Research Projects

Diphthamide (DPH) is a post translational modification of a conserved histidine residue in elongation factor 2 (eFE2) and is synthesized by seven enzymes (DPH1-7) and is crucial for maintaining the translation rate and fidelity. Two indirect evidences indicating presence of DPH in plants relate to an unusual amino acid that is ADP-ribosylated in wheat EF2 and ability of AtDPH4 to complement Scdph4 mutant, but conclusive evidence for presence of DPH on EF2 and its role in control of translation fidelity was lacking. Recently, the AtDPH1 protein was identified to regulate the first step of the DPH biosynthesis pathway and was shown to regulate plant growth. We identified homologues of yeast Dph genes in A.thaliana and observed that AtDPH5 can complement the yeast dph5 mutant. We procured A.thaliana T-DNA mutants for Dph2, Dph3, Dph5, Dph6 and Dph7 and identified their homozygous lines. Due to the importance of DPH modification in maintaining translational fidelity and regulating translation elongation together the following hypothesis will be tested Whether identified A.thaliana DPH genes (DPH2, DPH3, DPH5, DPH6, DPH7) are functional? Whether AtDph genes control translational fidelity and whether their loss results in phenotypic aberrations? What genes and proteins are regulated at the transcriptional and translational levels by DPH5? What are protein interactors of DPH5 gene? Which endogenous ADP-ribosylation factors and hydrolases are expressed during abiotic stresses and recovery phases respectively and whether they have the ability to ribosylate DPH? The approaches used are listed below Obj1: DPH genes will be used to complement the available yeast dph mutants. DPH modification in WT and mutant plants will be identified with the help of MS. An in-vitro ADP-ribosylation assay will be performed to test if eEF2 isolated from WT and dph mutant lines could be ADP-ribosylated, indicating the presence or absence of DPH respectively. Protoplasts generated from dph mutant lines will be checked for their survival in presence of diphtheria toxin, a DPH-ADP ribosylation factor causing cell death. Obj2: Dual luciferase reporter system will be used for finding the role of DPH genes in controlling translational fidelity. Plant DPH mutants will be phenotypically evaluated to understand their roles in plant growth and development. Obj3 & 4: Transcriptome and SWATH analysis will be performed with WT and dph5 mutant. For Y2H screening DPH5 protein will be used as a bait. Obj5: Study expression of endogenous ADP-ribosylation factors and hydrolases during abiotic stresses and recovery. ADP-ribosylation and hydrolase activity will subsequently be checked on purified eEF2. At the completion of the project we will identify the molecular switches that regulate translation. As protein translation is a key determinant of plant fitness in both normal and stressed conditions, regulatory checkpoints can be modulated by biotechnological approaches to improve plant fitness.