SUMMARY To analyse the parasitic behaviour of the plant-parasitic nematode Heterodera schachtii,
proteins secreted by this nematode were purified and separated by two-dimensional gel electrophoresis. Mass spectrometric analysis identified one of the spots as a
pectate lyase (EC 4.2.2.2). The corresponding gene was cloned from a cDNA library using primers derived from the
peptide tag. A second
pectate lyase was cloned based on similarity to known
pectate lyases of related
cyst nematodes. The predicted
proteins are only 29% identical. Despite the low homology, the
proteins have a similar secondary structure and it is likely that they fold into a similar right-handed beta-helix. Both
proteins have a putative
signal peptide for secretion, and in situ hybridization revealed that expression of the genes was limited to the subventral secretory glands. RT-PCR showed that both genes were expressed in the migratory preparasitic stage although the level of expression between the two genes was different. Post-transcriptional gene silencing by soaking the nematodes in
double-stranded RNA against the gene with the highest expression level affected the
infection process of the nematode, which is in agreement with the general idea that
pectate lyases are essential during migration of the nematode in the plant root.