The investigation of the detection of Plasmopara viticola by VRP1 receptors and the regulation of pathogen defense by the transcription factors VvWRKY33 and VvERF5 in the grapevine (Vitis sp.)

Funded by the German Research Foundation (DFG).

This project was part of a doctoral thesis by Dr. Patrick Merz edited.

Summary

Grapevine (Vitis vinifera L. ssp. vinifera) is one of the most important crop species in the world. Its high susceptibility to a large number of pathogens especially powdery (Erysiphe necator) and downy mildew (Plasmopara viticola) is a special economic threat for viticulture and can lead to substantial crop losses. Two-thirds of the total amount of fungicides in Europe are used in viticulture not least because of this fact. In order to reduce the application of fungicides new resistant cultivars (e.g. `Regent´) should be bred, however, the use of classical cross-breeding is a time consuming procedure. Mechanisms which mediate improved resistance in the resistant cultivars are to a great extent unknown. The identification as well as the functional characterization of genes involved in the regulation of resistance mechanisms could therefore help to improve and advance breeding of new resistant cultivars.

Using Microarrays a number of genes including receptors and transcription factors (TF) could be identified which showed a faster and higher induction in resistant cultivars compared to susceptible cultivars, but only a few of these genes could be functionally characterized so far. Therefore, in this project the function of a receptor family in the defence against P. viticola could be proven and also the differences in the transcriptional regulation of the defence response by TFs has been investigated between a susceptible and a resistant cultivar. In the first part of the project the genes of the VRP1 (Vitis resistance to Plasmopara 1) receptor family have therefore been characterized. The in silico comparison of the three chimeric VRP1 receptors between the susceptible cultivar `Lemberger´ and the resistant cultivar `Regent´ showed no sequence differences. The localization of the VRP1 proteins in protoplasts of a grapevine suspension cell culture (V. vinifera cv. `Chardonnay´) by using confocal microscopy after fusion with the green fluorescent protein (GFP) was able to demonstrate that all three VRP1 constructs were located in the cytoplasm. In addition, qPCR analysis detected that the receptors were specifically induced by infection with P. viticola in resistant `Regent´ compared to susceptible `Lemberger´, which was indicative of a function in the defence against P. viticola. Transient transformation of VRP1 genes into grapevine leaves with subsequent P. viticola infection revealed that the expression of VRP1-3 achieved an increase of resistance by up to 50 %. Additionally, the VRP1 genes were stably transformed into Arabidopsis thaliana. As a result of VRP1-3 overexpression the improved resistance of the transgenic Arabidopsis plants against Hyaloperonospora arabidopsidis up to 50 % could be detected.

In the second part of the project, the inducibility of the resistance gene VvPR10.1 (Pathogenesis Related 10.1) was investigated by TFs and their effect on the resistance. Using promoter induction analysis it could be shown that the TFs VvWRKY33, VvERF5 and VvCZF1 could induce the promoter of VvPR10.1, indicating a function in defence. It was additionally shown in vivo that an induction of VvWRKY33 in grapevine leaves of greenhouse plants caused by P. viticola infection led to an increase in the 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 caused an increase in resistance by 50-70 %. In addition, complementation of the A. thaliana knock-out mutant wrky33-1 with VvWRKY33 confirmed that the expression in the heterologous system Arabidopsis could restore the resistance phenotype of the wild-type. Moreover, it was even possible to achieve a significant increase in resistance to H. arabidopsidis and Botrytis cinerea compared to Col-0 wild-type. In the course of this thesis could be proven for the first time that the specific expression of the VRP1-3 receptor and the TFs VvWRKY33 or VvERF5 in grapevine, induced an increased resistance to P. viticola. This provides a basis for the elucidation of the underlying mechanisms of resistance, and thus for the development of new molecular markers to improve and advance the breeding of new resistant grapevine cultivars.

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): Die Untersuchung der Erkennung von Plasmopara viticola durch VRP1 Rezeptoren und der Regulation der Pathogenabwehr durch die Transkriptionsfaktoren VvWRKY33 und VvERF5 in der Weinrebe (Vitis sp.). Dissertation