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A novel protein factor, named PcF, has been isolated from the culture filtrate of Phytophthora cactorum strain P381 using a highly sensitive leaf necrosis bioassay with tomato seedlings. Isolated PcF protein alone induced leaf necrosis on its host strawberry plant. The primary structure and cDNA sequence of this novel phytotoxic protein was determined, and BLAST searches of Swiss-Prot, EMBL, and GenBank(TM)/EBI data banks showed that PcF shared no significant homology with other known sequences. The 52-residue PcF protein, which contains a 4-hydroxyproline residue along with three S-S bridges, exhibits a high content of acidic sidechains, accounting for its isoelectric 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 deduced amino acid sequence with cDNA sequencing. The cDNA sequence indicates that PcF is first produced as a larger precursor, comprising an additional N-terminal, 21-residue secretory signal peptide. Maturation of this protein involves the hydroxylation of proline 49, a feature that is unique among other known secreted fungal phytopathogenic proteins.