Molecular analysis of Arabidopsis thaliana LRR receptor protein kinases (LRR-RLK) implicated in pathogen defense

富含亮氨酸重复序列(LRR)蛋白(LRRP)和LRR受体样蛋白(LRR-RLK)与植物抗病活性密切相关。这些蛋白质被认为与迄今未知的相互作用蛋白质形成了大型信号感知复合体。由于编码真核生物中信号通路组件的基因经常被发现在转录水平上受到相应刺激的上调，我们将利用这一点来发现新的编码LRR-RLK和LRRP的基因，这些基因可能与疾病抗性有关。为了分析和分离介导植物防御反应激活的信号感知复合体，我们提出了如下策略：拟南芥COL-O植物将受到无毒细菌、卵菌和真菌病原体的侵染，并将被植物防御反应的电子受体渗透。来自受感染植物的RNA将被用于基因特异性引物(来自基因组数据库)的RT-PCR分析，或用于LRRP/LRR-RLK芯片上的cDNA微阵列分析。选定的基因将根据它们在植物防御信号中的功能进行表征(通过T-DNA插入或RNA干扰使基因失活，并随后在感染分析中分析突变体)。生化特征将包括分离与标记的LRRP/LRR-RLK诱饵蛋白相互作用的蛋白，并通过胰酶裂解/MALDI-TOF光谱鉴定相互作用蛋白。

Leucine-rich repeat (LRR) proteins (LRRP) and LRR receptor-like kinases (LRR-RLK) have been implicated in the acivation of plant disease resistance. These proteins are believed to form large signal perception complexes with so far unknown interacting proteins. Since genes encoding components of signaling pathways in eukaryotes are often found to be transcriptionally up-regulated by the corresponding stimulus, we will exploit this to identify new LRR-RLK and LRRP-encoding genes potentially involved in disease restistance. To analyze and isolate signal perception complexes mediating the activation of plant defense responses we propose the following strategy: Arabidopsis thaliana Col-O plants will be infected with avirulent bacterial, oomycete, and fungal pathogens and will be also infiltrated with elecitors of plant defense reactions. RNA from infected plants will be used in RT-PCR assays with gene-specific primers (derived from the genomic database) or in cDNA microarray assays on LRRP/LRR-RLK-chips. Selected genes will be characterized with respect to their function in plant defense signaling (gene inactivation by T-DNA insertion or RNA interference and subsequent analysis of the mutants in infection assays). Biochemical characterization will involve isolation of interacting proteins with tagged LRRP/LRR-RLK bait-protein and identification of interacting proteins by tryptic fragmentation/MALDI-TOF spectroscopy.