PcF protein from Phytophthora cactorum and its recombinant homologue elicit phenylalanine ammonia lyase activation in tomato.
The phytotoxic protein PcF (Phytophthora cactorum-Fragaria) is a 5.6-kDa cysteine-rich, hydroxyproline-containing protein that is secreted in limited amounts by P. cactorum, an oomycete pathogen of tomato, strawberry and other relevant crop plants. Although we have shown that pure PcF triggers plant reactivity, its mechanism of action is not yet understood. Here we show that PcF, like other known fungal protein elicitors involved in pathogen-plant interaction, stimulates the activity of the defense enzyme phenylalanine ammonia lyase (EC 4.3.1.5) in tomato seedlings. Recognizing that a key step in understanding the mechanism of action of PcF at a molecular level is knowledge of its three-dimensional structure, we overexpressed this protein extracellularly in Pichia pastoris. The preliminary structural and functional characterization of a recombinant PcF homologue, N4-rPcF, is reported. Interestingly, although N4-rPcF is devoid of proline hydroxylation and has four additional amino acid residues attached to its N terminus, its secondary structure and biological activity are indistinguishable from wild-type PcF.
Orsomando
G
G
Istituto di Biotecnologie Biochimiche, Università di Ancona, Via Brecce Bianche, 60131 Ancona, Italy.
Lorenzi
M
M
Ferrari
E
E
de Chiara
C
C
Spisni
A
A
Ruggieri
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism*; D010838:Phytophthora / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
D017931:DNA Primers; C088192:Pcf protein, Phytophthora cactorum; D010940:Plant Proteins; D011994:Recombinant Proteins; D010650:Phenylalanine Ammonia-Lyase