Isolation and characterisation of a polygalacturonase-inhibiting protein (PGIP) and its encoding gene from Vitis vinifera L.
Thesis (PhD)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: Polygalacturonase-inhibiting proteins (PGIPs) are present in the cell walls of a variety of plant species. These proteins have been shown to specifically inhibit endopolygalacturonases (endo-PGs) secreted by invading fungal pathogens as part of the induced disease resistance mechanism of plants. This is the first report on the isolation and characterisation of a pgip gene from Vitis vinifera L., designated grapevine pgip1. A single open reading frame encoding a deduced polypeptide of 333 amino acids with a predicted molecular mass of 37.1 kOa and a calculated isoelectric point of 8.61 was identified from a 5.6 kb subgenomic fragment of V. vinifera cv Pinotage. Nucleotide and derived amino acid sequence analysis of grapevine pgip1 showed significant homology with other characterised PGIP encoding genes and revealed features characteristic of PGIPs found in several other plant families. Genomic DNA analysis showed that grapevine pgip1 belongs to a small multigene family in Vitis cultivars. From Northern blot analysis it was evident that expression of the PGIP family is both tissue- and developmental stage specific. The grapevine pgip1 was transiently expressed in Nicotiana benthamiana L. with potato virus X (PVX) as a vector. Grapevine PGIP1 isolated from crude protein extracts of PVX-infected N. benthamiana were tested and showed inhibitory activity against polygalacturonases (PGs) from Botrytis cinerea. Grapevine PGIPs have not previously been purified and characterised. Molecular analyses have confirmed that PGIPs are typically encoded by multigene families and that the inhibitor specificities and kinetics of the isolated proteins differ within and among species. In this study, two PGIP isomers from V. vinifera berries were isolated. The one isomer, designated PGIP-A, was partially purified and had a molecular mass of 39 kOa, whereas the other PGIP, designated PGIP-B, was purified and had a molecular mass of 42 kOa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE) and Western blot analysis. Both proteins were cell wall-bound. Enzymatic deglycosylation confirmed that PGIP-B is a glycosylated protein. Grapevine PGIP-A showed strong inhibitory activity against a homogeneous PG from Aspergillus niger and to a lesser extent against PG from Fusarium moniliforme, but was unable to interact with a crude PG preparation from B. cinerea. Grapevine PGIP-B was able to strongly inhibit PGs from B. cinerea as well as from Colletotrichum gleosporoides, yet showed no inhibition towards PG from A. niger. The grapevine pgip1 gene was expressed under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in tobacco plants via Agrobacterium tumefaciensmediated transformation. Transgenic tobacco plants expressing the grapevine PGIP (gPGIP1) were used to demonstrate the effectiveness of this inhibitor against fungal PGs and to investigate whether gPGIP1 influences disease development. Northern blot analysis identified 19 transgenic plants expressing pgip1 transcript levels. Crude PGIP extracts from the transgenic tobacco plants inhibited PGs from B. cinerea and C. gleosporoides, but not PG from A. niger. Leaves from untransformed tobacco plants, from transgenic tobacco lines showing high and low PG inhibition, and from transgenic plants that did not express pgip1, were inoculated with B. cinerea. Transgenic leaves showed a reduction in the size of necrotic lesions of macerated tissues of approximately 45% relative to control and non-expressing transgenic leaves. The results from the heterologous expression of gPGIP1, together with the results from the protein purifications and inhibition studies, indicate that the isolated grapevine pgip1 gene encodes the isolated PGIP-B isomer. This work has ; established a good model system to study certain aspects of plant-pathogen interactions in grapevine. Heterologous expression of gPGIP1 has demonstrated that PGIP inhibition of fungal PGs slows disease development of B. cinerea in planta.
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