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 high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains 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. Southern 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 coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa 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 that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.
Bantignies
B
B
Department of Biology, Concordia University, Montréal, Québec, Canada.
Séguin
J
J
Muzac
I
I
Dédaldéchamp
F
F
Gulick
P
P
Ibrahim
R
R
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010946:Plants, Medicinal; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; C053376:pathogenesis-related proteins, plant; D012260:Ribonucleases