The sterol‐binding activity of PATHOGENESIS‐RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein

Abstract

SUMMARYPathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens.

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

“…Sterol-binding is important to secrete modified sterols, such as cholesteryl acetate, and required for growth in the presence of eugenol, a plant derived antimicrobial compound that binds to the caveolin-binding motif of Pry1, and whose toxic action is thereby neutralized (13,18). For the antimicrobial defense of plants, on the other hand, scavenging of sterols by PR-1 is likely important to inhibit the growth of sterol auxotrophic pathogens, such as oomycetes (16). Fatty acid binding, on the other hand, is required for the growth of yeast cells under conditions of elevated levels of intracellular free fatty acids, as occurs in mutants lacking major acyl-CoA synthetase, but is presumably also important in cells having reduced synthesis or uptake of CoA itself, or those displaying enhanced lipid remodeling induced by membrane perturbing agents.…”

“…The sterol-binding and export function of yeast Pry proteins appears to be a conserved function of members of the CAP protein superfamily since expression of the human CAP protein CRISP2 complements the defect in sterol export of a yeast mutant lacking Pry function, and purified CRISP2 binds sterols in vitro (13,14). The capacity to bind sterols is also conserved in SmVal4, a CAP protein from the human parasite Schistosoma mansoni, as well as in PR-1, the founding member of the CAP superfamily from plants (15,16). Consistent with the proposed defense function of PR-1, sterol sequestration by PR-1 inhibits growth of plant pathogenic oomycetes, which are auxotrophic for sterols (16).…”

“…The capacity to bind sterols is also conserved in SmVal4, a CAP protein from the human parasite Schistosoma mansoni, as well as in PR-1, the founding member of the CAP superfamily from plants (15,16). Consistent with the proposed defense function of PR-1, sterol sequestration by PR-1 inhibits growth of plant pathogenic oomycetes, which are auxotrophic for sterols (16). Sterol-binding by Pry1 requires a flexible loop containing aromatic amino acids, termed the caveolinbinding motif (17).…”

Plant pathogenesis–related proteins of the cacao fungal pathogen Moniliophthora perniciosa differ in their lipid-binding specificities

“…Importantly, this eugenol sensitivity is proportional to expression levels of Pry proteins and eugenol competes with cholesterol for binding to Pry in vitro, indicating that the flexible loop harboring the CBM can not only bind sterols but also other small hydrophobic compounds such as for example eugenol, thereby protecting cells from its toxicity (18,23). A possible sequestrationbased mode of action of CAP family members in plant innate immunity is supported by the fact that plant PR-1 binds sterols in vitro and addition of the purified PR-1 protein inhibits growth of the sterol-auxotrophic plant pathogenic oomycete Phytophthora (21). Thus, CAP family members secreted by both the host and the pathogen may serve to sequester small hydrophobic compounds required for the survival of either one (31,32).…”

“…The sterol binding activity is conserved among many CAP superfamily proteins, since expression of human CRISP2, parasitic SmVAL4, or plant PR-1 rescues the lack of Pry function in yeast, and these proteins bind cholesterol in vitro (18,20,21). Expression of tablysin-15, however, does not rescue the sterol export phenotype but rescues export of fatty acids (19).…”