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

该项目旨在研究AvrRpt2EA效应蛋白及其在寄主-病原-互作苹果×罗布斯塔5-淀粉欧文氏菌中的作用。该项目分为两个工作包(AP1和AP2)。工作包AP1的重点是评估AvrRpt2EA对寄主植物的一般和特殊影响。用一株AvrRpt2EA作为补充菌株进行了接种试验。重复上一个项目中启动的番茄PST DC3000(pZYR2-415-C)。在接种后，将研究是否能有效地将AvrRpt2EA蛋白分泌到苹果细胞中。随后，将对接种的植物进行筛选，看是否有因分泌AvrRpt2效应蛋白而引起的症状。同时，将利用热激诱导启动子驱动的表达avrRpt2EA基因的转基因苹果植株来研究AvrRpt2EA对寄主植物的影响。这样的转基因植物已经在前一个项目中生产出来，并可用于预期的研究。首先，将使用不同的分子方法如聚合酶链式反应、逆转录聚合酶链式反应和Southern杂交来检测转基因植株的转基因状态。热休克处理后，将在转录和翻译水平上检测avrRpt2EA的表达。将检测到由AvrRpt2EA表达引起的症状。此外，还应研究AvrRpt2EA是否能够阻断肉桂酸-4-羟基酶，以及如何影响次生代谢。在前一个项目期间获得的第一个结果表明肉桂酸-4-羟基酶是AvrRpt2EA的靶标，但最终的证据仍然缺乏。工作包AP2专注于在苹果基因组中检测AvrRpt2EA的进一步靶标。因此，将对最近公布的苹果品种‘金冠’的基因组以及几个公开可用的EST数据库进行筛选，以寻找含有推测的AvrRpt2EA裂解位点的蛋白质。确定的含有这种假定切割位点的开放阅读框将测试它们是否表达以及在哪里表达。随后，从cDNA中扩增出它们，并克隆到双元植物转化载体中。它们可能的切割位点的功能将通过与AvrRpt2EA在本氏烟草中的瞬时共表达来测试。根据AP2酵母双杂交(Y2H)实验的成功情况，还将进行其他实验(或类似实验)。这些实验将被用来确定AvrRpt2EA的进一步靶点，并研究它们与细菌效应蛋白的相互作用。这些实验是对上述其他实验的补充，只有在成功完成项目的其他部分后才会开始。

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.