34448157

RESUMO

Gene technology and editing are not only biotechnological techniques for creating new crop varieties but are also tools for researchers to discover gene functions. Field trial following laboratory experiments is an important step in order to evaluate new functions since many phenotypes, and combinations thereof, are difficult to detect in controlled environments and molecular analyses are nowadays possible to do in the field. Here we describe a standard protocol for creating new potato lines and producing seed tubers for field trials within 1 year.


Assuntos
Solanum tuberosum , Tubérculos , Plantas Geneticamente Modificadas , Solanum tuberosum/genética
8.
Sci Rep ; 11(1): 4487, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627728

RESUMO

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues.


Assuntos
Sistemas CRISPR-Cas/genética , Resistência à Doença/genética , Mutação/genética , Solanum tuberosum/genética , Citrus/genética , Edição de Genes/métodos , Solanum lycopersicum/genética , Oryza/genética , Fenótipo , Melhoramento Vegetal/métodos , Doenças das Plantas/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética
9.
Front Genome Ed ; 3: 795644, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35128523

RESUMO

Potato, Solanum tuberosum is a highly diverse tetraploid crop. Elite cultivars are extremely heterozygous with a high prevalence of small length polymorphisms (indels) and single nucleotide polymorphisms (SNPs) within and between cultivars, which must be considered in CRISPR/Cas gene editing strategies and designs to obtain successful gene editing. In the present study, in-depth sequencing of the gene encoding glucan water dikinase (GWD) 1 and the downy mildew resistant 6 (DMR6-1) genes in the potato cultivars Saturna and Wotan, respectively, revealed both indels and a 1.3-2.8 higher SNP prevalence when compared to the heterozygous diploid RH genome sequence as expected for a tetraploid compared to a diploid. This complicates guide RNA (gRNA) and diagnostic PCR designs. At the same time, high editing efficiencies at the cell pool (protoplast) level are pivotal for achieving full allelic knock-out in tetraploids. Furthermore, high editing efficiencies reduce the downstream cumbersome and delicate ex-plant regeneration. Here, CRISPR/Cas ribonucleoprotein particles (RNPs) were delivered transiently to protoplasts by polyethylene glycol (PEG) mediated transformation. For each of GWD1 and the DMR6-1, 6-10 gRNAs were designed to target regions comprising the 5' and the 3' end of the two genes. Similar to other studies including several organisms, editing efficiency of the individual RNPs varied significantly, and some generated specific indel patterns. RNP's targeting the 5' end of GWD1 yielded significantly higher editing efficiency as compared to targeting the 3' end. For DMR6-1, such an effect was not seen. Simultaneously targeting each of the two target regions with two RNPs (multiplexing) yielded a clear positive synergistic effect on the total editing when targeting the 3' end of the GWD1 gene only. Multiplexing of the two genes, residing on different chromosomes, yielded no or a slightly negative effect on editing from the single or combined gRNA/RNPs. These initial findings may instigate much larger studies needed for facilitating and optimizing precision breeding in plants.

10.
Plants (Basel) ; 9(6)2020 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-32486039

RESUMO

Potato (Solanum tuberosum) is among the best producers of edible biomass in terms of yield per hectare and a variety of different regional cultivars are used as a staple commodity in many countries. However, this crop is attacked by several diseases, with the worst being the late blight disease caused by Phytophthora infestans. Stacking of resistance (R) genes from wild Solanum relatives are interesting prospects for the sustainable control of late blight. Therefore, we optimized methods for the efficient generation and screening of R-gene-containing transformants in tetraploid and diploid hybrid potato genotypes. Using these methods, a high transformation efficiency was achieved for the transformation of tetraploid and diploid potato lines with a triple resistance (3R) gene construct. Transformation efficiencies were improved by optimizing several factors affecting regeneration, including the quality of the starting plant material, and the composition of the plant growth regulators used during selective regeneration. A refreshment protocol was designed to alleviate in vitro related stress in stock plants, which significantly improved the growth vigor and resulted in a 4- to 10-fold increase in transformation efficiency. Furthermore, long-term exposure to exogenous Indole-3-butyric acid that is usually used for the initiation of roots in vitro, was found to cause aberrant morphological phenotypes in potato.

12.
Int J Mol Sci ; 20(19)2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31554174

RESUMO

Plants have a variety of ways to defend themselves against pathogens. A commonly used model of the plant immune system is divided into a general response triggered by pathogen-associated molecular patterns (PAMPs), and a specific response triggered by effectors. The first type of response is known as PAMP triggered immunity (PTI), and the second is known as effector-triggered immunity (ETI). To obtain better insight into changes of protein abundance in immunity reactions, we performed a comparative proteomic analysis of a PTI and two different ETI models (relating to Phytophthora infestans) in potato. Several proteins showed higher abundance in all immune reactions, such as a protein annotated as sterol carrier protein 2 that could be interesting since Phytophthora species are sterol auxotrophs. RNA binding proteins also showed altered abundance in the different immune reactions. Furthermore, we identified some PTI-specific changes of protein abundance, such as for example, a glyoxysomal fatty acid beta-oxidation multifunctional protein and a MAR-binding protein. Interestingly, a lysine histone demethylase was decreased in PTI, and that prompted us to also analyze protein methylation in our datasets. The proteins upregulated explicitly in ETI included several catalases. Few proteins were regulated in only one of the ETI interactions. For example, histones were only downregulated in the ETI-Avr2 interaction, and a putative multiprotein bridging factor was only upregulated in the ETI-IpiO interaction. One example of a methylated protein that increased in the ETI interactions was a serine hydroxymethyltransferase.


Assuntos
Imunidade Vegetal , Folhas de Planta/imunologia , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Proteômica , Solanum tuberosum/imunologia , Solanum tuberosum/metabolismo , Biologia Computacional/métodos , Bases de Dados Genéticas , Espectrometria de Massas , Metilação , Mapeamento de Interação de Proteínas , Proteoma
13.
Int J Mol Sci ; 19(2)2018 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-29439444

RESUMO

Plants have evolved different types of immune reactions but large-scale proteomics about these processes are lacking, especially in the case of agriculturally important crop pathosystems. We have established a system for investigating PAMP-triggered immunity (PTI) and two different effector-triggered immunity (ETI; triggered by Avr2 or IpiO) responses in potato. The ETI responses are triggered by molecules from the agriculturally important Phytophthora infestans interaction. To perform large-scale membrane protein-based comparison of these responses, we established a method to extract proteins from subcellular compartments in leaves. In the membrane fractions that were subjected to quantitative proteomics analysis, we found that most proteins regulated during PTI were also regulated in the same way in ETI. Proteins related to photosynthesis had lower abundance, while proteins related to oxidative and biotic stress, as well as those related to general antimicrobial defense and cell wall degradation, were found to be higher in abundance. On the other hand, we identified a few proteins-for instance, an ABC transporter-like protein-that were only found in the PTI reaction. Furthermore, we also identified proteins that were regulated only in ETI interactions. These included proteins related to GTP binding and heterotrimeric G-protein signaling, as well as those related to phospholipase signaling.


Assuntos
Resistência à Doença , Proteínas de Membrana/química , Proteínas de Plantas/química , Proteômica/métodos , Solanum tuberosum/imunologia , Membranas Intracelulares/química , Espectrometria de Massas/métodos , Proteínas de Membrana/metabolismo , Phytophthora/patogenicidade , Folhas de Planta/química , Proteínas de Plantas/metabolismo , Solanum tuberosum/química , Solanum tuberosum/microbiologia
14.
Theor Appl Genet ; 129(1): 105-15, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26518573

RESUMO

KEY MESSAGE: We show the usefulness of integrating effector screening in a breeding program and in resistance gene cloning, with Phytophthora resistance in the Swedish potato breeding clone SW93-1015 as an example. Phytophthora infestans is one of the most devastating plant pathogens worldwide. We have earlier found that the SW93-1015 potato breeding clone has an efficient resistance against P. infestans under field conditions in Sweden, which has an unusually high local diversity of the pathogen. This potato clone has characteristics that are different from classical R-gene-mediated resistance such as elevated levels of hydrogen peroxide (H2O2) under controlled conditions. Analysis of 76 F1 potato progenies from two individual crosses resulted in nearly 50% resistant clones, from both crosses. This result suggests that the SW93-1015 clone has a simplex genotype for this trait. Screening with over 50 different P. infestans effectors, containing the conserved motif RXLR (for Arg, any amino acid, Leu, Arg), revealed a specific response to Avr2, which suggests that SW93-1015 might contain a functional homolog of the R2 resistance gene. We cloned eight R2 gene homologs from SW93-1015, whereof seven have not been described before and one gene encoded a protein identical to Rpi-ABPT. Expression of this gene in potato cultivar Désirée provided R2-specific resistance, whereas other homologues did not. Using RNAseq analyses we designed a new DNA marker for the R2 resistance in SW93-1015. In summary, we have demonstrated the use of effector screening in practical breeding material and revealed the key resistance mechanism for SW93-1015.


Assuntos
Resistência à Doença/genética , Genes de Plantas , Phytophthora infestans , Doenças das Plantas/genética , Solanum tuberosum/genética , Sequência de Aminoácidos , Cruzamento , Clonagem Molecular , Marcadores Genéticos , Genótipo , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Solanum tuberosum/microbiologia
15.
BMC Genomics ; 15: 497, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24947944

RESUMO

BACKGROUND: In order to get global molecular understanding of one of the most important crop diseases worldwide, we investigated compatible and incompatible interactions between Phytophthora infestans and potato (Solanum tuberosum). We used the two most field-resistant potato clones under Swedish growing conditions, which have the greatest known local diversity of P. infestans populations, and a reference compatible cultivar. RESULTS: Quantitative label-free proteomics of 51 apoplastic secretome samples (PXD000435) in combination with genome-wide transcript analysis by 42 microarrays (E-MTAB-1515) were used to capture changes in protein abundance and gene expression at 6, 24 and 72 hours after inoculation with P. infestans. To aid mass spectrometry analysis we generated cultivar-specific RNA-seq data (E-MTAB-1712), which increased peptide identifications by 17%. Components induced only during incompatible interactions, which are candidates for hypersensitive response initiation, include a Kunitz-like protease inhibitor, transcription factors and an RCR3-like protein. More secreted proteins had lower abundance in the compatible interaction compared to the incompatible interactions. Based on this observation and because the well-characterized effector-target C14 protease follows this pattern, we suggest 40 putative effector targets. CONCLUSIONS: In summary, over 17000 transcripts and 1000 secreted proteins changed in abundance in at least one time point, illustrating the dynamics of plant responses to a hemibiotroph. Half of the differentially abundant proteins showed a corresponding change at the transcript level. Many putative hypersensitive and effector-target proteins were single representatives of large gene families.


Assuntos
Interações Hospedeiro-Parasita , Phytophthora infestans , Doenças das Plantas/genética , Proteoma , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Transcriptoma , Resistência à Doença/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Parasita/genética , Doenças das Plantas/parasitologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteômica/métodos , Solanum tuberosum/parasitologia
16.
Biochem J ; 446(2): 271-8, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22631074

RESUMO

MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. In the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that At1g80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of At1g80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of At1g80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Biologia Computacional/métodos , Cotilédone/enzimologia , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Cinética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Moleculares , Proteínas Mutantes/metabolismo , Biblioteca de Peptídeos , Fosforilação , Estômatos de Plantas/enzimologia , Estômatos de Plantas/crescimento & desenvolvimento , Estômatos de Plantas/metabolismo , Plantas Geneticamente Modificadas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Especificidade por Substrato , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Nicotiana/metabolismo
17.
Plant Signal Behav ; 7(3): 400-8, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22476463

RESUMO

Phytophthora is the most devastating pathogen of dicot plants. There is a need for resistance sources with different modes of action to counteract the fast evolution of this pathogen. In order to better understand mechanisms of defense against P. infestans, we analyzed several clones of potato. Two of the genotypes tested, Sarpo Mira and SW93-1015, exhibited strong resistance against P. infestans in field trials, whole plant assays and detached leaf assays. The resistant genotypes developed different sizes of hypersensitive response (HR)-related lesions. HR lesions in SW93-1015 were restricted to very small areas, whereas those in Sarpo Mira were similar to those in Solanum demissum, the main source of classical resistance genes. SW93-1015 can be characterized as a cpr (constitutive expressor of PR genes) genotype without spontaneous microscopic or macroscopic HR lesions. This is indicated by constitutive hydrogen peroxide (H2O2) production and PR1 (pathogenesis-related protein 1) secretion. SW93-1015 is one of the first plants identified as having classical protein-based induced defense expressed constitutively without any obvious metabolic costs or spontaneous cell death lesions.


Assuntos
Phytophthora/patogenicidade , Solanum tuberosum/genética , Solanum tuberosum/microbiologia , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Genótipo , Doenças das Plantas/genética
18.
Mol Plant Microbe Interact ; 21(10): 1275-84, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18785823

RESUMO

Protein phosphorylation is a key biological process that regulates reactions involved in plant-microbe interactions. The phosphorylated form of a protein often represents only a small fraction of the total population and can be problematic to analyze in a mass spectrometer. We demonstrate how a titanium dioxide (TiO(2)) resin can be employed for the enrichment of phosphoproteins, as well as a method to derivatize TiO(2)-purified phosphopeptides to facilitate determination of the exact site of phosphorylation. The use of these methods was exemplified by the identification of two plant proteins that were shown to be phosphorylated after the elicitation of Arabidopsis cells with Phytophthora infestans zoospores and xylanase. Both of the proteins that were identified, At5g54430.1 and At4g27320.1, were found to contain a universal stress protein domain with conserved residues for ATP binding.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Arabidopsis/citologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Células Cultivadas , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Fosfopeptídeos/química , Fosfopeptídeos/genética , Fosfopeptídeos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Phytophthora/fisiologia , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Titânio/química , Titânio/farmacologia
19.
J Biol Chem ; 280(41): 34626-34, 2005 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-16020547

RESUMO

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.


Assuntos
Aciltransferases/química , Arabidopsis/genética , Arabidopsis/metabolismo , Ésteres/química , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/química , Esterol O-Aciltransferase/fisiologia , Esteróis/química , Aciltransferases/fisiologia , Sequência de Aminoácidos , Clonagem Molecular , DNA Bacteriano/química , DNA Complementar/metabolismo , Vetores Genéticos , Membranas Intracelulares/metabolismo , Lipídeos/química , Microssomos/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Plantas/genética , Plantas/metabolismo , Regiões Promotoras Genéticas , RNA/química , RNA/metabolismo , Esterol O-Aciltransferase/química , Especificidade por Substrato
20.
Plant Physiol ; 135(3): 1324-35, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15247387

RESUMO

A new pathway for triacylglycerol biosynthesis involving a phospholipid:diacylglycerol acyltransferase (PDAT) was recently described (Dahlqvist A, Stahl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S, [2000] Proc Natl Acad Sci USA 97: 6487-6492). The LRO1 gene that encodes the PDAT was identified in yeast (Saccharomyces cerevisiae) and shown to have homology with animal lecithin:cholesterol acyltransferase. A search of the Arabidopsis genome database identified the protein encoded by the At5g13640 gene as the closest homolog to the yeast PDAT (28% amino acid identity). The cDNA of At5g13640 (AtPDAT gene) was overexpressed in Arabidopsis behind the cauliflower mosaic virus promoter. Microsomal preparations of roots and leaves from overexpressers had PDAT activities that correlated with expression levels of the gene, thus demonstrating that this gene encoded PDAT (AtPDAT). The AtPDAT utilized different phospholipids as acyl donor and accepted acyl groups ranging from C10 to C22. The rate of activity was highly dependent on acyl composition with highest activities for acyl groups containing several double bonds, epoxy, or hydroxy groups. The enzyme utilized both sn-positions of phosphatidylcholine but had a 3-fold preference for the sn-2 position. The fatty acid and lipid composition as well as the amounts of lipids per fresh weight in Arabidopsis plants overexpressing AtPDAT were not significantly different from the wild type. Microsomal preparations of roots from a T-DNA insertion mutant in the AtPDAT gene had barely detectable capacity to transfer acyl groups from phospholipids to added diacylglycerols. However, these microsomes were still able to carry out triacylglycerol synthesis by a diacylglycerol:diacylglycerol acyltransferase reaction at the same rate as microsomal preparations from wild type.


Assuntos
Aciltransferases/genética , Aciltransferases/metabolismo , Aciltransferases/química , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA , Diacilglicerol O-Aciltransferase , Cinética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade por Substrato
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