Molecular and phenotypic characterisation of grapevines expressing non-vinifera PGIP encoding genes

[摘要] ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten theirexistence. Biotic factors such as pathogens are the cause of huge yield losses to crop plantsworldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogenssuch as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymescalled endopolygalacturonases (ePGs) during plant infection. These ePGs break down thepectin component of the cell wall, thus providing an entry route, as well as nutrients for thefungus.Plants have evolved mechanisms to counteract and suppress the action of the ePGs.This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibitingproteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongsthe existence of longer chain oligogalacturonide residues which are believed to elicit a cascadeof defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolatedand characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have beenexpressed in tobacco and shown to be potent antifungal proteins that caused the transgenictobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays.Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of thesusceptible Vitis vinifera. Characterisation of the putative transgenic population showed thattransgene integration was successful, the transgenes were being expressed and there were atleast 29 transgenic lines with independent integration events. The transgenic lines wereconfirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs).The plant lines with positive transgene integration, expression, independent integrationevents and exhibiting 100% transgene-derived PGIP activity were further selected for wholeplant and detached leaf antifungal assays where they were challenged with B. cinerea. Thewhole plant infection assay showed that expression of the non-vinifera PGIPs in V. viniferapromotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps beexplained by a quicker and stronger recognition between the pathogen and the host and thestronger activation of defence responses in the host. A more active hypersensitive response inthe host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speedof lesion development observed on the transgenics lines versus the wild type support thispossibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonviniferaPGIPs during infection of grapevine tissue; the potential changes that might be causedby expressing PGIPs in a grapevine host with a native PGIP with high homology to thetransgenes (including potential gene silencing) and the potential impact on defence signallingand defence responses all provides further avenues of study to elucidate this very interestingphenotype further. Overall, this study provides a comprehensively characterised population oftransgenic plants that provides useful resources for in vivo analysis of PGIP function in defence,where the host plant harbours a native copy of the PGIP encoding gene.

[发布机构] Stellenbosch University