Arabidopsis 2010: Functional Analysis and Phosphorylation Site Mapping of Leucine-Rich Repeat Receptor-Like Kinases in Arabidopsis

National Science Foundation MCB-0419819

Project Abstract

The Arabidopsis genome encodes more than 200 Leucine-Rich Repeat Receptor- like kinases (LRR RLKs) with an organization of functional domains similar to that of animal receptor kinases. Several LRR RLKs are known to be critical elements in signaling pathways regulating plant development and response to the environment, but the biological functions of most members of this large family of putative receptors remain unknown. This project will acquire fundamental biochemical knowledge of the kinase domains of all Arabidopsis LRR RLKs by using Gateway cloning and high-throughput liquid robotics for in vitro analysis of autophosphorylation activity, substrate preference and pair-wise interactions. A proteomic analysis of membrane proteins isolated from Arabidopsis plants grown under a variety of physiological conditions will be used to identify a subset of 30 LRR RLKs for detailed analysis of in vivo autophosphorylation sites by mass spectrometry. The functional significance of selected phosphorylation sites will be examined using genetic and biochemical approaches, and interaction between selected LRR RLKs in planta will also be examined. Information about the project, including a list of genes studied can be obtained below and at http://www.cals.ncsu.edu/hort_sci/faculty/clouse.html. Significance to 2010 Project Objectives-- Identification of specific phosphorylation sites and their functional significance will advance our understanding of RLK signaling mechanisms and provide a comparative database of phosphorylation sites for membrane localized kinases. The family-wide analysis of LRR RLK kinase domains may reveal new aspects of the evolution of function in multigene families and studies of in vivo interactions may uncover novel heterodimers, suggesting possible cross-talk between signaling pathways. Broader Impact of the Proposed Activity--Phosphorylation sites determined in this study will be posted regularly on the PlantsP database ( http://plantsp.genomics.purdue.edu), creating a unique community resource for comparative study. A variety of tagged constructs in Gateway vectors for in vitro and in vivo studies of Arabidopsis LRR RLKs will be deposited in the Arabidopsis Biological Resource Center, along with numerous lines of transgenic plants. Given the known importance of several LRR RLKs to plant development, it is likely that increased knowledge of this gene family could have practical agricultural impact. The project will provide excellent training in protein biochemistry, mass spectrometry, and Arabidopsis molecular genetics at all levels from high school student interns through postdoctoral scientists in an environment that encourages the participation of underrepresented minorities.

Project Objectives

1. Generation of bacterial and plant expression vectors for all Arabidopsis LRR RLKs. As a foundation for this study, and as a resource for the Arabidopsis community in general, we will use high-throughput liquid handling robots and the efficient Gateway cloning system to generate a complete set of expression vectors yielding tagged proteins for in vitro and in vivo functional analysis, including both full-length proteins and cytoplasmic kinase domains only, of all LRR RLKs with Flag, His, and GFP epitope tags.
2. Biochemical studies of kinase domain function in the complete LRR RLK family. Using high-throughput technology, the biochemical properties of recombinant kinase domains will be examined including assessing possible autophosphorylation sites, substrates and in vitro interactions in pairwise combinations of all LRR RLK members.
3. Selection of 30 LRR RLKs for in depth studies. A variety of different growth conditions and treatments will be surveyed for protein abundance and protein phosphorylation using proteomic approaches involving isotope coded affinity tagging and mass spectrometry. A total of 30 proteins will be selected for further analysis as described below.
4. Autophosphorylation site mapping. We will generate Flag-tagged proteins in transgenic plants for the 30 selected LRR RLKs and perform immunoprecipitation from appropriate tissues. Sites of in vivo autophosphorylation will be determined using a variety of approaches involving mass spectrometry.
5. Functional analysis of autophosphorylation sites. The function of the identified sites will be assessed in a selected subset of up to 12 LRR RLKs by in vitro mutagenesis of each site followed by observing the effect of loss of specific Ser or Thr residues on the ability of the construct to rescue knock-out mutants. Changes in phosphorylation status of specific sites under different growth conditions will be monitored by isotope dilution techniques followed by mass spectrometry.
6. in vivo interaction studies – Guided by the results from in vitro interaction studies, we will examine in planta interactions between selected LRR RLKs by co-immunoprecipitation experiments in transgenic plants expressing both proteins with different tags.

Deliverables

Database

This project will be closely coordinated with a second NSF Arabidopsis 2010 project on LRR RLKs (0418946) directed by Frans Tax, John Walker and Erin Dolan. Both of these projects will also be integrated with two German AFGN projects on LRR RLKs directed by Kay Schneitz and Thorsten Nuernberger.

Genes to be Studied