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.Phytophthora capsici is a soilborne pathogenic protist (phylum Oomycota) that infects aerial and subterranean structures of many solanaceous plants. Diseases caused by P. capsici are polycyclic in that multiple cycles of infection and inoculum production occur in a single growing season (20). The pathogen survives unfavorable conditions in soil by forming thickwalled oospores, while dissemination and infection are achieved through the production of motile biflagellate zoospores from oospores. The zoospores swim through water in the soil and are chemotactically attracted to the exudates released by the roots of potential host plants (11,14). After the zoospores have adhered to the root surface, they encyst and produce a precisely oriented germ tube that grows into adjacent host plant tissue (3). The progression from zoospores to germlings is triggered by environmental signals, some of which are produced by the plant root. Receptors on the surfaces of the zoospores, cysts, and germ tubes detect the environmental signals that trigger or orient each developmental event.Control of Phytophthora infection remains an ongoing agricultural problem and is most commonly accomplished by the application of biocides, such as methyl bromide or metalaxyl, to the soil. The ability of oospores to persist in soil for long periods obviates the use of crop rotation as an antipest strategy. Alternative, more environmentally benign methods of control will likely have to target host-specific stages of the infectious cycle, since the pathogen is so persistent.In the present study, we explore the possibility of using specific peptide ligands to interfere with the normal developmental progression of the pathogen. We reasoned that, since zoospores are chemotactic toward the plant surface and since the development of the pathogen involves interaction with the plant surface, surface receptor molecules on the zoosp...

Combinatorially selected defense peptides protect plant roots from pathogen infection

“…Peptides Pc42 and Pc87 were shown previously to be speciesspecific in their zoospore encystment effect (7). Similarly, combinatorial peptides selected against the human bacterial pathogen, Haemophilus influenzae, were shown to be strainspecific in their effects on bacterial cell growth (8).…”

“…To induce sporangium formation, cultures were incubated 2 additional days at 25°C under fluorescent lights (12 h light͞ dark). To release zoospores from sporangia, cultures were flooded with sterile deionized water and incubated at room temperature for 30 min (7). To determine zoospore density, an aliquot of a zoospore suspension was vortexed for 30 s to induce encystment, and the number of cysts was determined with a hemocytometer.…”

“…These bioactive peptides were demonstrated previously to induce premature Ph. capsici zoospore encystment (7). A stop codon was located immediately after the peptide insertion site.…”

“…Peptides that we have selected as ligand mimics have come from combinatorial libraries and are typically species-or strain-specific (7,8). These defense peptides differ from numerous naturally occurring antimicrobial peptides that function via disruption of cell membranes and that affect a broad range of prokaryotic and some eukaryotic microorganisms (9,10).…”

“…capsici (7). We selected peptides from a combinatorial phage-display library (14) and chose from the affinity-selected peptide population those peptides that induced premature zoospore encystment.…”