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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., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

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., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

* 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.

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.

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.

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.

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.