Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.
Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, beta-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.
Park
Sang-Wook
SW
Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, Colorado 80523-1173, USA.
Lawrence
Christopher B
CB
Linden
James C
JC
Vivanco
Jorge M
JM
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D022081:Cell Surface Extensions / Q000502:physiology; D002473:Cell Wall / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002688:Chitinases / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005030:Ethylenes / Q000494:pharmacology; D043326:Glucan 1,3-beta-Glucosidase; D008853:Microscopy; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D029604:Phytolacca americana / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010940:Plant Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D001617:beta-Glucosidase / Q000378:metabolism
D000935:Antifungal Agents; D018076:DNA, Complementary; D005030:Ethylenes; D000073718:Pancreatitis-Associated Proteins; D010940:Plant Proteins; C000612141:REG3A protein, human; C036216:ethylene; D002688:Chitinases; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase; D010450:Endopeptidases