Cloning and Characterization of Two Disease Lesion Mimic Genes in Maize

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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. ***