Direct evidence for ribonucleolytic activity of a PR-10-like protein from white lupin roots

An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a hi gh sequence similarity to pathogenesis-related proteins found in other spec ies. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid seq uence similarity to a number of the PR-10 family proteins. The clone contai ns an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16 905 Da and an isoelectric point of 4.66. Sou thern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia co li, and its protein product was purified to near homogeneity. Both the nati ve and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity agains t several RNA preparations, including lupin root total RNA. Characterizatio n of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kD a protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs th at are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.