Characterizing Arabidopsis G Protein Interactors By A Reverse Ras Recruitment System

Heterotrimeric G proteins are signal transducers which are conserved in eukaryotes ranging from yeast to humans. Heterotrimeric G proteins are GTPases, composed ofα,βandγsubunits. Studies with mammalian cells established that heterotrimeric G proteins associated with plasma membrane receptors which contain 7 transmembrane domains (GPCRs). In mammals, more than 20 Gα,5 distinct Gβand at least 12 Gγsubunits have been identified.In contrast, our understanding to the molecular mechanisms of activation, signaling and regulation of plant heterotrimeric G proteins is rudimentary. It is currently thought that plant heterotrimeric G proteins contain a single Gα-subunit (GPA1), a single Gβ(AGB1) and two Gγ-subunits (AGG1&2). Both GPA1 and AGB1 have been shown to be located at the plant plasma membrane. A wide range of biochemical, pharmacological, and genetic studies conducted over the last 20 years have shown that plant G proteins are involved in responses to a number of hormone, developmental and environmental signals. The involvement of heterotrimeric G proteins in plant hormone signaling is complex. In particular, recent genetic studies with Arabidopsis have revealed direct, indirect, tissue specific effects on auxin, ABA, GA, brassinosteroid, sphingolipid and D-glucose, Blue light, pathogen signaling. Although plant heterotrimeric G proteins are involved in this wide range of signaling events, there are only few known downstream effectors that physically interact with either the plant Gα, or the Gβγdimmer. Attempts to identify other components of plant G protein signaling by database mining, genetics and pharmacology have not been conclusive. Because conventional methods have not provided answers to these important questions, an innovative approach is required.In this subject, the yeast two-hybrid reverse Ras Recruitment system (rRRS) had been used to identify proteins that interact with GPA1. The putative ineracrors is ADLC1, ACC1, AtXB31 and so on. We focused on the AtXB31. Interactions had been confirmed by Pull down, BiFC, CoIP. AtXB31 mutants will be isolated and double mutants generated with gpa1. The phenotype and response of these to known G protein mediated events will be determined. The main results are as follow: 1. Constructing an Arabidopsis cDNA library of 2-week seedling, 3-weekseedling, leaf, stem, flower, which is 1×105 colonines.2. Constructing two kind of bait, which is pMet-WtGPA1 and pMet-Q222L, through reverse Ras Recruitment system, we got some putative interactors.3. The interaction is confirmed by yeast two hybridization, Pull down, BiFC, CoIP in vitro and in vivo. The N-Myristoylation site of AtXB31 is necessary for the interaction of GPA1 and AtXB31.4. The strcture analysis of AtXB31 suggests that, there is three conserved domain, such as N-Myristoylation site, ankyrin repeats, ring type Zinc figure of C3HC4. There is no transmembrane motif by Tmhmm software. The expression of AtXB31 can be detected in all major tissue of plant by RT-PCR.. Subcellular localization of the transiently expressed pBI121-AtXB31-GFP fusion protein in tobacco epidermal cells shows AtXB31-GFP fluorescence is concentrated to the intracellular membrane of cell. The expression of AtXB31 is enhanced treated with ABA and Xcc pathogen.5.AtXB31 and AtGPA1 may coregulate the signal transduction of Xcc8004 pathogens. From morphology, ectopic over-expression and RNA interference plants of AtXB31 showed no difference comparing with wild type. The mutant gpa1-4 and AtXB31RNAi is susceptible to the infection of Xcc8004. In other hand,we had proved AtXB31 response to the Xcc on transcription. So, AtXB31 and AtGPA1 may coregulate the signal transduction of Xcc8004 pathogens.As a bacterial pathogen, Xanthomonas campestris could cause serious disease to plant, such as black rot. Therefore, investigating the signaling pathways between palnt and pathogen and detecting the key regulary genes, will generate important theory against the pathogens.In conclusion, it would have tremendous potential for identifying components of plant GPA signaling pathways and, in combination with the use of biochemistry and the genetic screens, will generate novel information that will enhance our understanding of signal transduction mechanisms in plants. It would do a great good to upgrade of crops.