The transcriptome of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots.

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.

Myo-inositol oxygenase genes are involved in the development of syncytia induced by Heterodera schachtii in 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 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.

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.

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.

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

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.

Starch serves as carbohydrate storage in nematode-induced syncytia.

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.

Expansins are involved in the formation of nematode-induced syncytia in roots of Arabidopsis thaliana.

Parasitism of the cyst nematode Heterodera schachtii is characterized by the formation of syncytial feeding structures in the host root. Syncytia are formed by the fusion of root cells, accompanied by local cell wall degradation, fusion of protoplasts and hypertrophy. Expansins are cell wall-loosening proteins involved in growth and cell wall disassembly. In this study, we analysed whether members of the expansin gene family are specifically and developmentally regulated during syncytium formation in the roots of Arabidopsis thaliana. We used PCR to screen a cDNA library of 5-7-day-old syncytia for expansin transcripts with primers differentiating between 26 alpha- and three beta-expansin cDNAs. AtEXPA1, AtEXPA3, AtEXPA4, AtEXPA6, AtEXPA8, AtEXPA10, AtEXPA15, AtEXPA16, AtEXPA20 and AtEXPB3 could be amplified from the library. In a semi-quantitative RT-PCR and a Genechip analysis AtEXPA3, AtEXPA6, AtEXPA8, AtEXPA10 and AtEXPA16 were found to be upregulated specifically in syncytia, but not to be transcribed in surrounding root tissue. Histological analyses were performed with the aid of promoter::GUS lines and in situ RT-PCR. Results from both approaches supported the specific expression pattern. Among the specifically expressed genes, AtEXPA3 and AtEXPA16 turned out to be of special interest as they are shoot-specific in uninfected plants. We conclude that syncytium formation involves the specific regulation of expansin genes, indicating that the encoded expansins take part in cell growth and cell wall disassembly during syncytium formation.