Becraft 96-04426 Plant leaves are covered by a highly specialized epidermal layer which functions in development to delimit organs and prevent organ fusions. The endosperm of grass seeds also contains an epidermis-like cell layer called the aleurone which secretes hydrolytic enzymes required for remobilization of storage compounds during germination. The crinkly4 (cr4) gene of maize is involved in the differentiation of leaf epidermis and aleurone. cr4 encodes a receptor kinase with extracellular similarity to the ligand binding domain of tumor necrosis factor receptors (TNFRs) from mammals. Tumor necrosis factors (TNFs) are small protein signal molecules that are involved in developmental processes in the immune system and other tissues. The similarity between CR4 and TNFR suggests that the ligand for CR4 might also be a protein of the TNF class. Genomic southern hybridization suggests that cr4 is a member of a gene family. The proposed research has three specific aims: 1. Complete a molecular genetic analysis of the maize cr4 gene. This includes generating an allelic series of cr4 mutants and analyzing the expression pattern of the cr4 gene. The expression pattern will be studied using immunohistochemistry and genetic mosaic analyses. 2. Isolate a ligand for the extracellular domain of CR4. Three different approaches are proposed: A. molecular identification of a cloned gene that encodes a protein able to bind the extracellular domain of CR4 (i.e. interaction cloning), B. genetic identification of an interacting protein by analysis of mutants with similar phenotypes to cr4, or by identification of second site mutations that enhance or suppress the cr4 mutant phenotype, and C. a transgenic approach to introduce specifically engineered mutations that can be used in genetic suppressor screens. 3. Characterize the cr4 gene family in arabidopsis to determine the extent of the family, whether all members encode receptor kineses and the expression profiles for each member.
