Regulation of the grapevine's defense against pathogens

The grapevine (Vitis vinifera L. ssp. vinifera) is one of the most important cultivated plants worldwide. Its high susceptibility to a wide range of pathogens, particularly powdery mildew (Erysiphe necator) and downy mildew (Plasmopara viticola), poses a significant economic threat to viticulture and can lead to substantial crop losses. Not least for this reason, two-thirds of the total amount of fungicides used in Europe is applied in viticulture. To reduce fungicide use, new resistant grapevine varieties (e.g., ‘Regent’) are being bred; however, the traditional cross-breeding method used is a lengthy process. However, the mechanisms that confer increased resistance in these resistant grapevine varieties remain largely unknown. The identification and functional characterization of genes involved in the regulation of resistance mechanisms could thus contribute to improving and accelerating the breeding of new resistant grapevine varieties.

Using microarrays, a number of genes were identified, including receptors and transcription factors (TFs), which showed faster and stronger induction in resistant grapevine varieties compared to susceptible ones; however, only a few of these genes have been functionally characterized to date. As part of this study, the function of a receptor family in the defense against P. viticola was therefore demonstrated, and subsequently, the differences in the transcriptional regulation of the resistance response by TFs between a susceptible and a resistant grapevine variety were investigated. To this end, the genes of the VRP1 (Vitis Resistance to Plasmopara 1) receptor family were characterized in the first part of the project. An in silico comparison of the three chimeric VRP1 receptors between the susceptible grapevine variety ‘Lemberger’ and the resistant grapevine variety ‘Regent’ revealed no sequence differences. The localization of the VRP1 proteins following fusion with the green fluorescent protein (GFP) in protoplasts from a suspension culture (V. vinifera cv. `Chardonnay`) using confocal microscopy demonstrated that all three VRP1 constructs were located in the cytoplasm. In addition, qPCR analysis demonstrated that the receptors in the resistant ‘Regent’ were specifically induced by infection with P. viticola compared to the susceptible ‘Lemberger,’ suggesting a role in defense against P. viticola. Transient transformation of the VRP1 genes into grapevine leaves followed by infection with P. viticola showed that the expression of VRP1-3 resulted in an increase in resistance of up to 50%. Furthermore, the VRP1 genes were stably transformed into Arabidopsis thaliana. As a result of the overexpression of VRP1-3, an improvement in the resistance of the transgenic Arabidopsis plants against Hyaloperonospora arabidopsidis of up to 50% was also detected.

In the second part of the study, the inducibility of the resistance gene VvPR10.1 (Pathogenesis Related 10.1) by transcription factors (TFs) and their effect on resistance were investigated. Using promoter induction analysis, it was demonstrated that the TFs VvWRKY33, VvERF5, and VvCZF1 could induce the VvPR10.1 promoter, suggesting a role in defense. Furthermore, it was demonstrated in vivo that P. viticola infection-induced expression of VvWRKY33 in grapevine leaves from greenhouse-grown vines, in turn, led to increased expression of VvPR10.1. Ectopic expression of the TFs in grapevine leaves followed by P. viticola infection showed that the expression of VvWRKY33 and VvERF5 resulted in a 50–70% increase in resistance. Furthermore, complementation of the A. thaliana knock-out mutant wrky33-1 with VvWRKY33 demonstrated that expression in the heterologous Arabidopsis system could restore the wild-type phenotype with regard to resistance. Furthermore, a significant increase in resistance to H. arabidopsidis and Botrytis cinerea was even achieved compared to the wild-type Col-0. This study thus demonstrated for the first time that the targeted expression of the VRP1-3 receptor as well as the TFs VvWRKY33 and VvERF5 in grapevines could induce increased resistance to P. viticola. This provides a basis for elucidating the underlying resistance mechanisms and, consequently, for developing new molecular markers to improve and accelerate the breeding of new resistant grapevine varieties.

Publications

Merz et al (2014): The transcription factor VvWRKY33 is involved in the regulation of grapevine (Vitis vinifera) defense against the oomycete pathogen Plasmopara viticola. In : Physiologia Plantarum Volume 153, Issue 3

Merz, Patrick (2014): An investigation of the recognition of Plasmopara viticola by VRP1 receptors and the regulation of pathogen defense by the transcription factors VvWRKY33 and VvERF5 in grapevines (Vitis sp.). Dissertation