Isolation And Characterization Of Arabidopsis Genes Interacting With COI1

Isolation and Characterization of Arabidopsis Genes Interacting with COIl Abstract Plant hormones are involved in all aspects of plaiit growth and development. Jasmonic acid and its derivates (here referred as to jasmonates (IA,)) are a new é‘œass of plant regulators that are synthesized from lipids through the octadecanoid pathway and widely distributed in plant kingdom. They influence many plant developmental processes including root growth, fruit ripening, tuberization and pollen formation. Jasmonates also act as a wounding signal to regulate plant response to stress, wounding, and pathogen infection and insect attack. Although the effects of jasmonates on plants are well studied, the molecular basis of JA action remains unknown. COIl gene was the first isolated regulatory gene in IA signal pathway and found to be required for plant fertility and defense. The coil mutant plants are insensitive to lAs and extremely susceptible to insects ,fungal pathogens and drought. They also display male sterile phenotype. The deduced COIl amino acid sequence showed that COIl has two important motifs: F-box domain and leucine rich repeats domain. The F-box domain was present in many yeast, C. elegan and human proteins which interact with Skpl and Cdc53 (cullin) to assemble ubiquitin ligase complexes, designated SCF. The function of SCF complex is to selectively recruit many short-lived proteins for degradation. The ubiquitin-rnediated degradation of regulatory proteins plays important roles in control of numerous processes, including cell cycle control, signal transduction, transcription regulation, receptor-down regulation, and endocytosis. The ubiquitin protein degradation system has been implicated in the immune response and programmed cell death. Because COIl contains this important F-box motif, we speculate that COIl might be a component of SCF complex that is involved in IA signal pathway. In this study, we drew on yeast two-hybrid system to identify the 4 proteins that interact with COIl. As a result, we have identified several proteins, one of COIl interacting proteins was found to be Arabidopsis SKPI-like protein ASK1. ASKI was expressed in yeast and purified for raising antibody in rabbit. We have demonstrated for the first time that COIl interacts with ASK 1, to form a complex that is required for iA action in planta. Furthermore, we showed that expression of antisense ASKI results in male sterility. 24 ASK1 associated genes were isolated and sequenced. The result of the study gave an important clue to study in molecular basis of IA action. The main results of this study are as follows: 1. Yeast two-hybrid system We used COIl as bait to screen Arabidopsis cDNA library in yeast two-hybrid system. More than 300 putative positive colonies have been isolated. They belonged to 12 distinct groups. Representative cDNAs from each group were sequenced. DNA database searching showed that one of these genes, represented by 59 positive colony, was identical to the previously identified Arabidopsis SKPI like protein ASK1 (formerly named as AtSkpl). Other putative COIl Associated Proteins were designated as COAP2, 3 to 12, respectively. COIl interacted strongly with ASK 1, COAP2 and COAP3. Presence of the F-box motif in COIl led us to p...