Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum

A novel protein factor, named PcF, has been isolated from the culture filtr ate of Phytophthora cactorum strain P381 using a highly sensitive leaf necr osis bioassay with tomato seedlings. Isolated PcF protein alone induced lea f necrosis on its host strawberry plant. The primary structure and cDNA seq uence of this novel phytotoxic protein was determined, and BLAST searches o f Swiss-Prot, EMBL, and GenBank(TM)/EBI data banks showed that PcF shared n o significant homology with other known sequences. The 52-residue PcF prote in, which contains a 4-hydroxyproline residue along with three S-S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectr ic point of 4.4. The molecular mass of isolated PcF is 5,622 +/- 0.5 Da as determined by mass spectrometry and matches that calculated from the deduce d amino acid sequence with cDNA sequencing. The cDNA sequence indicates tha t PcF is first produced as a larger precursor, comprising an additional N-t erminal, 21-residue secretory signal peptide. Maturation of this protein in volves the hydroxylation of proline 49, a feature that is unique among othe r known secreted fungal phytopathogenic proteins.