Cloning and Characterization of Two Disease Lesion Mimic Genes in Maize

摘要本研究的目的是标记和克隆一种致病模拟物（DLM），即致死性叶斑-1基因（lls1），其突变是隐性的。主要重点将放在克隆和表征的lls1基因，以及定义各种生理和生化条件，影响lls1突变的表达。我们将继续对DLM显性突变体Les* -2552进行遗传表征，并进一步推进它们的共分离分析；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元素。lls1的作用似乎是细胞自主的（局限于表达它的细胞）；在许多植物中，具有明显边界的体细胞无性系可以作为正向突变或逆转事件被观察到。基于这些体细胞扇区的利用，一种新的克隆策略“体细胞标记”正在研究中。简要地比较了相邻突变型和野生型扇区DNA的mu杂交模式的多态性。突变DNA中任何额外的条带都可能包含导致突变的Mu元素。利用这种方法，从其中一个突变体中鉴定出了6.5 kb的Mu-1杂交片段，共分离分析的初步结果表明，该条带与突变等位基因紧密相连。我们还将探讨各种生理因素对lls1表达的影响。我们的初步研究表明，lls1的表达完全依赖于光。当叶片受到光照保护时，ls1病变的发生和扩大都受到抑制。然而，在黑暗中保存约48小时的ls1突变体在返回光线后10-12小时内完全枯萎并死亡。正常的兄弟姐妹没有这种损伤。另一方面，持续的强光延迟了ls1病变的发生和扩散。另一个生理事实是，由针扎或病原体感染引起的伤害，会过早地诱发ll1病变。在还不能表达lls1病变的叶片上，当这些叶片以某种方式发展出表达lls1的潜力时，几乎所有的损伤或感染部位都转化为lls1病变。此外，温度低于24℃时，lls1病变的表达增强，并且对植物的遗传背景非常敏感。从这些观察结果来看，lls1似乎可能编码一种因子，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包括线虫和人类在内的许多动物身上克隆出来。在没有任何病原体的情况下，玉米中存在许多疾病病变模拟（DLM）突变，这些突变促进了类似疾病症状的产生。目前，对这些突变的生化性质以及这些位点上的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仿病原体攻击期间引起的病变，因此有人认为一些病变模仿表型可能是由疾病发展的突变引起的。所有病变模拟的一个共同特征是它们与细胞死亡有关。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绝佳工具。＊＊＊

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. ***