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

研究 AvrRpt2Ea 效应蛋白在宿主-病原体相互作用中的作用 Malus xRobusta 5-Erwinia amylovora

• 批准号: 168149487
• 负责人: Professor Dr. Henryk Flachowsky
• 金额: --
• 依托单位: Institut für Züchtungsforschung an Obst
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/168149487?language=en
• 关键词: Ivestigations; role; AvrRpt2Ea; effector; protein

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.

该项目的目标是 AvrRpt2EA 效应蛋白及其在宿主与病原体相互作用中的作用 Malus ×Robusta 5 - 梨火疫病菌。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定影响。使用 AvrRpt2EA 补充丁香假单胞菌 pv 菌株进行接种实验。番茄 Pst DC3000 (pZYR2-415-C) 已在上一个项目期间启动，将重复进行。接种后研究丁香假单胞菌是否能有效地将AvrRpt2EA蛋白分泌到苹果细胞内。随后，将筛选接种的植物是否存在由 AvrRpt2 效应蛋白分泌引起的症状。同时，将使用表达由热激诱导型启动子驱动的avrRpt2EA基因的转基因苹果植物来研究AvrRpt2EA对宿主植物的影响。这种转基因植物在之前的项目中已经生产出来，可用于预期的研​​究。首先，将使用不同的分子方法（如 PCR、RT-PCR 和 Southern blot）测试转基因植物的转基因状态。热休克处理后，将在转录和翻译水平上测试 avrRpt2EA 的表达。将检测 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-羟化酶以及对次生代谢的影响。上一个项目期间获得的第一个结果表明肉桂酸-4-羟化酶是 AvrRpt2EA 的靶标，但最终证据仍然缺失。工作包 AP2 的重点是检测苹果基因组内 AvrRpt2EA 的进一步靶标。因此，最近公布的苹果品种“Golden Delicious”的基因组以及几个公开的 EST 数据库将筛选包含假定的 AvrRpt2EA 切割位点的蛋白质。将测试包含此类推定切割位点的已识别开放阅读框是否以及在何处表达。随后，它们将从 cDNA 中扩增并克隆到二元植物转化载体中。将通过与本塞姆氏烟草中的 AvrRpt2EA 瞬时共表达来测试它们假定的切割位点的功能。根据 AP2 酵母双杂交 (Y2H) 实验（或类似实验）中预期实验的成功，将另外进行。这些实验将用于确定 AvrRpt2EA 的进一步靶标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。

项目成果

Inoculation of Malus genotypes with a set of Erwinia amylovora strains indicates a gene‐for‐gene relationship between the effector gene eop1 and both Malus floribunda 821 and Malus ‘Evereste’

用一组火梨欧文氏菌菌株接种苹果基因型表明效应基因 eop1 与苹果 floribunda 821 和苹果“Evereste”之间存在基因-基因关系

• DOI: 10.1111/ppa.12784
• 发表时间: 2018
• 期刊: Plant Pathology
• 影响因子: 2.7
• 作者: Wöhner TW;Richter K;Sundin GW;Zhao Y;Stockwell VO;Sellmann J;Flachowsky H;Hanke M-V;Peil A
• 通讯作者: Peil A

Homologs of the FB_MR5 fire blight resistance gene of Malus ×robusta 5 are present in other Malus wild species accessions

Malus årobusta 5 的 FB_MR5 火疫病抗性基因的同源物存在于其他 Malus 野生物种种质中

• DOI: 10.1007/s11295-015-0962-y
• 发表时间: 2016
• 期刊: Tree Genetics & Genomes
• 影响因子: 2.4
• 作者: Wöhner T;Szentgyörgyi E;Peil A;Richter K;Hanke M-V;Flachowsky H
• 通讯作者: Flachowsky H