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Currently, I am working on several different projects involved in the ethylene signaling pathway. Two of these projects are aimed at understanding the previously uncharacterized protein, RTE1 (Reversion to Ethylene Sensitivity). RTE1 was isolated as a suppressor of a weak ethylene insensitive mutant, etr1-2. One project involves testing whether or not RTE1 physically interacts with the ethylene receptor ETR1. This will be approached by yeast two hybrid assays with full length RTE1 protein and various truncations of the ETR1 receptor. Future work will involve testing the possible interactions by coimmunoprecipitation assays. In addition to the yeast two hybrid tests, I am also working to characterize the RTE protein in C. elegans. We are interested in the RTE gene in animals because this gene was isolated as a suppressor of a signaling pathway not present in animals, which may indicate a broader role in these organisms. Furthermore, unlike plants, which have two or three homologues of the RTE gene, animals only have one copy of the RTE gene. We have obtained a loss of function mutant of RTE in C. elegans, and are currently at work to characterize and understand its function.
In addition to studying the RTE protein in both plants and animals, I am screening Arabidopsis seedlings to attempt to dissect the ethylene signaling pathway in even finer detail. In one screen, I am searching for more suppressors of the etr1-2 mutation. We are specifically looking at this mutation due to the different nature of the etr1-1 and etr1-2 mutations. The etr1-1 mutation is a strong insensitive mutation that prevents ethylene from binding to its site. However, the etr1-2 mutation is a weak insensitive mutation that still allows ethylene to bind in its site. We are looking for suppressors of the etr1-2 mutation in hopes of understanding how this mutation (and consequently the ETR1 receptor) works. Finally, I am also screening for enhancers of the ctr1-3 mutation. CTR1 is a Raf-like protein kinase that acts immediately downstream of ETR1 in the ethylene signaling pathway. CTR1 is a negative regulator in this pathway. That is, when CTR1 is active, ethylene responses are off, and vice versa. Therefore, the ctr1-3 mutation, which is a null, leads to a constitutive ethylene response. However, there are indications that this mutation does not confer a full constitutive response. One example is that ctr1 loss of function mutants still respond slightly to ethylene, on the basis of hypocotyl length assays. Furthermore, when both ETR1 and ERS1 (both ethylene receptors that contain all necessary residues for histidine kinase activity) are knocked out, the light grown phenotype of this mutant is much more severe compared to ctr1-3. This seems to indicate that there may be more than CTR1 involved in transmitting ethylene responses. Therefore, I am screening mutagenized ctr1-3 seedlings for a phenotype similar to the etr1-7/ers1-2 double null when grown in the light. In this way, we hope to find any additional members of the ethylene signaling pathway.
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