Characterization of Superoxide Dismutase from the Potato Cyst Nematode, Globodera pallida.

Potato cyst nematodes (PCNs), such as Globodera pallida and Globodera rostochiensis, are some of the most agriculturally and economically important pests of potato. Upon nematode infection, a principal component of plant defense is the generation of the reactive oxygen species (ROSs). ROSs are highly toxic molecules that cause damage to pathogens and host alike. To infect the plant, nematodes protect themselves from ROSs by activating their own antioxidant processes and ROS scavenging enzymes. One of these enzymes is a superoxide dismutase (SOD; EC 1.15.1.1), which prevents cellular damage by catalyzing conversion of the superoxide radical (O2-·) to hydrogen peroxide (H2O2) and molecular oxygen (O2). We have isolated a putatively secreted isoform of a Cu-Zn SOD (SOD-3) from G. pallida and localized the expression of this gene in the posterior region of the nematode. Furthermore, we studied the expression of the SOD-3 gene during early parasitic stages of infection (24 to 72 h) in the susceptible potato cultivar Desiree, the resistant potato cultivar Innovator, and an immune host, Solanum sisymbriifolium. The SOD-3 gene was significantly upregulated, regardless of the host type; however, the expression pattern differed between the susceptible and the resistant or immune hosts. This finding suggests that SOD-3 gene is responding to infection in plant roots differently depending on whether the nematode is experiencing a compatible or an incompatible interaction.

In situ Hybridization of Plant-parasitic Nematode Globodera pallida Juveniles to Detect Gene Expression.

In this study, we describe a standard whole mount in situ hybridization method which is used to determine the spatial-temporal expression pattern of genes from Globodera spp. Unlike more invasive radioactive labeling approaches, this technique is based on a safe, highly specific enzyme-linked immunoassay where a Digoxigenin (DIG)-tagged anti-sense probe hybridized to a target transcript is detected by anti-DIG antibodies conjugated with alkaline phosphatase enzyme (AP) (anti-DIG-AP). The hybrid molecules are visualized through an AP-catalyzed color reaction using as the substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium chloride (NBT). This method can be applied to both free-living pre-parasitic juveniles and early endoparasitic stages of cyst nematodes.