Synergistic antifungal activity of two chitin-binding proteins from spindle tree (<Emphasis Type="Italic">Euonymus europaeus</Emphasis> L.)

Two structurally different chitin-binding proteins were isolated from bark and leaves of the spindle tree (Euonymus europaeus L.). Both the small hevein-like chitin-binding protein (Ee-CBP) and the classical class-I chitinase (Ee-chitinase) possess antifungal properties, Ee-CBP being far more potent than Ee-chitinase. In addition, Ee-CBP and Ee-chitinase display a pronounced synergistic effect when added together in the test medium. Determination of the biological activities indicates that the synergism between Ee-CBP and Ee-chitinase relies on a different mode of action. Cloning and sequencing of the corresponding genes further revealed that Ee-CBP and Ee-chitinase are simultaneously expressed in bark and leaf tissues, and hence can act synergistically in planta. Moreover, analysis of the deduced sequences allowed the exact relationship between the structurally different Ee-CBP and Ee-chitinase to be corroborated. Both proteins are synthesized as similar chimeric precursors consisting of an N-terminal hevein domain linked to a C-terminal chitinase-like domain by a hinge region. However, whereas in the case of Ee-chitinase the C-terminal chitinase domain remains linked to the N-terminal hevein domain, the corresponding domain is cleaved from the Ee-CBP-precursor resulting in the formation of the hevein-type Ee-CBP. Since both precursors are—apart from the hinge region between the hevein and chitinase domains—very similar, the Ee-CBP/Ee-chitinase system offers a unique opportunity to study the importance of sequence and/or structural information comprised in the hinge region for the posttranslational processing of the respective precursor proteins.Abbreviations AMP Antimicrobial protein - CBP Chitin-binding protein - GlcNAc N-Acetylglucosamine - HCA Hydrophobic cluster analysis - MeJA Methyl jasmonate - PDB Potato dextrose broth - RACE Rapid amplification of cDNA ends - SPR Surface plasmon resonance - UDA Urtica dioica agglutinin - WGA Wheat (Triticum aestivum) germ agglutinin