Identification of stable plant cystatin/nematode proteinase complexes using mildly denaturing gelatin/polyacrylamide...

The biochemical interactions between two cystatins from rice seeds, oryzacystatin I (OCI) and oryzacystatin II (OCII), and the cysteine proteinases from three plant parasitic nematodes, Meloidogyne hapla, M. incognita and M. javanica, were assessed using standard protease assays and mildly denaturing gelatin/polyacrylamide gel electrophoresis (gelatin/PAGE). Activity detected in extracts of preparasitic second-stage larvae (J2) from M. hapla was optimal at pH 5.5 and was inhibited in vitro by the cysteine proteinase inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, hen egg cystatin, OCI, and OCII. As demonstrated by class-specific activity staining, all the activity measured between pH 3.5 and pH 7.5 was accounted for by a major proteinase form, Mhp1, and two minor forms, Mhp2 and Mhp3. Mhps were also detected in extracts and excretions of parasitic J2 and adult females, indicating their continuous expression throughout development of M. hapla, and their possible involvement in the extracellular degradation of proteins. Interestingly, the two plant cysteine proteinase inhibitors OCI and OCII showed different degrees of affinity for the major proteinase form, Mhp1. Both inhibitors almost completely inactivated this proteinase in native conditions but, unlike OCII, OCI conserved a high affinity for Mhp1 during mildly denaturing gelatin/PAGE, showing the differential stabilities of the OCI/Mhp1 and OCII/Mhp1 complexes. In contrast to Mhp1, the major cysteine proteinases detected in the two closely related species M. incognita and M. javanica were strongly inhibited by OCII, while the inhibition of OCI was partly prevented during electrophoresis. This species-related efficiency of plant cystatins against nematode cysteine proteinases could have practical implications when planning their use to control nematodes of the genus Meloidogyne.