Functional and Comparative Genomics of Disease Resistance Gene Homologs

编码含有NBS-LRR蛋白的基因是植物基因组中最普遍的一类基因，估计占拟南芥所有基因的1%，在水稻中至少占同样多。然而，人们对它们的功能知之甚少。序列基序表明它们在信号通路的开始起作用。这些基因唯一被证明的功能是抗病或抗虫，但它们也可能涉及植物生物学的其他方面，包括发育和对环境的反应。本项目的目标是完成拟南芥中NBS-LRR基因的详细功能分析以及玉米和水稻中代表基因的比较分析。将建立NBS-LRR编码基因的广泛功能类别，并对这些类别中的一个子集的特定功能进行表征。鉴定NBS-LRR基因产物诱导的基因。在拟南芥基因组测序项目中鉴定的NBS-LRR编码基因将使用涉及过表达或表型获得变异的配体非依赖性方法以高通量格式进行表征。微阵列将用于确定由NBS-LRR基因表达引起的全局表达模式的变化，并将寻找总体发育和生理变化。表达阵列数据将为每个基因提供一个“诱导表达签名”，这将表明其功能，并允许将它们分配到功能类别中。已知抗性基因如RPM1、RPS2和RPP5的表达特征将被表征并与未知基因的表达特征进行比较。在拟南芥中鉴定的代表每个功能类的同源物将以类似的方式在水稻和玉米中进行研究。同源物将从数据库中获得或使用保守的寡核苷酸引物重新扩增。采用与拟南芥相似的方法，每个同源基因的诱导表达特征将使用诊断芯片对水稻和玉米进行测定。这些诊断阵列将通过选择数据库中可用的谷类序列克隆来开发，这些克隆使用来自拟南芥的表达数据以及来自这些物种的其他假定防御基因。每个NBS-LRR同源基因将被映射到每个物种相对于各种表型的共识遗传图谱上。这种全球方法将有效地提供模式植物拟南芥约1%基因的功能信息，并将此信息扩展到两个重要的作物物种。这项工作将定义不同类别的抗性基因，并识别具有其他功能的基因；这将确定有多少是抗性基因，有多少是与防御无关的控制过程。它还将识别由每个类别调节的下游基因组。这将为深入研究NBS-LRR特异性基因及其下游基因的作用提供基础。它将提供工具来促进植物抗病性的实际操作，并可能最终提供操纵植物发育和生理的各个方面的能力。

Genes encoding NBS-LRR containing proteins are one of the most prevalent classes in plant genomes, comprising an estimated 1% of all genes in Arabidopsis and at least as many in rice. However, little is known of their function. Sequence motifs indicate that they act at the beginning of signaling pathways. The only demonstrated function for these genes is in disease or pest resistance, but they may also be involved in other aspects of plant biology including development and response to the environment. The objectives of this project are to complete a detailed functional analysis of NBS-LRR genes in Arabidopsis and a comparative analysis of representatives in maize and rice. Broad functional classes of NBS-LRR encoding genes will be established and the specific functions of a subset of these classes will be characterized. Genes induced by the action of NBS-LRR gene products will be identified. NBS-LRR encoding genes identified in the Arabidopsis genome sequencing project will be characterized in a high-throughput format using ligand-independent approaches involving either over-expression or gain-of-phenotype variants. Microarrays will be used to determine changes in global expression patterns caused by expression of NBS-LRR genes and will also look for gross developmental and physiological changes. The expression array data will provide an 'induced expression signature' for each gene that will indicate its function as well as allow them to be assigned into functional classes. The expression signature of known resistance genes, such as RPM1, RPS2 and RPP5, will be characterized and compared to the expression signatures of the unknown genes. Homologs representing each functional class identified in Arabidopsis will be studied in an analogous fashion in rice and maize. Homologs will be obtained from databases or amplified de novo using conserved oligonucleotide primers. Using parallel approaches to those employed for Arabidopsis, induced expression signatures for each homolog will be determined using diagnostic microarrays for rice and maize. These diagnostic arrays will be developed by selecting clones of cereal sequences available in the database using expression data from Arabidopsis plus other putative defense genes from each of these species. Each NBS-LRR homolog will be mapped onto the consensus genetic map for each species relative to a variety of phenotypes. This global approach will efficiently provide functional information on approximately 1% of the genes in the model plant, Arabidopsis, and will extend this information to two important crop species. The work will define the different classes of resistance genes and identify those with other functions; this will establish how many act as resistance genes and how many control processes unrelated to defense. It will also identify sets of downstream genes regulated by each class. This will provide the basis for detailed studies into the action of specific NBS-LRR and downstream genes. It will provide tools to facilitate the practical manipulation of plant disease resistance and may ultimately provide the ability to manipulate diverse aspects of plant development and physiology.