Development of the polygalacturonase inhibiting protein (PGIP) for delivery of foreign proteins to the surfaces of plant cells

Abstract

Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases (endo-PGs) from phytopathogenic fungi. For proteins to confer resistance to invading plant pathogens, it is preferred that they are either associated with the plant cell wall or secreted into the intercellular spaces where they can act almost immediately upon pathogen attack. The bactericidal efficacy of the Hen Egg White Lysozyme (HEWL) has previously been unequivocally demonstrated in transgenic plants; however, most of the protein remains intracellular. It was hypothesized that bean PGIP1, that has previously been expressed correctly in transgenic tomato plants and was found to inhibit the endopolygalacturonase activity of Stenocarpella maydis in a reducing sugar assay, would deliver the HEWL protein to the intercellular spaces due to its inherent translocation to the plant cell wall by means of a translational fusion between bean pgip1 and hewl genes. In this study, the efficacy of such a translational fusion was determined. The bean pgip1-hewl fusion was inserted into the binary vector pCAMBIA2300 and transformed into Nicotiana tabacum cv. LA Burley 21 plants by Agrobacterium-mediated transformation. Phenotypically normal transgenic plants were produced. Stable transgene insertion into the transgenic N. tabacum genomes was verified by PCR and Southern blot analyses. To demonstrate the efficacy of the bean PGIP1-HEWL fusion, independent homogenate and intercellular fluid protein extracts were prepared from transgenic N. tabacum leaf material. Protein extracts prepared so as to enrich for PGIP activity were tested in vitro for inhibition of S. maydis endo-PGs whereas protein extracts for HEWL activity were tested for lysis of Micrococcus luteus cells. Biochemical assays showed that bean PGIP1-HEWL inhibited S. maydis endo-PGs and cleaved M. luteus cell walls sufficiently to suggest that the PGIP1- HEWL fusion was structurally and functionally stable. Total protein extracts from the PGIP-HEWL and HEWL transgenic plants showed similar levels of HEWL specific activity, whereas intercellular fluid samples from PGIP-HEWL transgenic plants showed high activity in contrast to HEWL plants. With the success of showing protein activity in vitro of HEWL in intercellular spaces, bean PGIP1 can be recommended as a vehicle for delivery of other proteins to cell surfaces. Copyright 2006, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Feltman, NR 2006, Development of the polygalacturonase inhibiting protein (PGIP) for delivery of foreign proteins to the surfaces of plant cells, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-07152008-111131 / > E416/gm