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1.
BMC Plant Biol ; 21(1): 402, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34470613

RESUMO

BACKGROUND: Plant-parasitic nematodes and herbivorous insects have a significant negative impact on global crop production. A successful approach to protect crops from these pests is the in planta expression of nematotoxic or entomotoxic proteins such as crystal proteins from Bacillus thuringiensis (Bt) or plant lectins. However, the efficacy of this approach is threatened by emergence of resistance in nematode and insect populations to these proteins. To solve this problem, novel nematotoxic and entomotoxic proteins are needed. During the last two decades, several cytoplasmic lectins from mushrooms with nematicidal and insecticidal activity have been characterized. In this study, we tested the potential of Marasmius oreades agglutinin (MOA) to furnish Arabidopsis plants with resistance towards three economically important crop pests: the two plant-parasitic nematodes Heterodera schachtii and Meloidogyne incognita and the herbivorous diamondback moth Plutella xylostella. RESULTS: The expression of MOA does not affect plant growth under axenic conditions which is an essential parameter in the engineering of genetically modified crops. The transgenic Arabidopsis lines showed nearly complete resistance to H. schachtii, in that the number of female and male nematodes per cm root was reduced by 86-91 % and 43-93 % compared to WT, respectively. M. incognita proved to be less susceptible to the MOA protein in that 18-25 % and 26-35 % less galls and nematode egg masses, respectively, were observed in the transgenic lines. Larvae of the herbivorous P. xylostella foraging on MOA-expression lines showed a lower relative mass gain (22-38 %) and survival rate (15-24 %) than those feeding on WT plants. CONCLUSIONS: The results of our in planta experiments reveal a robust nematicidal and insecticidal activity of the fungal lectin MOA against important agricultural pests which may be exploited for crop protection.


Assuntos
Aglutininas/farmacologia , Arabidopsis/parasitologia , Herbivoria , Marasmius/química , Nematoides/fisiologia , Aglutininas/química , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Mariposas/fisiologia , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas
2.
Front Plant Sci ; 12: 657451, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33897746

RESUMO

Coprinopsis cinerea lectin 2 (CCL2) is a fucoside-binding lectin from the basidiomycete C. cinerea that is toxic to the bacterivorous nematode Caenorhabditis elegans as well as animal-parasitic and fungivorous nematodes. We expressed CCL2 in Arabidopsis to assess its protective potential toward plant-parasitic nematodes. Our results demonstrate that expression of CCL2 enhances host resistance against the cyst nematode Heterodera schachtii. Surprisingly, CCL2-expressing plants were also more resistant to fungal pathogens including Botrytis cinerea, and the phytopathogenic bacterium Pseudomonas syringae. In addition, CCL2 expression positively affected plant growth indicating that CCL2 has the potential to improve two important agricultural parameters namely biomass production and general disease resistance. The mechanism of the CCL2-mediated enhancement of plant disease resistance depended on fucoside-binding by CCL2 as transgenic plants expressing a mutant version of CCL2 (Y92A), compromised in fucoside-binding, exhibited wild type (WT) disease susceptibility. The protective effect of CCL2 did not seem to be direct as the lectin showed no growth-inhibition toward B. cinerea in in vitro assays. We detected, however, a significantly enhanced transcriptional induction of plant defense genes in CCL2- but not CCL2-Y92A-expressing lines in response to infection with B. cinerea compared to WT plants. This study demonstrates a potential of fungal defense lectins in plant protection beyond their use as toxins.

3.
Planta ; 253(3): 74, 2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33620564

RESUMO

MAIN CONCLUSION: Manipulation of sugar metabolism upon S. indica root colonization triggers changes in sugar pools and defense responses in A. thaliana. Serendipita indica is an endophytic fungus that establishes mutualistic relationships with many different plants including important crops as well as the model plant A. thaliana. Successful root colonization typically results in growth promotion and enhanced tolerance against various biotic and abiotic stresses. The fungus delivers phosphorus to the host and receives in exchange carbohydrates. There are hints that S. indica prefers hexoses, glucose, and fructose, products of saccharose cleavage driven by invertases (INVs) and sucrose synthases (SUSs). Carbohydrate metabolism in this interaction, however, remains still widely unexplored. Therefore, in this work, the sugar pools as well as the expression of SUSs and cytosolic INVs in plants colonized by S. indica were analyzed. Using sus1/2/3/4 and cinv1/2 mutants the importance of these genes for the induction of growth promotion and proper root colonization was demonstrated. Furthermore, the expression of several defense-related marker genes in both multiple mutants in comparison to the wild-type plants was determined. Our results show that in colonized A. thaliana plants S. indica manipulates the sugar metabolism by altering the expression of host's INV and SUS and modulates both the sugar pools and plant defense in its favor. We conclude that the interaction A. thaliana-S. indica is a balancing act between cooperation and exploitation, in which sugar metabolism plays a crucial role. Small changes in this mechanism can lead to severe disruption resulting in the lack of growth promotion or altered colonization rate.


Assuntos
Arabidopsis/química , Basidiomycota/fisiologia , Metabolismo dos Carboidratos , Açúcares/análise , Arabidopsis/microbiologia , Endófitos/fisiologia , Raízes de Plantas/química , Raízes de Plantas/microbiologia
4.
Agronomy (Basel) ; 9(10): 595, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31857912

RESUMO

The endophytic fungi Serendipita indica and S. vermifera have recently gained increasing attention due to their beneficial effects on plant growth and plant health. Little is known about other species, such as S. williamsii and S. herbamans. To test their biocontrol and growth-promoting potential, susceptible and tolerant tomato cultivars (Kremser Perle and Micro-Tom, respectively) were inoculated with S. williamsii, S. herbamans, S. indica, or S. vermifera and challenged with the soilborne pathogen Fusarium oxysporum f. sp. lycopersici (Fol) in greenhouse experiments. Furthermore, in vitro assays on the direct inhibitory effects of Serendipita spp. against Fol were performed. Negative effects of Fol on phenological growth in the susceptible cultivar were alleviated by all four applied Serendipita spp. Apart from these similar effects on biometric parameters, disease incidence was only reduced by S. herbamans and S. vermifera. In the tolerant cultivar, disease parameters remained unaffected although shoot dry mass was negatively affected by S. vermifera. Direct effects of Serendipita spp. against Fol were not evident in the in vitro assays indicating an indirect effect via the host plant. Our results highlight the importance of identifying cultivar-specific effects in pathogen-endophyte-plant interactions to determine the most beneficial combinations.

5.
Plant Mol Biol Report ; 36(2): 284-294, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29875545

RESUMO

Serendipita indica is an axenically cultivable fungus, which colonizes a broad range of plant species including the model plant Arabidopsis thaliana. Root colonization by this endophyte leads to enhanced plant fitness and performance and promotes resistance against different biotic and abiotic stresses. The involvement of MPK6 in this mutualistic interaction had been previously shown with an mpk6 A. thaliana mutant, which failed to respond to S. indica colonization. Here, we demonstrate that mpk6 roots are significantly less colonized by S. indica compared to wild-type roots and the foliar application of plant hormones, ethylene, or jasmonic acid, restores the colonization rate at least to the wild-type level. Further, hormone-treated mpk6 plants show typical S. indica-induced growth promotion effects. Moreover, expression levels of several genes related to plant defense and hormone signaling are significantly changed at different colonization phases. Our results demonstrate that the successful root colonization by S. indica depends on efficient suppression of plant immune responses. In A. thaliana, this process relies on intact hormone signaling in which MPK6 seems to play a pivotal role.

6.
J Exp Bot ; 67(1): 107-18, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26438412

RESUMO

Plant-parasitic cyst nematodes infect plants and form highly sophisticated feeding sites in roots. It is not known which plant cell signalling mechanisms trigger plant defence during the early stages of nematode parasitism. Mitogen-activated protein kinases (MAPKs) are central components of protein phosphorylation cascades transducing extracellular signals to plant defence responses. MAPK phosphatases control kinase activities and the signalling outcome. The involvement and the role of MPK3 and MPK6, as well as the MAPK phosphatase AP2C1, is demonstrated during parasitism of the beet cyst nematode Heterodera schachtii in Arabidopsis. Our data reveal notable activation patterns of plant MAPKs and the induction of AP2C1 suggesting the attenuation of defence signalling in plant cells during early nematode infection. It is demonstrated that the ap2c1 mutant that is lacking AP2C1 is more attractive but less susceptible to nematodes compared with the AP2C1-overexpressing line. This implies that the function of AP2C1 is a negative regulator of nematode-induced defence. By contrast, the enhanced susceptibility of mpk3 and mpk6 plants indicates a positive role of stress-activated MAPKs in plant immunity against nematodes. Evidence is provided that phosphatase AP2C1, as well as AP2C1-targeted MPK3 and MPK6, are important regulators of plant-nematode interaction, where the co-ordinated action of these signalling components ensures the timely activation of plant defence.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/parasitologia , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Fosfoproteínas Fosfatases/genética , Tylenchoidea/fisiologia , Animais , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Imunidade Vegetal , Análise de Sequência de DNA , Transdução de Sinais
7.
J Exp Bot ; 66(22): 7005-17, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26324462

RESUMO

Above- and belowground plant parts are simultaneously attacked by different pests and pathogens. The host mediates these interactions and physiologically reacts, e.g. with local and systemic alterations of endogenous hormone levels coupled with coordinated transcriptional changes. This in turn affects attractiveness and susceptibility of the plant to subsequent attackers. Here, the model plant Arabidopsis thaliana is used to study stress hormone-based systemic responses triggered by simultaneous root parasitism by the cyst nematode Heterodera schachtii and shoot herbivory by the thrips Frankliniella occidentalis and the spider mite Tetranychus urticae. First, HPLC/MS and quantitative reverse transcriptase PCR are used to show that nematode parasitism strongly affects stress hormone levels and expression of hormone marker genes in shoots. Previous nematode infection is then demonstrated to affect the behavioural and life history performance of both arthropods. While thrips explicitly avoid nematode-infected plants, spider mites prefer them. In addition, the life history performance of T. urticae is significantly enhanced by nematode infection. Finally, systemic changes triggered by shoot-feeding F. occidentalis but not T. urticae are shown to make the roots more attractive for H. schachtii. This work emphasises the importance of above- and belowground signalling and contributes to a better understanding of plant systemic defence mechanisms against plant-parasitic nematodes.


Assuntos
Arabidopsis/imunologia , Arabidopsis/parasitologia , Herbivoria , Reguladores de Crescimento de Plantas/fisiologia , Animais , Comunicação Celular , Células Vegetais/metabolismo , Raízes de Plantas/imunologia , Raízes de Plantas/parasitologia , Brotos de Planta/imunologia , Brotos de Planta/parasitologia , Tetranychidae/fisiologia , Tisanópteros/fisiologia , Tylenchoidea/fisiologia
8.
New Phytol ; 207(3): 778-89, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25825039

RESUMO

Heterodera schachtii, a plant-parasitic cyst nematode, invades host roots and induces a specific syncytial feeding structure, from which it withdraws all required nutrients, causing severe yield losses. The system H. schachtii-Arabidopsis is an excellent research model for investigating plant defence mechanisms. Such responses are suppressed in well-established syncytia, whereas they are induced during early parasitism. However, the mechanisms by which the defence responses are modulated and the role of phytohormones are largely unknown. The aim of this study was to elucidate the role of hormone-based defence responses at the onset of nematode infection. First, concentrations of main phytohormones were quantified and the expression of several hormone-related genes was analysed using quantitative real-time (qRT)-PCR or GeneChip. Further, the effects of individual hormones were evaluated via nematode attraction and infection assays using plants with altered endogenous hormone concentrations. Our results suggest a pivotal and positive role for ethylene during nematode attraction, whereas jasmonic acid triggers early defence responses against H. schachtii. Salicylic acid seems to be a negative regulator during later syncytium and female development. We conclude that nematodes are able to impose specific changes in hormone pools, thus modulating hormone-based defence and signal transduction in strict dependence on their parasitism stage.


Assuntos
Arabidopsis/fisiologia , Arabidopsis/parasitologia , Reguladores de Crescimento de Plantas/metabolismo , Imunidade Vegetal , Estresse Fisiológico , Tylenchoidea/fisiologia , Animais , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Bioensaio , Cromatografia Líquida de Alta Pressão , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Espectrometria de Massas , Oxilipinas/farmacologia , Parasitos/fisiologia , Doenças das Plantas/parasitologia , Imunidade Vegetal/efeitos dos fármacos , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/parasitologia , Ácido Salicílico/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Genética/efeitos dos fármacos , Tylenchoidea/efeitos dos fármacos
9.
Plant Sci ; 234: 38-49, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25804808

RESUMO

Gall forming phylloxera may compete for nutrients with meristematic tissues and develop heterotrophic structures that act as carbon sinks. In this work, we studied the underlying starch metabolism, sink-source translocation of soluble sugars towards and within root galls. We demonstrated that nodosities store carbohydrates by starch accumulation and monitored the expression of genes involved in the starch metabolic. Thereby we proved that the nodosity is symplastically connected to the source tissues through its development and that the starch metabolism is significantly affected to synthesize and degrade starch within the gall. Genes required for starch biosynthesis and degradation are up-regulated. Among the carbohydrate transporters the expression of a glucose-6-phosphate translocater, one sucrose transporter and two SWEET proteins were increases, whereas hexose transporters, tonoplast monosaccharide transporter and Erd6-like sugar transporters were decreased. We found general evidence for plant response to osmotic stress in the nodosity as previously suggested for gall induction processes. We conclude that nodosities are heterogenous plant organs that accumulate starch to serve as temporary storage structure that is gradually withdrawn by phylloxera. Phylloxera transcriptionally reprograms gall tissues beyond primary metabolism and included downstream secondary processes, including response to osmotic stress.


Assuntos
Regulação da Expressão Gênica de Plantas , Hemípteros/fisiologia , Interações Hospedeiro-Parasita , Vitis/parasitologia , Animais , Metabolismo dos Carboidratos , Perfilação da Expressão Gênica , Hemípteros/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/parasitologia , Raízes de Plantas/ultraestrutura , Tumores de Planta/genética , Tumores de Planta/parasitologia , Amido/metabolismo , Vitis/genética , Vitis/metabolismo , Vitis/ultraestrutura
10.
Bio Protoc ; 5(18)2015 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-29082278

RESUMO

This protocol describes an effective method of in situ RT-PCR that was developed to localize specific gene expression directly in thin cross-sections of nematode feeding sites induced by the cyst nematode Heterodera schachtii (H. schachtii) or the root-knot nematode Meloidogyne incognita (M. incognita) in Arabidopsis roots using DIG (Digoxigenin-11dUTP) labeling coupled with AP (alkaline phosphatase) and nitro-blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate-based detection. This method is applicable to any other Arabidopsis root tissue.

11.
Bio Protoc ; 5(18)2015 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-29085856

RESUMO

Plant parasitic nematodes are devastating pests on many crops. Juveniles (J2) of cyst nematodes invade the roots to induce a syncytium. This feeding site is their only source of nutrients. Male nematodes leave the roots after the fourth molt to mate with females. The females stay attached to their syncytia throughout their life and produce hundreds of eggs, which are contained in their bodies. When the females die their bodies form the cysts, which protect the eggs. Cysts can survive for many years in the soil until favorable conditions induce hatching of the juveniles. The beet cyst nematode Heterodera schachtii (H. schachtii)is a pathogen of sugar beet (Beta vulgaris) but can also complete its life cycle on Arabidopsis roots growing on agar plates under sterile conditions. We present here protocols for a stock culture of H. schachtii and an infection assay on agar plates.

12.
PLoS One ; 9(7): e102360, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25033038

RESUMO

Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is the only source of nutrients throughout their life. A recent transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that thousands of genes are up-regulated or down-regulated in syncytia as compared to root segments from uninfected plants. Among the down-regulated genes are many which code for WRKY transcription factors. Arabidopsis contains 66 WRKY genes with 59 represented by the ATH1 GeneChip. Of these, 28 were significantly down-regulated and 6 up-regulated in syncytia as compared to control root segments. We have studied here the down-regulated genes WRKY6, WRKY11, WRKY17 and WRKY33 in detail. We confirmed the down-regulation in syncytia with promoter::GUS lines. Using various overexpression lines and mutants it was shown that the down-regulation of these WRKY genes is important for nematode development, probably through interfering with plant defense reactions. In case of WRKY33, this might involve the production of the phytoalexin camalexin.


Assuntos
Arabidopsis/metabolismo , Células Gigantes/metabolismo , Raízes de Plantas/metabolismo , Fatores de Transcrição/biossíntese , Tylenchoidea/patogenicidade , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Indóis/metabolismo , Doenças das Plantas/parasitologia , Raízes de Plantas/genética , Raízes de Plantas/parasitologia , Regiões Promotoras Genéticas/genética , Tiazóis/metabolismo , Fatores de Transcrição/genética , Tylenchoidea/metabolismo
13.
Plant J ; 74(5): 852-66, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23480402

RESUMO

The beet cyst nematode Heterodera schachtii induces syncytia in the roots of Arabidopsis thaliana, which are its only nutrient source. One gene, At1g64110, that is strongly up-regulated in syncytia as shown by RT-PCR, quantitative RT-PCR, in situ RT-PCR and promoter::GUS lines, encodes an AAA+-type ATPase. Expression of two related genes in syncytia, At4g28000 and At5g52882, was not detected or not different from control root segments. Using amiRNA lines and T-DNA mutants, we show that At1g64110 is important for syncytium and nematode development. At1g64110 was also inducible by wounding, jasmonic acid, salicylic acid, heat and cold, as well as drought, sodium chloride, abscisic acid and mannitol, indicating involvement of this gene in abiotic stress responses. We confirmed this using two T-DNA mutants that were more sensitive to abscisic acid and sodium chloride during seed germination and root growth. These mutants also developed significantly smaller roots in response to abscisic acid and sodium chloride. An in silico analysis showed that ATPase At1g64110 (and also At4g28000 and At5g52882) belong to the 'meiotic clade' of AAA proteins that includes proteins such as Vps4, katanin, spastin and MSP1.


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ácido Abscísico/farmacologia , Animais , Arabidopsis/citologia , Arabidopsis/parasitologia , Ciclopentanos/farmacologia , Secas , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Células Gigantes/parasitologia , Interações Hospedeiro-Parasita , Manitol/farmacologia , Mutação , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/parasitologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácido Salicílico/farmacologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/parasitologia , Cloreto de Sódio/farmacologia , Estresse Mecânico , Temperatura , Tylenchoidea/fisiologia
14.
Plant Physiol Biochem ; 49(10): 1100-7, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21813283

RESUMO

The beet cyst nematode Heterodera schachtii induces a feeding site, called syncytium, in roots of host plants. In Arabidopsis, one of the genes whose expression is strongly induced in these structures is Pdf2.1 which codes for an antimicrobial plant defensin. Arabidopsis has 13 plant defensin genes. Besides Pdf2.1, the Pdf2.2 and Pdf2.3 genes were strongly expressed in syncytia and therefore the expression of all three Pdf genes was studied in detail. The promoter of the Pdf2.1 gene turned out to be an interesting candidate to drive a syncytium-specific expression of foreign genes as RT-PCR showed that apart from the feeding site it was only expressed in siliques (seeds). The Pdf2.2 and Pdf2.3 genes were in addition expressed in seedlings, roots, leaves, stems, and flowers. These results were supported by the analysis of promoter::GUS lines. After infection with H. schachtii all GUS lines showed a strong staining in syncytia at 5 and 15 dpi. This expression pattern was confirmed by in situ RT-PCR.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/parasitologia , Defensinas/metabolismo , Proteínas de Homeodomínio/metabolismo , Nematoides/patogenicidade , Raízes de Plantas/parasitologia , Regiões Promotoras Genéticas , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clonagem Molecular , Defensinas/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Células Gigantes/metabolismo , Células Gigantes/parasitologia , Proteínas de Homeodomínio/genética , Infecções por Nematoides/parasitologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Sementes/genética , Sementes/metabolismo
15.
New Phytol ; 184(2): 457-472, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19691674

RESUMO

* In plants, UDP-glucuronic acid is synthesized by the oxidation of UDP-glucose by UDP-glucose dehydrogenase or the oxygenation of free myo-inositol by myo-inositol oxygenase (MIOX). In Arabidopsis, myo-inositol oxygenase is encoded by four genes. Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots revealed that MIOX genes are among the most strongly upregulated genes. * We have used beta-glucuronidase (GUS) analysis, in situ reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR to study the expression of all four MIOX genes in syncytia induced by H. schachtii in Arabidopsis roots. All these methods showed that MIOX genes are strongly induced in syncytia. GeneChip data were analysed for the expression of genes related to the MIOX pathway (mapman). * Two complementary double mutants were used to study the importance of MIOX genes. Results of the infection assay with double mutants in two combinations (Deltamiox1+2, Deltamiox4+5) showed a significant reduction (P < 0.05) in the number of females per plant when compared with the wild-type. Furthermore, syncytia in double mutants were significantly smaller than in wild-type plants. * Our data demonstrate an important role of the MIOX genes for syncytium development and for the development of female nematodes.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Expressão Gênica , Genes de Plantas , Células Gigantes/enzimologia , Inositol Oxigenase/genética , Nematoides , Animais , Arabidopsis/citologia , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Feminino , Perfilação da Expressão Gênica , Glucuronidase , Inositol Oxigenase/metabolismo , Mutação , Raízes de Plantas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
16.
J Exp Bot ; 60(11): 3085-95, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19487386

RESUMO

The plant-parasitic nematode Heterodera schachtii stimulates plant root cells to form syncytial feeding structures which synthesize all nutrients required for successful nematode development. Cellular re-arrangements and modified metabolism of the syncytia are accompanied by massive intra- and intercellular solute allocations. In this study the expression of all genes annotated as sugar transporters in the Arabidopsis Membrane Protein Library was investigated by Affymetrix gene chip analysis in young and fully developed syncytia compared with non-infected Arabidopsis thaliana roots. The expression of three highly up-regulated (STP12, MEX1, and GTP2) and three highly down-regulated genes (SFP1, STP7, and STP4) was analysed by quantitative RT-PCR (qRT-PCR). The most up-regulated gene (STP12) was chosen for further in-depth studies using in situ RT-PCR and a nematode development assay with a T-DNA insertion line revealing a significant reduction of male nematode development. The specific role of STP12 expression in syncytia of male juveniles compared with those of female juveniles was further shown by qRT-PCR. In order to provide evidence for sugar transporter activity across the plasma membrane of syncytia, fluorescence-labelled glucose was used and membrane potential recordings following the application of several sugars were performed. Analyses of soluble sugar pools revealed a highly specific composition in syncytia. The presented work demonstrates that sugar transporters are specifically expressed and active in syncytia, indicating a profound role in inter- and intracelluar transport processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Células Gigantes/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Nematoides/fisiologia , Doenças das Plantas/parasitologia , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Células Gigantes/parasitologia , Proteínas de Transporte de Monossacarídeos/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/parasitologia
17.
Plant J ; 57(5): 771-84, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18980640

RESUMO

Arabidopsis thaliana is a host for the sugar beet cyst nematode Heterodera schachtii. Juvenile nematodes invade the roots and induce the development of a syncytium, which functions as a feeding site for the nematode. Here, we report on the transcriptome of syncytia induced in the roots of Arabidopsis. Microaspiration was employed to harvest pure syncytium material, which was then used to prepare RNA for hybridization to Affymetrix GeneChips. Initial data analysis showed that the gene expression in syncytia at 5 and 15 days post-infection did not differ greatly, and so both time points were compared together with control roots. Out of a total of 21 138 genes, 18.4% (3893) had a higher expression level and 15.8% (3338) had a lower expression level in syncytia, as compared with control roots, using a multiple-testing corrected false discovery rate of below 5%. A gene ontology (GO) analysis of up- and downregulated genes showed that categories related to high metabolic activity were preferentially upregulated. A principal component analysis was applied to compare the transcriptome of syncytia with the transcriptome of different Arabidopsis organs (obtained by the AtGenExpress project), and with specific root tissues. This analysis revealed that syncytia are transcriptionally clearly different from roots (and all other organs), as well as from other root tissues.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Perfilação da Expressão Gênica , Células Gigantes/metabolismo , Nematoides/fisiologia , Raízes de Plantas/genética , Animais , Arabidopsis/metabolismo , Arabidopsis/parasitologia , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Células Gigantes/parasitologia , Análise de Sequência com Séries de Oligonucleotídeos , Raízes de Plantas/metabolismo , Raízes de Plantas/parasitologia , Análise de Componente Principal , RNA de Plantas/metabolismo
18.
Proc Natl Acad Sci U S A ; 105(42): 16374-9, 2008 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-18922781

RESUMO

The dsRNA-binding protein Staufen was the first RNA-binding protein proven to play a role in RNA localization in Drosophila. A mammalian homolog, Staufen1 (Stau1), has been implicated in dendritic RNA localization in neurons, translational control, and mRNA decay. However, the precise mechanisms by which it fulfills these specific roles are only partially understood. To determine its physiological functions, the murine Stau1 gene was disrupted by homologous recombination. Homozygous stau1(tm1Apa) mutant mice express a truncated Stau1 protein lacking the functional RNA-binding domain 3. The level of the truncated protein is significantly reduced. Cultured hippocampal neurons derived from stau1(tm1Apa) homozygous mice display deficits in dendritic delivery of Stau1-EYFP and beta-actin mRNA-containing ribonucleoprotein particles (RNPs). Furthermore, these neurons have a significantly reduced dendritic tree and develop fewer synapses. Homozygous stau1(tm1Apa) mutant mice are viable and show no obvious deficits in development, fertility, health, overall brain morphology, and a variety of behavioral assays, e.g., hippocampus-dependent learning. However, we did detect deficits in locomotor activity. Our data suggest that Stau1 is crucial for synapse development in vitro but not critical for normal behavioral function.


Assuntos
Alelos , Dendritos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Regulação da Expressão Gênica , Hipocampo/metabolismo , Homozigoto , Locomoção , Camundongos , Mutação/genética , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/genética
19.
Plant Physiol ; 148(1): 358-68, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18599655

RESUMO

During the interaction between sedentary plant-parasitic nematodes and their host, complex morphological and physiological changes occur in the infected plant tissue, finally resulting in the establishment of a nematode feeding site. This cellular transformation is the result of altered plant gene expression most likely induced by proteins injected in the plant cell by the nematode. Here, we report on the identification of a WRKY transcription factor expressed during nematode infection. Using both promoter-reporter gene fusions and in situ reverse transcription-polymerase chain reaction, we could show that AtWRKY23 is expressed during the early stages of feeding site establishment. Knocking down the expression of WRKY23 resulted in lower infection of the cyst nematode Heterodera schachtii. WRKY23 is an auxin-inducible gene and in uninfected plants WRKY23 acts downstream of the Aux/IAA protein SLR/IAA14. Although auxin is known to be involved in feeding site formation, our results suggest that, during early stages, auxin-independent signals might be at play to activate the initial expression of WRKY23.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Comportamento Alimentar/fisiologia , Interações Hospedeiro-Parasita , Fatores de Transcrição/metabolismo , Tylenchoidea/fisiologia , Animais , Arabidopsis/parasitologia , Ácidos Indolacéticos/metabolismo , Transdução de Sinais , Regulação para Cima
20.
Plant J ; 53(2): 336-51, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18069944

RESUMO

Cyst nematodes induce root syncytia with specific features such as hypertrophy, increased metabolic activity and fusion with adjacent cells. Cell walls of the syncytia undergo massive changes such as thickening, local dissolution and formation of ingrowths. Cell wall degrading and modifying proteins are apparently involved in syncytium formation but detailed knowledge of this is still limited. Therefore, we studied the regulation and function of the entire Arabidopsis endo-1,4-beta-glucanase gene family in syncytia induced by Heterodera schachtii. Endo-1,4-beta-glucanases hydrolyze the 1,4-beta-glucosidic linkages between glucose residues. Using semi-quantitative and quantitative approaches we identified seven genes that are upregulated in syncytia. Two of these genes, coding for secreted AtCel2 and membrane-bound KOR3, are shoot-specific but show high expression in syncytia at different developmental stages. In silico analysis of the promoter regions of both genes compared with other genes with modified regulation in nematode feeding sites did not reveal specific cis-acting elements that could be related to specific transcription in syncytia. However, motifs responsive to sugar and different plant hormones were identified. Accordingly, treatments with sucrose, gibberellic acid and NAA induced upregulation of AtCel2, whereas ABA triggered downregulation of both AtCel2 and KOR3 in roots. As AtCel2 is related to degradation of the cell wall matrix, we analysed the hemicellulose content in syncytia. The measured values resembled the expression pattern of AtCel2. A distinctly reduced number of females developed in cel2 and kor3 T-DNA mutants, and we therefore conclude that endo-1,4-beta-glucanases play an important role in the formation and function of syncytia.


Assuntos
Arabidopsis/enzimologia , Celulase/metabolismo , Células Gigantes/citologia , Raízes de Plantas/citologia , Tylenchoidea/fisiologia , Animais , Arabidopsis/genética , Arabidopsis/parasitologia , Celulase/genética , Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Regulação da Expressão Gênica de Plantas , Células Gigantes/enzimologia , Células Gigantes/parasitologia , Raízes de Plantas/enzimologia , Raízes de Plantas/parasitologia
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