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Plant Physiol, September 2002, Vol. 130, pp. 164-178
Isolation and Characterization of a Novel Ribosome-Inactivating
Protein from Root Cultures of Pokeweed and Its Mechanism of Secretion
from Roots1
Sang-Wook
Park,
Christopher B.
Lawrence,
James C.
Linden, and
Jorge
M.
Vivanco*
Departments of Horticulture and Landscape Architecture (S.-W.P.,
J.M.V.), Bioagricultural Sciences and Pest Management (C.B.L.),
Microbiology (J.C.L.), and Chemical Engineering (J.C.L.), Colorado
State University, Fort Collins, Colorado 80523-1173
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,  -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.
1
This work was supported by the National Science
Foundation (CAREER award no. MCB-0093014 to J.M.V.) and by the
Colorado State University Agricultural Experiment Station (to
J.M.V.).
*
Corresponding author; e-mail jvivanco{at}lamar.colostate.edu; fax
970-491-7745.
© 2002 American Society of Plant Physiologists
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