Effects of a Novel Pathogenesis-Related Class 10 (PR-10) Protein from Crotalaria pallida Roots with Papain Inhibitory Activity against Root-Knot Nematode Meloidogyne incognita

A novel pathogenesis-related class 10 (PR-10) protein with papain inhibitory activity, named CpPRI, was purified from Crotalaria pallida roots by ammonium sulfate precipitation followed by three reverse-phase high-performance liquid chromatographies (HPLCs). CpPRI is made up of a single polypeptide chain with a M-r of 15 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This protein exhibited a K-i value of 1.8 X 10(-9) M and operates via a noncompetitive inhibition mechanism. The alignment of the N-terminal amino acid sequence of CpPRI with other proteins revealed its identity with PR-10 proteins. CpPRI acts against digestive proteinase from root-knot nematode Meloidogyne incognita and demonstrated nematostatic and nematicide effects on this parasite in bioassays. In a localization study, fluorescein-5-isothiocyanate (FITC) CpPRI was observed to internalize and diffuse over the entire J2 body after 6 h of incubation. This fact could explain the natural tolerance of this plant species to nematodes.