Diversity and activity of sugar transporters in nematode-induced root syncytia

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

Transcriptome profiling of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots : a model system to study plant-nematode interactions at a global view

Die Modellpflanze Arabidopsis ist eine ideale Wirtspflanze für den wirtschaftlich bedeutenden Rübenzystenematoden H. schachtii. Anand dieses Modell-Systems wurde das Transkriptom der Synzytien mit Hilfe des ATH1 Genchips der Firma Affymetrix analysiert. Es wurde Mikroaspiration angewandt, um möglichst reines Untersuchungsmaterial aus Synzytien zu gewinnen. Unsere Analyse zeigte, dass sich die Expressionsprofile von 5 und 15 Tage alten Synzytien nicht stark unterscheiden. Ein Vergleich zwischen Kontrollwurzeln und Synzytien von beiden Infektionszeitpunkten zusammen genommen hat gezeigt, dass 34.2 % der insgesamt 21,138 durch den Chip repräsentierten Arabidopsis Gene unterschiedlich exprimiert sind, wobei etwa die eine Hälfte der Gene signifikant aufreguliert (18.4 %) war und die andere abreguliert (15.8 %). Eine Hauptkomponenten-Analyse, in welcher auch Chip-Daten von diversen anderen Wurzelgeweben und Organen von Arabidopsis integriert wurden, hat gezeigt, dass sich die beiden Transcriptome der Synzytien klar von denen aller anderen Organe und Wurzelproben unterscheiden. Da die Zellwand drastischen Veränderungen während der Synzytienbildung unterworfen ist, wurden zwei Genfamilien, die für zellwandmodifizierende Proteine und Enzyme kodieren, detailierteren Untersuchungen unterzogen. Innerhalb der Familien der Expansine und der Endo-1,4--Glukanasen wurden je zwei synzytial induzierte Gene entdeckt, die normalerweise nur sproßspezifisch exprimiert werden und möglicherweise eine funktionale Rolle bei zellwandaufbauenden- und -abbauenden Prozessen spielen; u.zw. die beiden Expansin-Gene AtEXPA3 und AtEXPA1 sowie die Glukanasen-Gene AtCel2 und KOR3. Zwei der am stärksten aufregulierten Gene gehören zur MIOX Genfamilie, welche normalerweise pollenspezifisch exprimiert werden. Zwecks einer effizienten Produktion von transgenen Pflanzen mittels Agrobakterium wurde ein verbesserter binärer Vektor namens pPZP3425 konstruiert. Er leitet sich vom weit verbreiteten pPZP111 ab und beinhaltet ein gus-Gen, das von einem starken konstitutiven Promoter gesteuert wird. Er kann daher sowohl zur Überexpression von Genen als auch zur Erzeugung von Promotor:gus Fusionen verwendet werden und möglicherweise auch dazu beitragen, nematodenresistente Pflanzen zu erzeugen.Plant-parasitic nematodes are obligate biotrophic pathogens causing major economic impacts worldwide. Sedentary endoparasites such as cyst nematodes have evolved very specialized and complex feeding sites that become their permanent source of nutrients. To date, our knowledge about the processes of feeding site formation and function is still limited. We therefore used Arabidopsis as a host for the beet cyst nematode Heterodera schachtii to investigate the complex changes via transcriptome analysis using the Affymetrix GeneChips ATH1. We employed microaspiration to harvest pure material from the induced feeding site that starts from a single root cell and grows by incorporation of adjacent cells into an enlarged, multi-cellular syncytium, thereby undergoing drastic morphological and ultrastructurally changes. Our analysis showed that between 5 and 15 days after infection the expression profile did not differ greatly. However, the combined syncytial tissue compared to uninfected roots revealed that 34.2% of 21,138 genes are differentially expressed. Genes belonging to gene ontology categories related to metabolic activity were preferentially upregulated. A principal component analysis including chip data from several other root tissues and organs demonstrated that the transcriptomes of syncytia are clearly distinct from all other organs including roots. We examined the role of two entire gene families coding for cell wall modifying proteins in more detail. Within the expansin and endo-1,4--glucanase gene family two usually shoot-specifically expressed genes are induced in syncytia and are supposed to play a functional role in cell wall assembly and disassembly processes; i.e. AtEXPA3, AtEXPA1 and AtCel2, KOR3, respectively. The most strongly upregulated genes belong to pollen-specific MIOX genes. There are also some first indications of an involvement in syncytium formation. Finally, we constructed an improved binary Agrobacterium vector termed pPZP3425 that derived from pPZP111. It can be used for multiple purposes and may help to construct resistant plants.submitted by Dagmar Elisabeth SzakasitsAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2009OeBB(VLID)193052

Starch Serves as Carbohydrate Storage in Nematode-Induced Syncytia1[W][OA]

The plant parasitic nematode Heterodera schachtii induces specific syncytial feeding sites in the roots of Arabidopsis thaliana from where it withdraws all required nutrients. Therefore, syncytia have to be well supplied with assimilates and generate strong sinks in the host plant's transport system. Import mechanisms and consequent accumulation of sucrose in syncytia were described recently. In this work, we studied the starch metabolism of syncytia. Using high-performance liquid chromatography and microscopic analyses, we demonstrated that syncytia store carbohydrates by starch accumulation. Further, we monitored the expression of genes involved in the starch metabolic pathway by gene chip analysis and quantitative reverse transcription-PCR. Finally, we provide functional proof of the importance of starch synthesis for nematode development using T-DNA insertion lines. We conclude that syncytia accumulate starch as a carbohydrate buffer to compensate for changing solute uptake by the nematode and as long-term storage during juvenile development

The promoter of a plant defensin gene directs specific expression in nematode-induced syncytia in Arabidopsis roots

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

Myo-inositol oxygenase genes are involved in the development of syncytia induced byHeterodera schachtiiin Arabidopsis roots

•  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 β-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 (Δmiox1+2, Δmiox4+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

Arabidopsis endo-1,4-ß-glucanases are involved in the formation of root syncytia induced by Heterodera schachtii

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