Molecular Genetic Analysis of lls1, An Inhibitor of Cell Death in Plants

Johal 9729608这项研究的重点是阐明玉米中一种新基因的功能，该基因似乎在植物中起到抑制细胞死亡的作用。该基因被命名为LLSL(致死叶斑病)，由一种隐性突变来鉴定，其特征是启动坏死性病变，扩展到杀死叶片。由于LLSL斑点在视觉上类似于由Cochliobolus Carbon小种1在感病玉米上引发的损伤，该突变已被归入称为疾病损伤模拟的一般突变类别。带有这些遗传缺陷的植物似乎经历了细胞的异常死亡，导致了自发损伤的形成，有时类似于已知的玉米疾病。LLSL的遗传和表型行为预测，该基因的正常功能是防止细胞死亡的扩散，这可能是由任何刺激引发的，包括病原体感染、身体损伤或遗传损伤。一旦LLSL植物上的一个细胞死亡，它就会引发细胞死亡的连锁反应，导致死亡细胞的区域不断扩大(以放射状方式)，最终吞噬整个叶片组织。有趣的是，LLSL损伤的表达是发育程序性的，细胞自主的，和光依赖的。看来，LLSL损伤只在光合作用活跃的组织中启动和传播。为了了解LLSL基因的性质，用Mutator对其进行标记，从玉米中克隆了LLSL基因。序列分析表明，LLSL编码一种在单子叶植物和双子叶植物之间高度保守的新蛋白。它在玉米和拟南芥中都以单拷贝形式存在。表型比较以及拟南芥同源基因的定位数据预测它是加速细胞死亡(ACDL)基因。至少有两个EMS诱导的ACDL等位基因在LLSL同源基因的保守残基中经历了单碱基对突变，这一假设得到了证实。在这项提案中，将使用遗传学、组织学和分子生物学工具的多方面方法来揭示LLSL在植物中的功能。该实验计划将利用两个优秀的植物模型系统--玉米和拟南芥。具体目标是确定：*LLSL在正常和病理情况下是如何调控的；*它在细胞和细胞内水平上的作用部位；*它在细胞中的靶标/底物是什么；以及*它与拟南芥ACDL突变体的关系7这项研究的结果很可能为植物细胞死亡的调控机制提供一个独特而重要的洞察。除了对基础研究做出贡献外，这些研究最终可能会为农业中细胞死亡的生物技术操作提供合理的新方法的构想。一些明显的领域是植物保护、转化、抗逆性、繁殖和雄性不育。

Johal 9729608 The focus of this research is to elucidate the function of a novel gene from maize that appears to act as a suppressor of cell death in plants. This gene, designated llsl (lethal leaf spot) is identified by a recessive mutation and characterized by the initiation of necrotic lesions that expand to kill leaves. Since llsl spots visually resemble lesions incited by race 1 of Cochliobolus carbonum on susceptible maize, this mutation has been placed in the general class of mutations called disease lesion mimics. Plants with these genetic defects appear to undergo aberrant cell death, resulting in the formation of spontaneous lesions that sometimes resemble known diseases of maize. The genetic and phenotypic behavior of llsl predicts that the normal function of this gene is to prevent the spread of cell death, which may be triggered by any stimulus, including a pathogen infection, physical wounding or a genetic lesion. Once a cell dies on an llsl plant, it initiates a chain reaction of cell death, leading to the formation of a continually expanding (in a radial fashion) zone of dying cells that eventually consumes the entire leaf tissue. Interestingly, the expression of llsl lesions is developmentally programmed, cell autonomous, and light-dependent. It appears that llsl lesions only initiate and propagate in photosynthetically active tissues. In order to understand the nature of the llsl gene, it has been cloned from maize following tagging with Mutator. Sequence analysis of Llsl reveals that it encodes a novel protein highly conserved between monocots and dicots. It is present in a single copy in both maize and Arabidopsis. Phenotypic comparison along with mapping data of the Arabidopsis homolog predict it to be the acdl (accelerated cell death) gene. This hypothesis is strengthened by the demonstration that at least two EMS-induced acdl alleles have undergone single base-pair mutations in conserved residues of the llsl homolog. In this proposal, a multi-faceted approach employing genetic, histological and molecular biology tools, will be used to unveil the function of Llsl in planta. The experimental plan will take advantage of two excellent plant model systems - maize and Arabidopsis. Specific goals are to determine: * how llsl is regulated during both normal and pathological situations; * where its site of action is, both at the cellular and intracellular levels; * what its target/substrate is in the cell; and * what its relationship is to the acdl mutant of Arabidopsis7 It is highly likely that the results obtained from this study will provide a unique and important insight into the mechanisms of regulation of cell death in plants. In addition to contributing to basic research, these studies may eventually permit the conception of rational new approaches for the biotechnological manipulation of cell death in agriculture. Some obvious areas are plant protection, transformation, stress resistance, propagation and male sterility.