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.
OBJECTIVE 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.
OBJECTIVE 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.
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 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.
OBJECTIVE 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.
OBJECTIVE 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.
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.
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.
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.
|
AGI
Identifier |
LRR
RLK Subfamily |
Gene
Name |
PlantsP
ID |
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LRR I
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LRR I
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LRR I |
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LRR I |
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LRR I
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I
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LRR I
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LRR I
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LRR I |
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LRR I
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I
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LRR I
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LRR I
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LRR I
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I
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LRR I
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LRR I |
LRRPK
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR I |
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LRR II |
SERK2 |
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LRR II |
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LRR II |
SERK1 |
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LRR II |
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LRR II |
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LRR II |
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LRR II |
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LRR II |
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LRR II |
BAK1 |
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LRR II |
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LRR II |
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LRR II |
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LRR II |
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LRR II |
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LRR III
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LRR III
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LRR III |
RKL1 |
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LRR III
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LRR III
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LRR III |
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III |
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III |
TMKL1 |
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LRR III
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LRR III
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LRR III
|
IMK2 |
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LRR III
|
MRLK |
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LRR III
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LRR III |
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LRR III
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LRR III
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LRR III |
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR III
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LRR IV |
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LRR IV
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LRR IV
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LRR IV
|
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LRR V |
Scrambled/SRF9/SUB |
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LRR V |
SRF6 |
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LRR V |
SRF5 |
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LRR V |
SRF1 |
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LRR V |
SRF4 |
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LRR V |
SRF7 |
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LRR V |
SRF3 |
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LRR V |
SRF8 |
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LRR V |
SRF2 |
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VI
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LRR VII
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LRR VII
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LRR VII
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LRR VII
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LRR VII
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LRR VII
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LRR VII
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LRR VII
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LRR VII |
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LRR VII
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LRR VIII-1 |
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LRR-VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-2 |
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LRR VIII-1 |
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LRR VIII-2 |
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LRR VIII-1 |
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LRR VIII-1 |
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LRR VIII-1 |
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LRR VIII-1 |
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LRR VIII-1 |
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LRR VIII-1 |
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LRR IX
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LRR IX |
TMK1 |
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LRR IX
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LRR IX
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LRR X
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LRR X
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LRR X
|
BRL1 |
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LRR X
|
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LRR X
|
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LRR X
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LRR X
|
VH1/BRL2 |
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LRR X
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LRR X |
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LRR X |
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LRR X
|
BRL3 |
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LRR X
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LRR X |
BRI1 |
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LRR X |
EMS1 |
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LRR X
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LRR X
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LRR XI
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LRR XI
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LRR XI
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LRR XI
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LRR XI
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LRR XI
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LRR XI
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LRR XI
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LRR XI |
CLV1 |
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LRR XI
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LRR XI
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LRR XI
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LRR XI |
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LRR XI
|
BAM2 |
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LRR XI
|
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LRR XI
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LRR XI
|
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LRR XI |
HAESA |
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LRR XI
|
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LRR XI
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LRR XI
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LRR XI |
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LRR XI
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LRR XI
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LRR XI
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LRR XI
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LRR XI
|
BAM1 |
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LRR XI
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LRR XII |
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LRR XII
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LRR XII
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LRR XII
|
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LRR XII
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LRR XII
|
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LRR XII
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LRR XII |
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LRR XII
|
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LRR XII |
FLS2 |
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LRR XIII
|
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LRR XIII |
ERECTA |
||
|
LRR XIII |
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LRR XIII
|
Erectalike-ERL2 |
||
|
LRR XIII
|
ERL1(Torii,K) |
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|
LRR XIII
|
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|
LRR
XIV |
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LRR XIV |
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LRR XIV |
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