Isolation and characterisation of a polygalacturonase-inhibiting protein (PGIP) and its encoding gene from Vitis vinifera L.
Date
2001-12
Authors
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Publisher
Stellenbosch : Stellenbosch University
Abstract
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.
AFRIKAANSE OPSOMMING: Sien volteks vir opsomming
AFRIKAANSE OPSOMMING: Sien volteks vir opsomming
Description
Thesis (PhD)--Stellenbosch University, 2001.
Keywords
Grapes -- Disease and pest resistance, Fungal diseases of plants, Polygalacturonase, Proteins, Dissertations -- Biochemistry, Theses -- Biochemistry