Towards identification of oesophageal gland proteins in Globodera rostochiensis

Secretory proteins from the dorsal and subventral oesophageal glands of potato cyst- nematodes (Globodera rostochiensis and G.pallida ) are considered to play an important role in the induction and exploitation of the specialized feeding cells (syncytia) that these parasites establish in the roots of their host plant. Identification of these secretory proteins will provide insight into the mechanisms involved in the formation of these syncytia, and is likely to aid in the development of novel forms of (artificial) host plant resistance to potato cyst-nematode infection. This thesis presents the data which were collected during a search for the oesophageal gland secretory proteins of G.rostochiensis.In the first part of the investigations (Chapters 2 to 5) the proteins of G.rostochiensis are examined using two-dimensional gel electrophoresis (2D-GE) followed by silver staining. Chapter 2 compares the protein patterns of two motile (2nd-stage juveniles (J2) and males) and two sedentary (4th-stage females and adult females) developmental stages using conventional (2D-GE) in large gels. This demonstrated very large differences in protein composition between these four developmental stages, and it was estimated that 74% of the polypeptides visible in these gels showed variations in their presence or abundance. The highest similarity index values were calculated for the protein patterns of both motile stages (0.674) and of both sedentary stages (0.767) with decreased values for all other pairwise comparisons.Chapter 3 introduces a miniature 2D-GE technique of which the high sensitivity is demonstrated by its ability to genotype single females of G. pallida . In Chapter 4 this mini 2D-GE technique is used to analyse the proteins of the complete range of post-embryonic developmental stages of G. rostochiensis. This analysis demonstrated large changes in protein pattern during the transformation of preparasitic J2 into sedentary J3 and during the metamorphosis of males into adults. The changes in abundance of three muscle proteins showed that during these two events the body-wall musculature is respectively broken down and restored. It is concluded that 4th-stage male juveniles complete their metamorphosis to adults using protein resources that they have aqcuired while feeding as 2nd- and 3rd-stage juveniles. From the occurrence of several characteristic dominant proteins in the male juvenile stages it is postulated that cyst-nematodes may use a system of storage proteins to complete the different phases of their life-cycle.Because of the large differences between the 2D-GE patterns of the developmental stages examined in Chapters 2 and 4, it was not possible to identify putative oesophageal gland proteins in these gels. Therefore, in Chapter 5 adult males and J2 are dissected into anterior parts (containing the oesophageal glands) and posterior parts (without the glands) and the protein composition of these body fragments. is analysed using mini 2D- GE. This identified a single polypeptide of 34.4 kDa which was characteristic of the anterior part of both J2 and males. Although this protein may originate from an oesophageal gland, this could not be confirmed with certainty, and other explanations for its origin remain as yet equally plausible.The second part of the investigations (Chapters 6 and 7) describes the production of monoclonal antibodies (MAbs) against antigens from G.rostochiensis with the aim to identify oesophageal gland proteins. In Chapter 6 mice were immunized with antigen samples derived from preparasitic J2 and fourthstage females, and MAbs produced by hybridoma cell lines were screened with immunofluorescence microscopy for reactivity with structures within J2. This identified MAbs with various binding specificities, including 5 MAbs which reacted strongly with the subventral oesophageal glands. Unfortunately, these latter MAbs did not identify oesophageal gland proteins on Western blots of J2, indicating that their epitopes were sensitive to denaturation by the electrophoresis detergent SDS.In Chapter 7 mice were therefore immunized with partially purified SDS-denatured proteins from J2, which had been collected by preparative continuous flow electrophoresis. This produced 12 new MAbs against the subventral glands, of which 11 reacted with proteins on Western blots of J2. Together these MAbs identified 4 proteins of respectively 30, 31, 39 and 49 kDa. Immunoelectron microscopy with one of these MAbs showed a specific binding to the contents of the secretory granules within the subventral glands of J2, indicating that these proteins are indeed the secretory products of these gland cells. It is discussed in Chapter 7 that the antibodies to the subventral gland proteins may be useful for engineering resistance to cyst-nematodes into potato by means of 'plantibody' technology.The MAbs to the oesophageal gland proteins from Chapter 7 will be valuable tools in future studies relating to the nature of these antigens and their possible function in the host-parasite interaction. In a following thesis by colleague G. Smant results of such further studies will be presented, and this will include the identification of the abovementioned subventral gland proteins in 2D-gels of G. rostochiensis.