Phage-displayed peptides as developmental agonists for Phytophthora capsici zoospores

Abstract

As part of its pathogenic life cycle, Phytophthora capsici disperses to plants through a motile zoospore stage. Molecules on the zoospore surface are involved in reception of environmental signals that direct preinfection behavior. We developed a phage display protocol to identify peptides that bind to the surface molecules of P. capsici zoospores in vitro. The selected phage-displayed peptides contained an abundance of polar amino acids and proline but were otherwise not conserved. About half of the selected phage that were tested concomitantly induced zoospore encystment in the absence of other signaling agents. A display phage was shown to bind to the zoospore but not to the cyst form of P. capsici. Two free peptides corresponding to active phage were similarly able to induce encystment of zoospores, indicating that their ability to serve as signaling ligands did not depend on their exact molecular context. Isolation and subsequent expression of peptides that act on pathogens could allow the identification of receptor molecules on the zoospore surface, in addition to forming the basis for a novel plant disease resistance strategy.

FIG. 1.

Micrograph (negative of original dark-field image) showing a mixture of nonmotile and motile zoospores. Encysted zoospores do not move during the period of exposure (1 to 2 s), and their images are period shaped, while the tracks of the motile zoospores are elongated. The large spherical objects are sporangia and are disregarded in the measurements.

FIG. 2.

Premature encystment of P. capsici zoospores induced by phage-displayed peptides. (a) Effectiveness of different selected phage. A 20-μl droplet containing 400 zoospores was incubated with phage-displayed peptides (∼5 × 10¹⁰ particles) for 20 min at room temperature. The number of zoospores encysted was counted and expressed as a percentage. The percentages represent the means of two experiments. The percent encystment for the control zoospore population that contained no phage varied between 0 and 10%. (b) Concentration dependence of encystment. Phage (in water) were mixed with zoospores, and the amount of encystment was determined visually after 5 min at room temperature. The error bars indicate standard deviations.

FIG. 3.

Zoospore encystment induced by free peptides. The percentages of encystment induced by peptide 87 (•), peptide 42 (▪), and peptide 56 (▴) are shown. The error bars represent the calculated Poisson standard deviation as determined from the number of cysts and zoospores counted in each determination. These varied, since zoospores were able to swim in and out of the field during the experiment while cysts were necessarily fixed.