Ivestigations of the role of the AvrRpt2Ea effector protein within the host-pathogen interaction Malus x robusta 5-Erwinia amylovora

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The project is aimed on the AvrRpt2EA effector protein and its role within the host-pathogen-interaction Malus × robusta 5 - Erwinia amylovora. The project is divided into two work packages (AP1 and AP2). Work package AP1 is focused on evaluating the general and specific effects of AvrRpt2EA onto the host plant. The inoculation experiments using an AvrRpt2EA complemented strain of Pseudomonas syringae pv. tomato Pst DC3000 (pZYR2-415-C), which have been started during the previous project, will be repeated. After inoculation it will be studied whether the AvrRpt2EA protein will be effectively secreted into the apple cell by P. syringae or not. Subsequently, inoculated plants will be screened for symptoms resulting from the secretion of the AvrRpt2 effector protein. In parallel the effects of AvrRpt2EA onto the host plant will be investigated using transgenic apple plants expressing the avrRpt2EA gene driven by a heat-shock inducible promoter. Such transgenic plants were produced already during the previous project and are available for the intended study. At first, the transgenic plants will be tested on their transgenic status using different molecular methods like PCR, RT-PCR and Southern blot. After heat-shock treatment they will be tested on the expression of avrRpt2EA at the transcriptional and translational level. Symptoms resulting from the AvrRpt2EA expression will be detected. Furthermore, it should be studied whether AvrRpt2EA is able to block the cinnamic acid-4-hydroxylase and how the secondary metabolism will be affected. First results obtained during the previous project period suggest the cinnamic acid-4-hydroxylase being a target of AvrRpt2EA, but the final proof is still missing.Work package AP2 is focused on detecting further targets of AvrRpt2EA within the apple genome. Therefore, the recently published genome of the apple cultivar 'Golden Delicious' as well as several publically available EST databases will be screened for proteins containing putative AvrRpt2EA cleavage sites. Identified open reading frames containing such putative cleavage sites will be tested whether and where they are expressed. Subsequently, they will be amplified from cDNA and cloned into binary plant transformation vectors. The function of their putative cleavage site will be tested by transient co-expression together with AvrRpt2EA in Nicotiana benthamiana. Depending on the success of the intended experiments in AP2 yeast-two-hybrid (Y2H)-experiments (or similar experiments) will be carried out in addition. These experiments will be performed to identify further targets of AvrRpt2EA and to study their interaction with the bacterial effector protein. These experiments are in addition to the other aforementioned experiments and will be only started after completing the other parts of the project successfully.