Cloning and Characterization of Two Disease Lesion Mimic Genes in Maize

该建议的目的是标记和克隆一种疾病病变模拟物（DLM），基因致死性叶斑-1（lls 1），其突变是隐性的。 重点将放在克隆和表征lls 1基因，以及定义各种生理和生化条件，影响lls 1突变的表达。 我们将继续对DLM显性突变体Les*-2552进行遗传特征分析，并进一步进行共分离分析;还将尝试对一些突变体进行共分离分析，以确定可能导致这些突变的Mu元件。 lls 1的作用似乎是细胞自主的（仅限于其表达的细胞）;在许多植物上，观察到具有尖锐边界的体细胞克隆区段作为正向突变或逆转事件。 目前正在研究一种新的克隆战略，即基于使用这些体细胞部分的“体细胞标记”。 简言之，比较来自相邻突变体和野生型区段的DNA的Mu-杂交模式的多态性。 突变体DNA中任何额外的条带都可能含有引起突变的Mu元素。 使用这种策略，已经从一个lls 1突变体中鉴定出6.5kb的Mu-1杂交片段，并且来自共分离分析的初步结果表明，该条带与突变等位基因紧密连锁。 我们也将研究各种生理因素对lls 1表达的影响。 我们的初步研究表明lls 1的表达完全依赖于光。 当叶片避光时，lls 1病斑的发生和扩大都受到抑制。 然而，lls 1突变体在黑暗中保持约48小时后完全枯萎，并在返回光照后10-12小时内死亡。 正常的兄弟姐妹没有这种损伤。 另一方面，持续的强光会延迟lls 1病变的发生和扩散。 另一个生理因素，受伤，无论是由针刺或病原体感染引起的，诱导lls 1病变过早。 在还不能表达lls 1病变的叶片上，当表达lls 1的潜力在这些叶片中以某种方式发展时，几乎所有的创伤或感染部位都转化为lls 1病变。 此外，lls 1损伤的表达通过低于24 ℃的温度增强，并且对植物的遗传背景非常敏感。 从这些观察结果来看，lls 1可能编码了一种因子，可以防止玉米细胞受到可能由自由基毒性引起的细胞损伤。 类似的基因最近已经从许多动物身上克隆出来，包括线虫和人类。 在玉米中存在许多疾病病变模拟（DLM）突变，其在不存在任何病原体的情况下促进疾病样症状的产生。 目前，对这些突变的生物化学性质或这些基因座上野生型等位基因的功能一无所知。 由于它们被认为在生理上模拟病原体攻击期间引起的病变，因此已经表明某些病变模拟表型可能是由疾病发展的突变引起的。 所有病变模拟物的一个共同特征是它们与细胞死亡相关。 因此，DLM突变可能是研究控制植物细胞死亡的遗传和细胞机制的极好工具。 ***

Johal Abstract 9317052 The objective of this proposal is to tag and clone a disease lesion mimic (DLM), gene lethal leaf spot-1 (lls1), mutations of which are recessive. Major emphasis will be placed of cloning and characterizing the lls1 gene as well as defining various physiological and biochemical conditions that affect the expression of the lls1 mutation. We will continue to genetically characterize Les* -2552, a DLM dominant mutant and to advance them further for co-segregation analyses; co-segregation analysis on some mutants to identify Mu-elements that may have caused these mutations will also be attempted. The action of lls1 appear to be cell autonomous (confined to cells where it is expressed); on a number of plants, somatic clonal sectors with sharp boundaries were observed either as forward mutations or reversion events. A new cloning strategy, "somatic tagging", based on the use of these somatic sectors, is being investigated. Briefly, the Mu-hybridizing pattern of DNA from adjacent mutant and wild type sectors is compared for polymorphism. Any extra band in the mutant DNA may harbor the Mu element responsible for the mutation in question. Using this strategy a 6.5 kb Mu-1 hybridizing fragment has been identified from one of the lls1 mutants and preliminary results from a co- segregation analysis have indicated that this band is tightly linked with the mutant allele. We will also investigate the effect of various physiological factors on the expression of lls1. Our preliminary studies have indicated that the expression of lls1 is completely dependent on light. Both the initiation and enlargement of lls1 lesions are inhibited when the leaves are protected from light. However, lls1 mutants kept in the dark for about 48 hours completely wilt and die within 10-12 hours upon return to light. Normal siblings show no such damage. On the other hand, continuous bright light delays the onset and spread of lls1 lesions. Another physiological facto r, wounding, caused either by pin pricks or by pathogen infections, induces lls1 lesions prematurely. On leaves which are not yet competent to express lls1 lesions, almost all wounding or infection sites transform into lls1 lesions when the potential to express lls1 somehow develops in these leaves. Additionally, the expression of lls1 lesions is enhanced by temperatures below 24 C and is very sensitive to the genetic background of the plant. From these observations, it seems likely that lls1 may encode a factor that prevents maize cells from cellular damage, possibly caused by free radical toxicity. Similar genes have recently been cloned from a number of animals, including nematodes and humans. %%% A number of disease lesion mimic (DLM) mutations exist in maize that promote the production of disease like symptoms in the absence of any pathogen. At present, nothing is known about the biochemical nature of these mutations, or the function of the wild- type alleles at these loci. Since they are considered to physiologically mimic the lesions caused during pathogen attack, it has been suggested that some lesion mimic phenotypes may result from mutations of disease development. One feature that is common to all lesion mimics is their association with cell death. Accordingly, DLM mutations may represent excellent tools for studying the genetic and cellular mechanisms controlling cell death in plants. ***