By performing cDNA AFLP on pre- and early parasitic juveniles, we identified genes encoding a novel type of ubiquitin extension proteins secreted by the dorsal pharyngeal gland in the cyst nematode Heterodera schachtii. The proteins consist of three domains, a signal peptide for secretion, a mono-ubiquitin domain, and a short C-terminal positively charged domain. A gfp-fusion of this protein is targeted to the nucleolus in tobacco BY-2 cells. We hypothesize that the C-terminal peptide might have a regulatory function during syncytium formation in plant roots.

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

SUMMARY The presence of different types of cytokinins was analysed in exudates and lysates of stage-2 juveniles of Heterodera schachtii and Meloidogyne incognita and in mixed stages of Caenorhabditis elegans. For all species, cytokinins were detected in lysates and exudates in which benzyladenine and zeatin-type cytokinins were the most prominent forms. The production of cytokinins by Meloidogyne was much higher than by Heterodera, and the detected levels were in a range which interfered with the physiological activities of the host plant. The presence of 5-methoxy-N,N-dimethyltryptamine hydrogen oxalate did not affect hormone production by H. schachtii, whereas resorcinol slightly stimulated hormone production by M. incognita. The exuded cytokinins may play a role in feeding site induction, more particularly in cell cycle activation and in establishing the feeding site as a nutrient sink.

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