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

本研究的重点是阐明来自玉米的一个新基因的功能，该基因似乎可以抑制植物细胞死亡。该基因被命名为llsl（致命叶斑病），通过隐性突变确定，其特征是开始坏死病变，扩大到杀死叶子。由于lsl斑点在视觉上类似于由1种玉米上的玉米锥虫引起的病变，因此这种突变被归入称为疾病病变模拟的突变的一般类别。具有这些遗传缺陷的植物似乎经历了异常的细胞死亡，导致自发损伤的形成，有时类似于已知的玉米疾病。llsl的遗传和表型行为预示着该基因的正常功能是防止细胞死亡的扩散，细胞死亡可能由任何刺激触发，包括病原体感染、物理损伤或遗传病变。在lsl植物中，一旦细胞死亡，就会引发细胞死亡的连锁反应，导致死亡细胞形成一个不断扩大的（以放射状的方式）区域，最终消耗整个叶片组织。有趣的是，llsl病变的表达是发育程序化的，细胞自主的，和光依赖的。llsl病变似乎只在光合作用活跃的组织中发生和繁殖。为了了解llsl基因的性质，利用Mutator标记从玉米中克隆了该基因。对Llsl的序列分析表明，它编码了一个在单子房和双子房之间高度保守的新蛋白。它存在于玉米和拟南芥的单一拷贝中。表型比较以及拟南芥同源定位数据预测它是acdl（加速细胞死亡）基因。至少有两个ems诱导的acdl等位基因在llsl同源物的保守残基上发生了单碱基对突变，这一证明加强了这一假设。本研究将采用遗传学、组织学和分子生物学等多种方法，揭示Llsl在植物中的功能。该实验计划将利用两种优秀的植物模式系统——玉米和拟南芥。具体目标是确定：*在正常和病理情况下，llsl是如何调节的；*其作用部位在细胞和细胞内水平；*细胞中的靶物/底物是什么；以及它与拟南芥acdl突变体的关系。本研究的结果极有可能为研究植物细胞死亡的调控机制提供独特而重要的见解。除了有助于基础研究之外，这些研究可能最终允许对农业中细胞死亡的生物技术操纵的合理新方法的概念。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.