Isolation and characterisation of a polygalacturonase-inhibiting protein (PGIP) and its encoding gene from Vitis vinifera L.

[摘要] ENGLISH ABSTRACT:Polygalacturonase-inhibiting proteins (PGIPs) are present in the cell walls of a varietyof plant species. These proteins have been shown to specifically inhibitendopolygalacturonases (endo-PGs) secreted by invading fungal pathogens as partof the induced disease resistance mechanism of plants. This is the first report on theisolation and characterisation of a pgip gene from Vitis vinifera L., designatedgrapevine pgip1. A single open reading frame encoding a deduced polypeptide of333 amino acids with a predicted molecular mass of 37.1 kOa and a calculatedisoelectric point of 8.61 was identified from a 5.6 kb subgenomic fragment ofV. vinifera cv Pinotage. Nucleotide and derived amino acid sequence analysis ofgrapevine pgip1 showed significant homology with other characterised PGIPencoding genes and revealed features characteristic of PGIPs found in several otherplant families. Genomic DNA analysis showed that grapevine pgip1 belongs to asmall multigene family in Vitis cultivars. From Northern blot analysis it was evidentthat expression of the PGIP family is both tissue- and developmental stage specific.The grapevine pgip1 was transiently expressed in Nicotiana benthamiana L. withpotato virus X (PVX) as a vector. Grapevine PGIP1 isolated from crude proteinextracts of PVX-infected N. benthamiana were tested and showed inhibitory activityagainst polygalacturonases (PGs) from Botrytis cinerea.Grapevine PGIPs have not previously been purified and characterised. Molecularanalyses have confirmed that PGIPs are typically encoded by multigene families andthat the inhibitor specificities and kinetics of the isolated proteins differ within andamong species. In this study, two PGIP isomers from V. vinifera berries wereisolated. The one isomer, designated PGIP-A, was partially purified and had amolecular mass of 39 kOa, whereas the other PGIP, designated PGIP-B, waspurified and had a molecular mass of 42 kOa as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SOS-PAGE) and Western blot analysis.Both proteins were cell wall-bound. Enzymatic deglycosylation confirmed that PGIP-Bis a glycosylated protein. Grapevine PGIP-A showed strong inhibitory activity againsta homogeneous PG from Aspergillus niger and to a lesser extent against PG fromFusarium moniliforme, but was unable to interact with a crude PG preparation fromB. cinerea. Grapevine PGIP-B was able to strongly inhibit PGs from B. cinerea aswell as from Colletotrichum gleosporoides, yet showed no inhibition towards PG fromA. niger.The grapevine pgip1 gene was expressed under the control of the Cauliflowermosaic virus (CaMV) 35S promoter in tobacco plants via Agrobacterium tumefaciensmediatedtransformation. Transgenic tobacco plants expressing the grapevine PGIP(gPGIP1) were used to demonstrate the effectiveness of this inhibitor against fungalPGs and to investigate whether gPGIP1 influences disease development. Northernblot analysis identified 19 transgenic plants expressing pgip1 transcript levels. CrudePGIP extracts from the transgenic tobacco plants inhibited PGs from B. cinerea andC. gleosporoides, but not PG from A. niger. Leaves from untransformed tobaccoplants, from transgenic tobacco lines showing high and low PG inhibition, and fromtransgenic plants that did not express pgip1, were inoculated with B. cinerea.Transgenic leaves showed a reduction in the size of necrotic lesions of maceratedtissues of approximately 45% relative to control and non-expressing transgenicleaves. The results from the heterologous expression of gPGIP1, together with theresults from the protein purifications and inhibition studies, indicate that the isolatedgrapevine pgip1 gene encodes the isolated PGIP-B isomer. This work has; established a good model system to study certain aspects of plant-pathogeninteractions in grapevine. Heterologous expression of gPGIP1 has demonstrated thatPGIP inhibition of fungal PGs slows disease development of B. cinerea in planta.

[发布机构] Stellenbosch University