Molecular characterization and functional importance of pectate lyase secreted by the cyst nematode Heterodera schachtii

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.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.A