Feuerbrand: Identifikation, Klonierung und funktionelle Charakterisierung korrelierender Gene bei <i>Malus</i> ×<i>robusta</i>

Abstract

Institute for Breeding Research on Fruit

The bacterial plant disease fire blight caused by Erwinia amylovora is one of the main threats of apple production worldwide. Effective control can be only achieved by the use of the antibiotic streptomycin, but the application is restricted or even prohibited in Europe. Since most of the commercially successful apple cultivars are susceptible, fire blight resistance is one of the main goals of many apple breeding programs. Most described sources for fire blight resistance can be found among wild apple species, which, however, are characterised by bad fruit quality and small fruit size. The introgression of these resistances by classical breeding requires, therefore, several backcrosses to get rid of the undesired traits. Molecular methods like marker assisted breeding can help to speed up this process. However, it is essential to determine related genes and to understand the mechanism of resistance. This had let to the aim of the study: the identification, cloning and functional characterisation of fire blight related genes of Malus ×robusta.

Beside the identification of new genes, the study also focused on a closer description of already known genes. One gene with an essential role in the pathogenesis of fire blight is the effector AvrRpt2_EA. First, the avrRpt2_EA gene of twenty-two different E. amylovora strains differing in origin and isolated from different hosts was sequenced. Apart of one single nucleotide polymorphism (SNP), which resulted in an exchange from cysteine to serine in the amino acid sequence, all genes were identical. The two different alleles were later used for the complementation of the avrRpt2_EA deletion mutant strain ZYRKD3-1. The established strains were evaluated by virulence analysis and Real-Time qRT-PCR.

AvrRpt2_EA of E. amylovora is homologue to the effector AvrRpt2 of P. syringae pv. tomato. In A. thaliana AvrRpt2 interacts with the guard protein RIN4, which mediates resistance by activation of the R protein RPS2. The similarity between the two effectors raised the question, if there is a similar resistance mechanism between the two plant species. This would imply it is possible to identify RIN4 in Mr5. In addition to the genetic characterisation, the analysis was also focused on the proteomic level, which was done by a western blot and a yeast two-hybrid assay to prove protein-protein interactions.

Another major objective of this study was a RNA-seq analysis of the transcriptome of Mr5, to identify new genes related to the resistance mechanism. Therefore, Mr5 was inoculated with the virulent avrRpt2_EA deletion mutant strain ZYRKD3-1 or with the non-virulent wild type strain Ea1189. The comparison of the transcriptomes enabled the identification of differentially expressed genes (DEGs), which are related to the disease response. The significant DEGs were assigned to pathways, whereby the focus was on genes belonging to the biotic stress. Selected genes were further analysed with the BioMark™ HD system.