Florian M. W. Grundler

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.

PYK10, a β-glucosidase located in the endoplasmatic reticulum, is crucial for the beneficial interaction between Arabidopsis thaliana and the endophytic fungus Piriformospora indica

Piriformospora indica, an endophyte of the Sebacinaceae family, promotes growth and seed production of many plant species, including Arabidopsis. Growth of a T-DNA insertion line in PYK10 is not promoted and the plants do not produce more seeds in the presence of P. indica, although their roots are more colonized by the fungus than wild-type roots. Overexpression of PYK10 mRNA did not affect root colonization and the response to the fungus. PYK10 codes for a root- and hypocotyl-specific beta-glucosidase/myrosinase, which is implicated to be involved in plant defences against herbivores and pathogens. Expression of PYK10 is activated by the basic helix-loop-helix domain containing transcription factor NAI1, and two Arabidopsis lines with mutations in the NAI1 gene show the same response to P. indica as the PYK10 insertion line. PYK10 transcript and PYK10 protein levels are severely reduced in a NAI1 mutant, indicating that PYK10 and not the transcription factor NAI1 is responsible for the response to the fungus. In wild-type roots, the message level for a leucine-rich repeat protein LRR1, but not for plant defensin 1.2 (PDF1.2), is upregulated in the presence of P. indica. In contrast, in lines with reduced PYK10 levels the PDF1.2, but not LRR1, message level is upregulated in the presence of the fungus. We propose that PYK10 restricts root colonization by P. indica, which results in the repression of defence responses and the upregulation of responses leading to a mutualistic interaction between the two symbiotic partners.

Studies on the nutrient uptake by the beet cyst nematode Heterodera schachtii by in situ microinjection of fluorescent probes into the feeding structures in Arabidopsis thaliana.

A method was developed, which enables substances to be injected into the feeding structure (syncytium) established by Heterodera schachtii in roots of Arabidopsis thaliana. The technique was used to study the uptake if nutrients by the feeding nematode. The fluorescent dye lucifer yellow CH (LOCH) and fluorescence-labelled dextrans of different molecular weights were injected into the thin and translucent roots of A. thalana. Such roots are a feature of this plant and they provide optimal conditions for microinjection. Injected LOCH was taken up by feeding juveniles and adults, indicated by the staining of the alimentary duct and the digestive system. Fluorescent dextrans of 3, 10 and 20 kDa but not of 40 and 70 kDa were ingested, suggesting that molecules of a maximum Stokes radius of 3.2 to 4.4 nm are taken up

A Role for AtWRKY23 in Feeding Site Establishment of Plant-Parasitic Nematodes

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.

Cellular and molecular aspects of plant-nematode interactions

Root Parasitic Nematodes: An Overview U. Wyss. A: Cellular and Molecular Aspects of the Interaction. Root Anatomy and Development, The Basis for Nematode Parasitism B. Scheres, et al. Plant Signals in Nematode Hatching and Attraction R.N. Perry. Invasion and Migration Behaviour of Sedentary Nematodes N. von Mende. The Biology of Giant Cells T. Bleve-Zacheo, M.T. Melillo. The Structure of Syncytia W. Golinowski, et al. Nematode Secretions J.T. Jones, W.M. Robertson. Physiology of Nematode Feeding and Feeding Sites F.M.W. Grundler, A. Bockenhoff. Cell Cycle Regulation in Nematode Feeding Sites G. Gheysen, et al. Regulation of Gene Expression in Feeding Sites C. Fenoll, et al. B: Natural Resistance. Natural Resistance: The Assessment of Variation in Virulence in Biological And Molecular Terms C. Zijlstra, et al. Genetic and Molecular Strategies for the Cloning of (A)Virulence Genes in Sedentary Plant-Parasitic Nematodes P. Castagnone-Sereno, et al. Breeding for Nematode Resistance in Sugarbeet: A Molecular Approach M. Kleine, et al. Resistance to Root-Knot Nematodes in Tomato T.B. Liharska, V.M. Williamson. Biochemistry of Plant Defence Responses to Nematode Infection G. Zacheo, et al. C: Engineered Resistance. Engineering Resistance Against Plant Parasitic Nematodes Using Anti-Nematode Genes P.R. Burrows, D. de Waele. Engineering Plant Nematode Resistance by Antifeedants M.J. McPherson, et al. Anti-Feeding Structure Approaches to Nematode Resistance S.A. Ohl, et al. Towards Plantibody-Mediated Resistance Against Nematodes W.J. Stiekema, et al. Epilogue P.R. Burrows.

Formation of wall openings in root cells of Arabidopsis thaliana following infection by the plant-parasitic nematode Heterodera schachtii

The induction and differentiation of feeding structures (syncytia) of the cyst nematode Heterodera schachtii in roots of Arabidopsis thaliana is accompanied by drastic cellular modifications. We investigated the formation of cell wall openings which occurred during syncytium differentiation. At the beginning of syncytium induction, a callose-like layer was deposited inside of the wall of the initial syncytial cell (ISC). First wall dissolutions developed by gradual widening of plasmodesmata between the ISC and neighbouring cells. As a general thickening of syncytial cell walls blocked existing plasmodesmata, other large openings were formed by enzymatic dissolution of intact walls by putative cellulase activity.

Induction of Phloem Unloading in Arabidopsis thaliana Roots by the Parasitic Nematode Heterodera schachtii

Phloem unloading of both the fluorescent probe carboxyfluorescein (CF) and 14C-labeled solutes was induced in Arabidopsis thaliana L. roots by the parasitic nematode Heterodera schachtii Schmidt. Confocal laser scanning microscopy demonstrated that anomalous unloading of CF from the sieve element companion cell complexes occurred specifically into the syncytium, the nematode- induced feeding structure located within the stele of the root. From this syncytial complex of modified root cells, both fluorescent and radioactive labels were withdrawn by feeding nematodes. Movement of CF was unidirectional from the phloem to the syncytium. A range of low-molecular-weight fluorescent probes (including CF) microinjected into the syncytium stayed in this structure, demonstrating that it is symplastically isolated from the surrounding root tissue. The mechanism of unloading in this host-pathogen relationship therefore appears to be apoplastic. Our results provide unequivocal evidence that sedentary cyst-forming nematodes have direct access to phloem-derived solutes.

The Companion Cell-Specific Arabidopsis Disaccharide Carrier AtSUC2 Is Expressed in Nematode-Induced Syncytia

Cyst nematodes induce a metabolically highly active syncytial cell complex in host roots. The syncytia are symplastically isolated. Because they form a strong sink, assimilates must be imported via the apoplast, thus suggesting that specific membrane-bound sugar transport proteins are expressed and activated. To identify possible candidate genes, transgenic Arabidopsis plants expressing different reporter genes under the control of different promoters from Arabidopsis sugar transporter genes were infected with the beet cyst nematode (Heterodera schachtii). With polymerase chain reaction, 13 additional sugar transporters were tested for their presence in the syncytia through the use of a syncytium-specific cDNA library. Analysis of the infected roots showed that the promoter of the sucrose (Suc) transporter AtSUC2 gene that codes for a companion cell-specific Suc transporter in noninfected plants was found to be expressed in syncytia. Its expression patterns in β-glucuronidase and green fluorescent protein plants were monitored. Syncytium-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction. Results support the idea that AtSUC2 mediates the transmembrane transfer of Suc. AtSUC2 is the first disaccharide carrier described to be activated by pathogens.

Changes in the structure of Arabidopsis thaliana roots induced during development of males of the plant parasitic nematode Heterodera schachtii

Plant parasitic nematodes of the genus Heterodera show a high degree of sexual dimorphism, which is reflected by different nutritional demands and differences in the structure of the induced specific syncytial feeding site in the plant. The determination of the sex of the nematode Heterodera schachtii and other related species was repeatedly reported to be dependent on trophic factors, which are provided by the induced syncytia. The structural differences of syncytia induced by H. schachtii in roots of Arabidopsis thaliana were analysed at the anatomical and ultrastructural level. Syncytia of males were induced in the root pericycle. The developing syncytium then expanded into procambial or cambial cells of the vascular cylinder. Differentiated vascular elements were not included. The expansion of the syncytium triggered the proliferation of cambial and peridermal tissues, in a manner similar to secondary growth, and the formation of additional xylem and phloem elements. In comparison to syncytia associated with females, syncytia associated with males were less hypertrophied and were composed of more cells. Distinct cell wall openings were mostly found between the few strongly hypertrophied syncytial elements at the actual feeding site in the pericycle. The ultrastructure was very similar to female-associated syncytia but showed conspicuous differences in the structure and localization of cell wall ingrowths. These ingrowths were rare and weakly developed and occurred not only at the interface with xylem elements but also at the internal and external walls of the syncytia. After feeding had ceased at the end of the third developmental stage the syncytia degenerated.

Localization of hydrogen peroxide during the defence response of Arabidopsis thaliana against the plant-parasitic nematode Heterodera glycines

Hydrogen peroxide (H 2 O 2 ) production during the infection of Arabidopsis thaliana by the soybean cyst nematode Heterodera glycines was detected histochemically by the reaction of H 2 O 2 with cerium chloride producing four different patterns of electron-dense precipitates of cerium perhydroxides. As A. thaliana is not a regular host of H. glycines , the defence response is considerable, but does not completely inhibit the development of the nematode. H 2 O 2 was produced not only by cells mechanically damaged during invasion and feeding site induction by the nematode, but also by cells surrounding developing syncytia and cells which were neither in contact with the nematode nor with the syncytium. Die Lokalisation von Peroxid wahrend der Abwehrreaktion von Arabidopsis thaliana gegen den pflanzenparasitaren Nematoden Heterodera glycines - Die Bildung von Wasserstoffperoxid (H 2 O 2 ) im Rahmen der Infektion von Arabidopsis thaliana durch den Sojabohnen-Zystennematoden Heterodera glycines wurde histochemisch durch die Reaktion von H 2 O 2 mit Cerchlorid nachgewiesen, wobei vier verschiedene Muster elektronendichter Prazipitate von Cerperhydroxiden gebildet wurden. Da A. thaliana kein regularer Wirt von H. glycine s ist, kommt es zu einer betrachtlichen Abwehrreaktion, die jedoch die Entwicklung des Nematoden nicht vollstandig verhindert. H 2 O 2 wurde nicht nur von Zellen produziert, die im Laufe des Eindringens und der Induktion des Nahrzellensystems durch den Nematoden mechanisch beschadigt worden waren, sondern auch von Zellen, die sich entwickelnde Syncytien umgaben und von Zellen, die weder mit dem Nematoden noch mit dem Syncytium in Kontakt standen.