Investigation of the recognition of Plasmopara viticola by VRP1 receptors and the regulation of pathogen defense by the transcription factors VvWRKY33 and VvERF5 in grapevine (Vitis sp.)
Funded by the German Research Foundation (DFG)
This project was carried out as part of a doctoral thesis by Dr. Patrick Merz.
Summary
The grapevine(Vitis vinifera L. ssp. vinifera) is one of the most important cultivated plants worldwide. Its high susceptibility to a large number of pathogens, in particular powdery mildew(Erysiphe necator) and downy mildew(Plasmopara viticola), is a particular economic threat to viticulture and can lead to considerable crop losses. This is one of the reasons why two thirds of the total amount of fungicides are used in viticulture in Europe. In order to reduce the use of fungicides, new resistant grape varieties (e.g. 'Regent') are to be bred, whereby the classic cross-breeding method used is a lengthy process. However, the mechanisms that mediate the increased resistance in the resistant grape varieties are largely unknown. The identification and functional characterization of genes involved in the regulation of resistance mechanisms could therefore contribute to improving and accelerating the breeding of new resistant grape varieties.
Using microarrays, a number of genes could be identified, including receptors and transcription factors (TF), which showed a faster and stronger induction in resistant grape varieties compared to susceptible grape varieties, but of which only a few genes could be functionally characterized so far. In this project, the function of a receptor family in the defense against P. viticola was therefore demonstrated and the differences in the transcriptional regulation of the resistance response by TFs between a susceptible and a resistant grape variety were subsequently investigated. For this purpose, the genes of the VRP1(Vitis Resistance to Plasmopara 1) receptor family were characterized in the first part of the project. The in silico comparison of the three chimeric VRP1 receptors between the susceptible grape variety 'Lemberger' and the resistant grape variety 'Regent' showed no sequence differences. The localization of the VRP1 proteins after fusion with the green fluorescent protein (GFP) in protoplasts of a suspension culture(V. vinifera cv. `Chardonnay') by confocal microscopy showed 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 function in defense against P. viticola. Transient transformation of VRP1 genes in grapevine leaves followed by P . viticola infection showed that expression of VRP1-3 resulted in an increase in resistance of up to 50%. In addition, VRP1 genes were stably transformed in Arabidopsis thaliana. As a result of the overexpression of VRP1-3, an improvement in the resistance of the transgenic Arabidopsis plants to Hyaloperonospora arabidopsidis of up to 50 % was also detected.
In the second part of the work, the inducibility of the resistance gene VvPR10.1 (Pathogenesis Related 10.1) by TFs and its effect on resistance was investigated. Using promoter induction analysis, it was shown that the TFs VvWRKY33, VvERF5 and VvCZF1 were able to induce the promoter of VvPR10.1, indicating a role in defense. Additionally, it could be shown in vivo that induction of VvWRKY33 in grapevine leaves of greenhouse grapevine caused by P. viticola infection in turn led to an increase in the expression of VvPR10.1. Ectopic expression of TFs in grapevine leaves followed by P. viticola infection showed that expression of VvWRKY33 and VvERF5 resulted in a 50-70% increase in resistance. In addition, 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 in terms of resistance. In addition, a significant increase in resistance to H. arabidopsidis and Botrytis cinerea was achieved compared to the wild type Col-0. This work thus demonstrated for the first time that the targeted expression of the VRP1-3 receptor and the TFs VvWRKY33 and VvERF5 in grapevine could induce an increased resistance to P. viticola. This provides a basis for the elucidation of the underlying resistance mechanisms and thus for the development of 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): The investigation of Plasmopara viticola recognition by VRP1 receptors and the regulation of pathogen defense by the transcription factors VvWRKY33 and VvERF5 in grapevine(Vitis sp.). Dissertation