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2/7/2013
Autoregulation of Nodulation
Soybean (Glycine max) GmRIC1 regulates
nodulation in wild-type bean (Phaseolus
vulgaris), but not in a supernodulating line
found to be mutated in Nodule
Autoregulation Receptor Kinase (PvNARK)
Brett Ferguson,
Li D, Hastwell AH, Reid
DE, Li Y, Jackson S, and
Peter Gresshoff
• Nodule Autoregulation
(AON) is controlled by the
shoot and root
• Regulation is dependent
on NARK and systemic
signals travelling to and
from the roots
• NARK is a LRR receptor
kinase, similar to
CLAVATA1 in Arabidopsis
which perceives CLE
peptides and modulates
SAM activity
NARK
Q
SDI
Inoculated wild-type
NARK-dependent nodule regulation
A
Figure 1. Phaseolus vulgaris wildtype,
OAC
Rico,
and
its
supernodulation Pvnark mutant,
R32. A) Shoot and B) root
phenotypes of 2 month-old OAC
Rico (left) and R32 (right),
inoculated with Bradyrhizobium
japonicum.
B
Reid et al, 2011. Ann Bot
Two weeks
Two months
1
2/7/2013
A
PvNARK
GmNARK
B
C
MRNGVCYRLL
WKFSTSLSAH
LTITQNNLTG
DAYDNNFIGQ
STNSLSGKIP
SCNLSGEIPP
GLTGEIPQSF
SFVLPPNLGQ
QIPDDIGNCK
PPEISGDSLG
VFVLPMLTTV
NLTDLSILNV
VFSDKSFEGN
IATAALLVAV
IGKGGAGIVY
NIMRLLGYVS
AARGLCYLHH
SQSMSSIAGS
FGDGVDIVGW
MMCVREMGPA
FLVLVWFSVA
CLFPGVTCDQ
QLPKELAALT
LPEEFVKLEK
ESLSRLKTLK
SLSSLKKLDT
SQLRNLTLMN
NGRLKFFDVT
SLVKIRASNN
ILTLSNNLLT
NISGNNLTGA
SRNLITGPIP
PNLCSSRSCP
TVYMMRRRML
RGSMPNGTDV
NKETNLLLYE
DCSPLIIHRD
YGYIAPAEYA
INKTRLEISP
RPTMREVVHM
KCSSFSDMDA
DLRVVAINVS
SLKLLNISHN
LRYLKLDGNY
YLKLGYNNAY
LFLQMNNLTG
FFHNKLSGSV
TNHFTGLIPP
YLSGAIPSGI
GRIPPALKNL
IPTTLIHCVS
DEIRFMASLT
NSSMYPDDAF
HRAMTWKLTA
AIKRLVGAGS
YMPNGSLGEW
VKSNNILLDE
YTLKVDEKSD
PSDAALVLAV
LTNPPHSTTH
LRKLKDSMKG
FVPLFGNLPP
SFSHYFPGQS
FSGSIPESYS
EGGIPPEFGA
TIPSEFSAME
PAFVGELPNL
GLCKSGRLQT
FKLPSVKIIE
RALQTLSLDA
LSSVDLSRNM
TLDLSYNNFK
RRRRGPWSSK
FQRLNWKAED
GRNDYGFRAE
LHGAKGGHLR
NFEAHVADFG
VYSFGVVLLE
VDPRLSGYPL
THNHNLINL*
AKAKDDALHD
EIGHFDKLQN
FLPITQLEVF
EFKSLEFLSL
MKSLIYLDLS
SLMSLDLSFN
ETLQLWENNF
FLITDNFFHG
LANNRFDGEL
NELVGEIPGE
LVGEIPKGIK
GKLPTGGQFF
QTRAIITVIA
VVECLKEENI
IETLGKIRHR
WEMRFKIAVE
LAKFLHDPGA
LIIGRKPVGE
TSVIYMFNIG
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
990
GmNARK
PvNARK
LjHAR1
MtSUNN
PsSYM29
Figure 2. The Nodulation Autoregulation Receptor Kinase of Phaseolus vulgaris. A) Phytozome cluster analysis of the
genomic environments of PvNARK and GmNARK reveal a well conserved region of synteny existing between the two
species. A truncated version of NARK (dark purple) is located directly upstream of PvNARK, but not GmNARK. B) PvNARK is
comprised of four domains: leucine-rich repeat-containing N-terminal signal peptide (red), leucine-rich repeats (blue),
transmembrane (yellow) and serine/threonine kinase (green). C) Phylogenetic tree of PvNARK and its orthologues in
soybean (GmNARK), Lotus japonicus (LjHAR1), Medicago truncatula (MtSUNN) and pea (PsSYM29).
PvNARK cDNA sequence (2970 bp)
ATGGATGCTTTGCGGAAGCTGAAGGACTCCATGAAAGGAGCCAAAGCCAAAGACGATGCTCTTCACGATTGGAAGTTCTCCACCTCGCTTT
CCGCACACTGTTTATTTCCTGGTGTTACGTGTGATCAAGACCTTCGAGTCGTTGCTATCAACGTCTCCTTCGTTCCACTCTTCGGCAACCTT
CCGCCGGAGATTGGACACTTCGACAAACTCCAAAACCTAACCATCACGCAGAACAACCTCACCGGCCAGCTTCCCAAGGAGCTCGCTGCCCT
CACTTCCCTCAAGCTCCTCAACATCTCTCACAACTCCTTCTCCCACTATTTTCCCGGCCAAAGCTTTCTTCCCATCACCCAACTCGAGGTTT
TCGACGCCTACGACAACAACTTCATCGGACAGCTTCCGGAGGAATTCGTGAAACTGGAGAAACTCCGATACCTCAAGCTAGACGGAAATTAC
TTTTCCGGCAGCATACCGGAGAGCTACTCCGAGTTTAAGAGCTTGGAGTTTTTGAGCTTAAGCACTAACAGTTTGTCGGGGAAGATTCCCGA
GAGTTTGTCTCGGTTGAAGACGCTGAAGTATCTAAAACTC
High conservation.
Structure and Function
GGATACAACAACGCTTACGAAGGTGGAATCCCGCCGGAGTTCGGCGCCA
TGAAATCTCTTATATACCTTGACCTATCTAGCTGCAACCTCAGCGGCGAGATTCCACCGAGCTTGAGCAGTTTGAAGAAACTTGACACGTTG
TTCTTGCAAATGAACAACCTTACCGGAACCATTCCTTCTGAATTCTCGGCGATGGAGAGCCTCATGTCACTGGATCTCTCCTTCAACGGTCT
CACTGGAGAGATCCCGCAGAGTTTCTCTCAGCTCAGAAACCTCACTCTCATGAACTTCTTCCACAACAAGCTTAGCGGCTCTGTTCCCGCCT
TCGTCGGCGAGCTTCCGAATCTGGAAACGCTGCAACTCTGGGAAAACAACTTCTCCTTCGTGCTGCCCCCGAATCTGGGCCAAAACGGGAGG
TTAAAGTTTTTCGACGTCACGACCAATCACTTCACCGGGTTGATCCCTCCGGGTTTGTGCAAGAGTGGGAGGTTACAAACCTTCCTCATCAC
CGACAACTTCTTTCACGGTCAAATCCCTGATGATATTGGAAACTGCAAGTCTCTCGTCAAGATCCGAGCCTCCAATAACTACCTCAGCGGCG
CCATTCCCTCAGGAATTTTCAAACTGCCTTCGGTCAAGATAATCGAACTCGCGAACAACCGTTTCGACGGCGAACTGCCACCGGAGATTTCC
GGCGATTCTCTTGGGATTCTCACGCTTTCCAACAACTTGCTGACGGGGAGAATTCCCCCGGCGTTGAAGAACTTGAGGGCACTGCAGACACT
CTCACTCGACGCGAACGAGTTGGTTGGAGAAATCCCGGGGGAGGTTTTTGTCTTACCAATGCTAACCACCGTCAACATAAGCGGCAACAATC
TGACTGGAGCAATCCCAACGACGCTGATTCACTGCGTTTCACTCTCCTCCGTGGACCTCAGCAGGAACATGCTCGTCGGGGAGATTCCGAAA
GGCATAAAAAACCTCACGGACTTAAGCATTTTGAATGTCTCCAGAAACCTGATAACAGGGCCAATCCCCGACGAGATTCGGTTCATGGCAAG
CCTCACAACGCTGGATCTCTCCTACAACAATTTCAAAGGCAAGCTCCCTACGGGTGGCCAGTTTTTTGTGTTCAGCGACAAGTCCTTTGAAG
GGAACCCCAATCTCTGTTCCTCCCGCTCTTGCCCTAATTCCTCCATGTACCCTGACGACGCCTTCAGAAGAAGGCGCGGCCCTTGGAGTTCT
AAGCAAACGAGGGCGATAATCACCGTGATCGCAATAGCCACAGCGGCGCTGCTGGTGGCGGTGACGGTGTACATGATGCGAAGGAGGATGCT
GCACCGGGCGATGACGTGGAAGCTGACGGCGTTCCAGCGGCTGAACTGGAAAGCGGAGGACGTGGTGGAGTGTCTGAAGGAAGAGAACATCA
TAGGAAAAGGAGGGGCGGGAATCGTGTACCGCGGCTCCATGCCAAATGGAACCGACGTGGCTATAAAGCGGTTGGTTGGGGCGGGTAGCGGG
AGAAACGATTACGGTTTCAGGGCTGAGATAGAAACGCTGGGGAAGATAAGGCACAGAAACATAATGAGGCTTCTGGGTTACGTGTCGAACAA
GGAGACAAATCTGCTGCTTTACGAGTACATGCCGAATGGGAGCTTGGGAGAGTGGCTGCATGGGGCGAAGGGAGGGCACTTAAGGTGGGAGA
TGAGGTTCAAGATTGCAGTGGAAGCTGCTAGAGGACTCTGCTACTTGCACCATGATTGTTCCCCTCTCATCATTCACAGAGACGTCAAATCC
AACAATATATTGCTCGACGAAAACTTCGAAGCCCATGTCGCCGATTTCGGACTTGCCAAGTTCCTCCACGACCCAGGTGCCTCTCAGTCCAT
GTCCTCCATTGCTGGCTCCTACGGCTACATTGCTCCAGCAGAGTATGCTTACACGTTGAAAGTGGACGAGAAAAGCGATGTGTACAGCTTCG
GGGTGGTGCTGCTGGAGCTGATAATAGGGAGAAAACCAGTGGGGGAGTTTGGAGATGGAGTGGACATCGTTGGATGGATCAACAAAACGAGA
TTGGAGATCTCTCCGCCGTCGGATGCAGCGTTGGTGTTGGCAGTGGTGGATCCAAGGCTCAGTGGGTATCCATTGACAAGTGTGATTTACAT
GTTCAATATTGGGATGATGTGTGTTAGGGAAATGGGCCCAGCCAGGCCCACCATGAGGGAAGTGGTTCACATGCTCACCAATCCTCCTCACT
CCACCACTCACACTCACAACCACAACCTCATTAATCTC
TAA
Coding sequence of NARK in bean (Phaseolus vulgaris) and R32 mutant
A
Mutant change at amino-acid level
Intron
SP
Polar,
Uncharged
Q106*
nts1007
LRR
L346F
nod3-7
K115*
nts246
G198E
R32
B
Polar,
Acidic
G198E
V370D
nod4
TM
K606*
en6500
Kinase
W677*
& L829V
F262
G863D
nod1-3
Q920*
nts382
V837A
nts1116
Known NARK mutations in soybean (Glycine max) and bean
(Phaseolus vulgaris). A) Illustration of the NARK protein
structure depicting known mutations, including the location,
amino acid change and mutant name. Mutations are shown for
bean (orange arrow) and various lines of soybean (grey arrows),
including the F262 mutant (olive green arrows), which was
recently isolated from a TILLING population (Batley et al. 2012)
and contains two separate mutations. Not shown is the FN37
mutant of Glycine soja (wild soybean), which was generated using
fast neutron technology and is completely devoid of the NARK
gene (Searle et al. 2003). B) Phyre modeling (Kelley and
Strenberg 2009) (based on PDB: 1ZIW) of the NARK leucine-rich
repeat domain of bean. The site of the mutated amino acid
residue (G198E; highlighted in orange) of the supernodulation
mutant, R32, is depicted flanking the central cleft of the LRR
domain that likely defines the ligand binding site.
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2/7/2013
A
500
B
400
500
400
300
300
200
200
100
100
***
0
OAC
Rico
WT
Control
control
OAC
Rico
WT RIC1
GmRIC1 ox
***
0
R32 R32
Control
control
R32
R32
RIC1
GmRIC1 ox
Hairy-root over-expression of the soybean AON CLE peptide,
GmRIC1, in bean and its effect on nodule formation. Nodulation
of OAC Rico (wild type) hairy roots over-expressing GmRIC1 was
significantly inhibited compared with those induced with the
vector-only control (P < 0.001). In contrast, nodule numbers were
not reduced in R32 mutant plants. Error bars indicate mean ±
standard error (SE) (n = 10-12).
OAC
Rico
WT
Control
control
OAC
Rico
WT RIC1
GmRIC1 ox
R32 R32
Control
control
R32
R32
RIC1
GmRIC1 ox
Hairy-root over-expression of the soybean AON CLE peptide,
GmRIC1, in bean and its effect on nodule formation. Nodulation
of OAC Rico (wild type) hairy roots over-expressing GmRIC1 was
significantly inhibited compared with those induced with the
vector-only control (P < 0.001). In contrast, nodule numbers were
not reduced in R32 mutant plants. Error bars indicate mean ±
standard error (SE) (n = 10-12).
Conclusions:
1) CLE peptides related to Arabidopsis CLAVATA3 peptide
act as regulators of nodule development in legumes.
2) Peptide gene GmRIC1 when overexpressed in wild type
bean (Phaseolus vulgaris) suppresses nodule number.
3) Bean has an AON (Autoregulation of Nodulation) gene
called PvNARK, similar to other legume AON genes.
4) R32 is a PvNARK missense mutant (Gly to Glu) that
prevents CLE peptide suppression.
5) AON and its components (receptors, signals) are shared
in legumes.
3