Isolation and molecular analysis of the pepper Bs4C resistance gene that mediates recognition of the cognate Xanthomonas TAL effector protein AvrBs4

微生物疾病是对种植作物的持续威胁，而种植作物是我们全球粮食供应的基础。栽培作物的野生近亲代表着丰富的抗微生物免疫系统资源，然而，在育种计划中挖掘其潜力之前，需要对其进行基因特征描述。由于技术的限制，过去对植物免疫系统的研究主要是在小基因组的模式植物中进行的。然而，下一代测序(NGS)技术现在促进了从复杂的植物基因组和转录本中直接分离有价值的特征。在这一建议的框架内，我们想要分离和鉴定对野油菜黄单胞菌PV具有抗性的辣椒抗性(R)基因Bs4C。辣椒和番茄细菌性斑点病的病原菌--白粉菌(XCV)。采用基于NGS的转录组谱分析方法鉴定Bs4C候选基因(2.2.1.5)。我们将通过逆转录-聚合酶链式反应、基因定位和功能分析来明确哪些候选基因代表Bs4C(3.2.1.1-3.2.1.8)。如果我们未能在识别的转录本中识别Bs4C，我们将应用基于NGS的基因组重测序、遗传图谱和功能分析来识别Bs4C(3.2.1.12)。在分离后，我们将在分子水平上描述Bs4C基因的特征，以阐明它如何介导对XCV的识别和免疫反应的执行(3.2.1.9-3.2.1.11)。在技术层面，我们的目标是建立一种新的适用于复杂作物基因组的R基因克隆策略，并实现NGS技术。

Microbial diseases are a continuous threat to cultivated crops that provide the basis of our global food supply. Wild relatives of cultivated crops represent a rich resource of antimicrobial immune systems that however need to be characterized genetically before their potential can be exploited in breeding programs. Due to technical limitations, plant immune systems were studied in the past primarily in model plants with small genomes. Yet, next-generation sequencing (NGS) technologies facilitate now direct isolation of valuable traits from complex plant genomes and transcriptomes. In the framework of this proposal we want to isolate and characterize the pepper resistance (R) gene Bs4C that confers resistance to Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato. NGS-based transcriptome profiling was used to identify Bs4C candidate genes (2.2.1.5). We will apply reverse transcription (RT)-PCR, genetic mapping and functional analysis to clarify which of the identified candidate genes represents Bs4C (3.2.1.1 - 3.2.1.8). If we fail to identify Bs4C within the identified transcripts we will apply NGS-based genome re-sequencing, genetic mapping and functional analysis to identify Bs4C (3.2.1.12). Upon isolation we will characterize the Bs4C gene at the molecular level to clarify how it mediates recognition of Xcv and execution of an immune response (3.2.1.9 - 3.2.1.11). At the technical level we aim to establish a novel R gene cloning strategy that is applicable to complex crop genomes and that implements NGS technology.