|Ph.D Student||Ron Neta|
|Subject||Molecular Interactions between Tomato Homeodomain|
Proteins from the KNOX and the BEL Families
|Department||Department of Biology||Supervisor||Professor Emeritus Eliezer Lifschytz|
Plants generate new organs throughout their life cycle from special groups of cells called meristems. One of the most important groups of genes that determine the meristematic nature of cells encodes transcription factors with homeodomain of the class-I KNOX family (hereafter KNOX-I).
To understand the cellular functions of the KNOX-I proteins we have identified and studied four proteins of the BEL family that interact with TKn2, a KNOX-I protein. BEL proteins are putative transcription factors which, like KNOX-I contain homeodomain of the TALE super-family.
We describe the sequence organization of the four TBL proteins and their genetic localization. Subsequently, the expression patterns of the TBLs genes was found to overlap with that of TKn2, proving that interaction in vivo is posible. In transgenic plants, suppression of TBL1 elicits morphogenetic changes in all organs and alters the shoot growth habit.
The following results were obtained in a comprehensive analysis of the interactions between TBL and KNOX proteins. (i) In a two-hybrid screen, with TBL1 three proteins of the KNOX-I group were identified. TBL-KNOX-I interactions were also verified by in-vitro experiments. In addition to the KNOX-I proteins, two unexpected proteins were also found to interact with TBL1. The first is encoded by a KNOX-II gene, a non-meristematic class of KNOX genes. The second protein designated as TKD1, was a novel protein with unusual KNOX domain and lacking ELK and HD regions that characterize all known KNOX proteins. (ii) We have determined that all TBL proteins may interact with all KNOX-I proteins but only TBL1 interacts with KNOX-II proteins. It was further demonstrated that KNOX-I proteins may interact differentially with one another. (iii) Analysis of the exceptional TKD1 protein showed that it may form homodimers and also interacts with all TBLs. In addition TKD1 interacts with KNOX-II, but surprisingly, not with KNOX-I proteins. Deletion analysis suggests that TKD1 has different binding domains for TBLs and for the other interacting proteins. (iv) In vitro competition assays suggested that TBL1 and TBL3 may compete for binding to TKn2.This finding promoted an extensive analysis to find out if all TBLs recognize the same site in the KNOX proteins. The results showed that TBL proteins recognize a common binding site at the C- terminal end of the KNOX-domain in all KNOX proteins tested. (v) Using site-directed mutagenesis of conserved amino acids at the C-terminal part of the KNOX domain of TKn2, it was found that all conserved amino acids are important for the binding. In-addition, a deletion of the last eight amino acids of the KNOX domain in TKD1 completely eliminated its binding to TBLs proteins.
The results presented in this work suggest that to regulate the fate of cells and the diversity of developmental programs, plants exploit systems of interaction among HD proteins using principles employed also by animal cells.