title: A basic serine protease from Paecilomyces lilacinus with biological activity against Meloidogyne hapla eggs.
abstract: Scanning electron micrographs of the nematode-egg-parasitic fungus Paecilomyces lilacinus infecting eggs of the root-knot nematode Meloidogyne spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N- and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode Meloidogyne hapla induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33.5 kDa, a pH optimum of 10.3, a temperature optimum of 60 degrees C and an isoelectric point above pH 10.2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes.
authors.affiliation: DLO Research Institute for Plant Protection (IPO-DLO), Wageningen, The Netherlands.
authors.forename: P J
authors.initials: PJ
authors.lastname: Bonants
authors.forename: P F
authors.initials: PF
authors.lastname: Fitters
authors.forename: H
authors.initials: H
authors.lastname: Thijs
authors.forename: E
authors.initials: E
authors.lastname: den Belder
authors.forename: C
authors.initials: C
authors.lastname: Waalwijk
authors.forename: J W
authors.initials: JW
authors.lastname: Henfling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005260:Female; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010063:Ovum / Q000187:drug effects / Q000382:microbiology; D010143:Paecilomyces / Q000201:enzymology* / Q000254:growth & development / Q000502:physiology; D010572:Pest Control, Biological; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014431:Tylenchoidea / Q000187:drug effects / Q000382:microbiology*
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D012697:Serine Endopeptidases