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

 

National Science Foundation (MCB) 0419819

 

PI:                      Steve Clouse, North Carolina State University

Co-PI:                 Michael Goshe, North Carolina State University

Co-PI:                 Jia Li, University of Oklahoma

Co-PI:                 Steve Huber, University of Illinois

 

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

 

OBJECTIVE 1Generation 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.

OBJECTIVE 2Biochemical 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.

OBJECTIVE 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.

OBJECTIVE 4Autophosphorylation 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. 

OBJECTIVE 5Functional 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.

OBJECTIVE 6in 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

 

The Arabidopsis Biological Resource Center at Ohio State University will distribute the following Gateway vector constructs for the entire LRR RLK family when completed: 1. N-terminal-His7-Kinase Domain (bacterial expression), 2. N-terminal-Flag-Kinase Domain (bacterial expression), 3. N-Flag-mutant-Kinase Domain (bacterial expression, kinase inactive mutant created by substituting Glu for the invariant Lys in kinase subdomain II), 4. 35S promoter-full length cDNA-Flag C-terminal tag (plant expression), 5. 35S promoter-full length cDNA-GFP C-terminal tag (plant expression). In addition seeds of transgenic lines from a subset of these vectors as well as some with native promoters will be distributed.

 

Database

 

A database of phosphorylation sites in LRR LRKs we identify will be maintained at the PlantsP database (http://plantsp.genomics.purdue.edu/). The database is envisioned as protein sequences of LRR RLKs with specific Ser and Thr residues marked when phosphorylated. Clicking on these residues will link to the mass spectrum and alignments of these LRR RLKs with other receptor kinase phosphorylated at similar residues. The database will be coordinated with the developing plant phosphorylation database of the Scott Peck group. For a prototype of the phosphorylation database showing confirmed BRI1 in vivo phosphorylation sites go to http://biochem.ncsu.edu/faculty/goshe/Bri1.htm.

 

Coordination with other LRR RLK projects

 

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

AGI Identifier

LRR RLK Subfamily

Gene Name

PlantsP ID

At1g05700 

LRR I    

  

21076

At1g07550 

LRR I    

   

21013

At1g07560 

LRR I       

 

21012

At1g49100 

LRR I        

 

21093

At1g51790 

LRR I    

 

21125

At1g51800 

LRR I        

 

21112

At1g51810 

LRR I        

 

18122

At1g51820 

LRR I        

 

18127

At1g51830 

LRR I        

 

18132

At1g51850 

LRR I        

 

18167

At1g51860 

LRR I    

   

18137

At1g51870 

LRR I    

   

18142

At1g51880 

LRR I    

   

18147

At1g51890 

LRR I     

   

18152

At1g51910 

LRR I    

  

18157

At1g62090

LRR I

 

21145

At1g67720 

LRR I         

 

21011

At2g04300 

LRR I        

 

21567

At2g14440 

LRR I     

   

21631

At2g14510 

LRR I    

   

21629

At2g19190 

LRR I    

   

21584

At2g19210 

LRR I    

   

21585

At2g19230 

LRR I    

   

21598

At2g28960 

LRR I        

 

21649

At2g28970 

LRR I        

 

21650

At2g28990 

LRR I        

 

21651

At2g37050 

LRR I        

 

21534

AT3g21340 

LRR I        

 

21235

AT3g46330 

LRR I        

 

21741

AT3g46340 

LRR I        

 

21742

AT3g46350 

LRR I        

 

21743

AT3g46370 

LRR I        

 

21744

AT3g46400 

LRR I        

 

21745

AT3g46420 

LRR I        

 

21241

AT4g20450 

LRR I        

 

21838

AT4g29180 

LRR I    

   

21890

AT4g29450 

LRR I    

   

21891

AT4g29990 

LRR I 

LRRPK    

21893

AT5g16900 

LRR I        

 

22073

AT5g48740 

LRR I        

 

22012

AT5g59650 

LRR I        

 

21350

AT5g59660 

LRR I        

 

21351

AT5g59670 

LRR I        

 

21352

AT5g59680 

LRR I        

 

21353

At1g34210 

LRR II        

SERK2

21432

At1g60800 

LRR II        

 

21056

At1g71830 

LRR II       

SERK1

36635

At2g13790 

LRR II &n