Phytotoxic Protein PcF, Purification, Characterization, and cDNA Sequencing of a Novel Hydroxyproline-containing Factor Secreted by the Strawberry Pathogen Phytophthora cactorum

Abstract

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.In modern agriculture, the selection of pathogen-resistant cultivars remains of the utmost importance. Conventional breeding selection protocols are relatively inefficient as a consequence of the general lack of genetic variability in cultivated plants as well as the inability to keep pace with the rapid adaptation of pathogen genotypes. New strategies, aimed at achieving resistant plants through gene engineering, require an in depth knowledge of the mechanism of pathogenesis at the molecular level. Host-pathogen interactions can result in either "incompatibility" (resistance) or "susceptibility" (pathogenesis). In either case, mounting evidence suggests that the process is mediated by the production of so-called elicitor-and toxinsignaling molecules (1-3). Signal recognition at the plant cell surface triggers an ordered cascade of downstream events, leading to a range of host-cell responses (4 -7). Because elicitor and toxic mediators play a central role, elucidation of the mechanism of plant-pathogen interactions promises to provide insights about strategies for incorporating pathogen resistance in cultivated plants.The European cultivated strawberry plants (Fragaria vesca ϫ ananassa Duch.) are mostly susceptible to attack by Phytophthora cactorum (8, 9), a pathogenic oomycete for many herbaceous and woody plants because of its wide range host specificity. This pathogen is the causal agent of the "leather rot" and "root rot" diseases in strawberry plants, whose morphological symptoms are recognized by rotting of root, crown, and fruit tissues (10). Plich and Rudnicki (11) first reported that culture filtrates of P. cactorum possessed phytotoxins with action on tomato and involvement in the development of apple tree diseases. Most of these metabolites, however, have not been purified and characterized. The exception is "cactorein" from P. cactorum, a secreted pr...

A Domain-Centric Analysis of Oomycete Plant Pathogen Genomes Reveals Unique Protein Organization

“…The abundance of some domains in plant pathogens is too low to be identified as being overrepresented. For example, the PcF domain (IPR018570), which is present in a small, approximately 50-amino acid necrosis-inducing protein found in various Phytophthora species (Orsomando et al, 2001;Liu et al, 2005), was not identified in the initial overrepresentation analysis. Also in this cluster is the sugar fermentation stimulation domain (IPR005224), which is mainly found in bacteria and involved in the regulation of maltose metabolism (Kawamukai et al, 1991).…”

Comparative structural and functional characterization of putative protein effectors belonging to the PcF toxin family from Phytophthora spp

“…Mature PcF is a 52-residue protein monomer tightly-bound by three disulfide bridges, that is processed before secretion by removal of a N-terminal signal peptide and by a peculiar modification of one of its three Pro residues to 4-hydroxyproline. 17,21 Like most apoplastic proteins, PcF is acidic and resistant to heat denaturation and proteolysis, though it does not appear to inhibit most common proteases in vitro. 17 Its 3D structure recently solved by NMR unraveled a peculiar disulfide-stabilized helix-loophelix fold with a negative surface spot, highly suggestive of electrostatic interaction with yet unidentified target(s).…”

“…17,21 Like most apoplastic proteins, PcF is acidic and resistant to heat denaturation and proteolysis, though it does not appear to inhibit most common proteases in vitro. 17 Its 3D structure recently solved by NMR unraveled a peculiar disulfide-stabilized helix-loophelix fold with a negative surface spot, highly suggestive of electrostatic interaction with yet unidentified target(s). 22 The protein behaves as a necrosis-inducing factor, that is it triggers HR symptoms of localized cell-death when applied on leaves of both strawberry and tomato.…”

“…It has been originally named after the protein effector PcF (Phytophthora cactorum-Fragaria), discovered by us based on its toxic effect on both tomato and strawberry, and purified from the culture filtrates of a P. cactorum strain isolated from infected strawberry. 17 The other family members have been related to PcF mainly on the basis of their conserved sequences, cysteine patterns, and N-terminal secretory signals. 14,18 They are encoded by polymorphic genes and identified by the acronym small cysteinerich (SCR) followed by their residues number.…”

Purification and Characterization of AsES Protein

“…PcF is a secreted 52-amino acid peptide of Phytophthora cactorum that produces necrosis in tomato and strawberry. PcF was proposed to function as a toxin because it triggers responses that are similar to disease symptoms in host plants (21). Likewise, a dual function was found for CBEL (cellulose binding, elicitor, and lectin-like), a 34-kDa cell wall protein that was first isolated from Phytophthora parasitica var.…”