Interplay of dirgent and jacalin-proteins in broad-spectrum disease resistance of plants against fungal pathogens

融合蛋白由一个菠萝琳相关凝集素(JRL)和一个引导域组成，仅存在于禾本科植物中。Ulrich Schaffrath的研究小组已经证明，该家族不同成员分别在水稻、大麦和小麦中的结构性过度表达，导致了对不同病原体(包括细菌、卵菌和真菌)的广谱定量抗病。含有人工分离的JRL结构域的重组蛋白在被白粉菌攻击的大麦中表达时，定位于穿透部位。当共表达时，由分离的介导体结构域组成的蛋白质也与JRL-蛋白一起定位在穿透部位。根据这些结果，我们推测JRL-和Drigent蛋白是古老的植物防御系统的一部分，该系统由两个成分组成，一个作用于病原体识别，另一个作用于抗性的执行。各自蛋白质的融合是一项仅在禾本科物种中实现的发明。在拟议的项目中，我们将通过回答以下问题来挑战这一假设：i)在病原体攻击过程中暴露了哪些碳水化合物特征并与JRL-结构域结合；ii)在介导域蛋白的存在下产生了哪种抗菌物质；以及iii)在其他植物物种中是否存在以所建议的方式协同作用的JRL和导引蛋白对。特别是，将对不同Poaceae融合蛋白的结构域进行结构域交换实验，以揭示功能相似之处。生物化学家Thomas Classen的研究小组将致力于JRL结构域结合伙伴的鉴定和涉及介导性结构域的生化反应的表征。这将包括重组蛋白的合成、结晶学、JRL-凝集素与病原体和/或植物中的碳水化合物的下拉分析以及高效液相分析。拟南芥将作为模式植物，寻找与上述禾本科植物特有的融合蛋白类似的抗病蛋白对。利用生物信息学手段，我们已经确定了两个有希望的候选基因，它们位于拟南芥1号染色体上相邻的位置。这两个蛋白都将在大麦中表达，以阐明它们与侵染抗性的关系。有希望的JRL-和Drigent蛋白候选将被用于构建人工融合蛋白，并测试其是否能够提供广谱抗病能力。这最终将证明，在作物中表达人工融合蛋白的翻译方法是否是一种有前景的提高植物抗逆性的策略。沙夫拉斯和克拉森实验室在分子植物病理学和生物化学方面的专业知识将为该项目的实现奠定坚实的基础。

Fusion proteins consisting of a jacalin-related lectin (JRL) and a dirigent domain occur exclusively within the plant family Poaceae. The group of Ulrich Schaffrath has shown that constitutive over-expression of different members of this family in rice, barley and wheat, respectively, led to quantitative broad-spectrum disease resistance against different pathogens including bacteria, oomycetes and fungi. Recombinant proteins with an artificially separated JRL-domain localizes towards the site of penetration when expressed in barley attacked by the powdery mildew fungus. When co-expressed, also a protein consisting of the separated dirigent domain localizes at the penetration site together with the JRL-protein. The activity of both protein partners is required for enhanced pathogen resistance.Based on these results we hypothesize, that JRL- and dirigent proteins are part of an ancient plant defense system which consists of two components, one acting on pathogen recognition and the other on execution of resistance. Fusion of the respective proteins is an invention solely realized in Poaceae species. In the proposed project we will challenge this hypothesis by answering the following questions: i) which carbohydrate signature is exposed during pathogen attack and binds the JRL-domain; ii) which antimicrobial substance is produced in the presence of the dirigent-domain protein; and iii) are there pairs of JRL and dirigent proteins in other plant species that cooperatively act in the proposed manner. In particular, domain swap experiments will be performed with the domains of different Poaceae fusion proteins to unravel functional similarities. The identification of the JRL-domain binding partner and characterization of the biochemical reaction in which the dirigent-domain is involved will be targeted by the group of Thomas Classen, a biochemist. This will include synthesis of recombinant proteins, crystallography, pull-down assays of JRL-lectins with carbohydrates from pathogens and/or plants as well as HPLC-analyses.Arabidopsis will be used as model plant, to search for pairs of proteins that act in disease resistance in a similar manner as the above mentioned Poaceae-specific fusion proteins. Using bioinformatics, we already identified two promising candidate genes which are located next to each other on Arabidopsis chromosome 1. Both proteins will be expressed in barley to elucidate their involvement in penetration resistance. Promising candidates of JRL- and dirigent proteins will be used to construct an artificial fusion protein and test whether this is capable to provide broad-spectrum disease resistance. This finally will prove if translational approaches, in which artificial fusion proteins are expressed in crop plants, are a promising strategy to improve plant stress resistance.The combined expertise of the Schaffrath and Classen labs in molecular phytopathology and biochemistry will build a solid base for the realization of the project.