Cell wall biosynthesis in cyst nematode induced syncytia

After invading roots of host plants the cyst nematode Heterodera schachtii homes in on a defined set of procambial cells in the root vasculature and re-programs their developmental fate to form a syncytium that nurtures immobile juveniles into adulthood. Syncytium formation involves degradation of walls between adjacent pre-syncytial cells, thickening of syncytial outer walls and deposition of cell wall ingrowths at the interface between the syncytium and the host vasculature. Finally, internal turgor-pressure together with action of cell wall modifying factors triggers massive expansion of syncytium and leads to hypertrophy of the feeding site complex. In the present project we hypothesize that the wall thickening process and the formation of cell wall ingrowths in syncytia involves biosynthesis of new cell wall material. Further, we will test whether cell wall alteration might ultimately be important for feeding site formation and nematode development. This project comprises three main objectives. The first one is the in-depth analysis of the Affymetrix GeneChips made for several stages of syncytia induced by H. schachtii in roots of Arabidopsis focusing on genes involved in cell wall biosynthesis. These data together with expression (qRT-PCR) and localisation (in situ RT-PCR) studies will generate hypotheses on which cell wall components are made and allow us to select candidate genes. The second objective is to analyze the qualitative composition of the syncytial walls at the different developmental stages by use of cell wall carbohydrate specific immunoprobes. Finally, as a third objective a selected collection of A. thaliana cell wall defective mutants for interesting candidate genes will be tested based on expression analysis and immunocytochemistry. By testing our hypotheses we contribute to a further understanding of plant-nematode interactions and, if a deliberate modification of the cell wall biosynthetic machinery in crop species interferes with the nematode life cycle, new resistance breeding strategies can be developed.

Cyst (Heterodera spp.) and root-knot nematodes (Meloidogyne spp.) are root pathogens that cause severe losses in the crop production. In the host roots they induce specific feeding structures, so called syncytia and giant cells, respectively. One of the remarkable events occurring during their formation is the extensive remodelling of the plant cell wall resulting from the cell wall thickening, extension and dissolution. Thus, the main aim of this project was to get insights into the genetic background of these events as well as into the qualitative composition of cell walls in nematode feeding sites. In course of this project two groups of Arabidopsis cell wall enzymes were analysed: cellulose synthase genes (CesA genes) and pectate lyase-like genes (PLL genes). Members of CesA family are responsible for biosynthesis of the cell wall and were shown to be downregulated in syncytia induced by sugar beet cyst nematode Heterodera schachtii. These results were confirmed here by additional expression and localization studies in 5 and 15 dai syncytia. Further, functional tests with specific mutant lines showed an increase in nematode development. This suggests that nematode induces specific downregulation of the CesA genes in syncytia, which is beneficial for the development of the pathogen and the development of its feeding site. Previously, it was shown that two PLL genes, PLL18 and PLL19, are among the most upregulated genes in syncytia induced by Heterodera schachtii as well in galls induced by root-knot nematode Meloidogyne incognita. These results were confirmed with additional expression and localization studies, however specific differences in the expression pattern of both genes in the nematode-infected root tissue were observed. In roots infected with the root-knot nematode both PLLs are upregulated in giant cells. Tests with two homozygous TDNA-insertional mutants of both genes, pll19 and pll18, showed significant reduction in the parasitism of cyst but not root-knot nematodes. Thus, while loss of neither gene has an effect on the development of giant cells, they play a significant role during the formation of syncytia. In addition, we performed immunochemistry using specific antibodies to localize pectic HG with different degrees of methyl-esterification in cell walls of nematode feeding sites. These results provide interesting insights into the cell wall composition that can be correlated with specific stages of the feeding site development as well as the expression pattern of pectate lyases. This project shows that mechanisms regarding pectin and its role during the formation of syncytia and giant cells/galls are different. It indicates substantial developmental and structural differences regarding cell wall between in both feeding sites as well as functional differences between two closely related pectate lyases. This study not only sheds light on crucial cell wall processes in different nematode feeding sites but also expands our knowledge of the interaction between plant-parasitic nematodes and their host plants, which could be utilized in various resistance breeding strategies.