Symbiosis signaling &

Horizontal transfer of Rhizobia

Rujirek Noisangiam

1
• Rhizobium-legume symbiosis

• The studies on biology of symbiotic development

contribute to understanding both the bacterial and
the plant components of the symbiosis.

2
Symbiosis & Horizontal transfer of Rhizobia

Rhizobia Classification
Genome structure
Significant N-fixation
Assimilation

Symbiosis signaling

Horizontal transfer of Symbiosis Island Discovery of Symbiosis Island

Symbiosis Island transfer

3
 Overview

Rhizobia

• Classification Symbiosis island

• Genome structure
• Significant Rhizobia

Summary

4
 Rhizobia

• Nod factors Symbiosis signaling

• Type III and IV secretion system
• Receptors
Symbiosis island transfer
• Calcium spiking

Summary

5
Symbiosis island transfer in Mesorhizobia Rhizobia

• Some background on symbiosis island Symbiosis island transfer

• How does the island transfer?

Symbiosis signaling

Summary

6
Classification Rhizobia
Five genera of α-proteobacteria;
• Rhizobium
Symbiosis island
• Sinorhizobium
• Mesorhizobium
• Bradyrhizobium
Rhizobia
• Azorhizobium
Two genera of β- proteobacteria;
• Burkholderia Summary
• Ralstonia

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 Rhizobia

• Classification Symbiosis island

• Genome structure
Rhizobia
• Significant

Summary

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 Rhizobia
Genome structure
• Several species of rhizobia
Symbiosis island
Gene required for nodulation & N-fixation are on
large plasmids.
Rhizobia
• In Bradyrhizobium species & M. loti strain ICMP3153

Symbiosis genes are encoded on chromosome Summary

( Symbiosis island ).

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Genome structure Rhizobia
Rhizobium sp. NGR234
• Chromosome 3.5 Mb Symbiosis island
• > 2 mb megaplasmid (pNGR234b)
• 536,165 bp (a smaller plasmid pNGR234a)
• The small plasmid carries most of gene used Rhizobia
for symbiosis

R. Leguminosarum bv. viciae Summary

• A circular chromosome of 5,047,142 bp
• Six plasmids (pRL10 carries genes for symbiosis)

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Genome structure Rhizobia
B. japonicum
• A single chromosome 9,105,828 bp Symbiosis island

Sinorhizobium meloloti
• 3.65 Mb chromosome Rhizobia
• 1.35 Mb megaplasmid (pSymA)
• 1.68 Mb megaplasmid (pSymB)
Summary

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 Rhizobia
Genome structure
Mesorhizobium loti MAFF303099
Symbiosis island
• single chromosome 7,036,071 bp
• pMLa (351,911 bp)
• pMLb (208,315 bp) Rhizobia
• Symbiosis island on chromosome

Summary

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 Rhizobia

• Classification Symbiosis island

• Genome structure
• Significant Rhizobia

Summary

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 N2 N2O
Significant 6
NH3 5
NO
• Rhizobia can fix amount of atmospheric
1
nitrogen which become available to plants Plants NO2

7
• Reduce the need for nitrogen fertilizer & 4

decreasing nitrogen leaching from the soil 1 2 NO3

leached
NH4 3

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 N2 1. Uptake of NH4 or NO3 by
N2O
Significant 6
NH3 5
organism
NO
• Nitrogen cycle 2. Release of NH4 by
1 decomposition
Plants NO2

7 3, 4 Microbial oxidation of
4
NH4 (yields energy in
1 2 NO3
leached aerobic conditions)
NH4 3
1. Denitrification (NO3)
respiration) by microbes
• Nitrogen fixation in anaerobic conditions

N2 + 8e- + 8H+ + 16ATP  2NH3 + H2 + 16ADP 1. Nitrogen fixation

This is catalyzed by bacterial enzyme “ Nitrogenase” 2. Nitrate leaching from soil

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Significant
• Nitrogenase Rhizobia
• 2 components
• Fe protein & MoFe protein
Symbiosis island
• Reduce N2
• Sensitive to O2
Rhizobia
• Leghaemoglobin
• Oxygen carrying molecule
• Regulate the supply of oxygen to the nodule Summary

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Significant
• Assimilation

• NO3-

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Significant
• Assimilation
NH4+ is incorporated into
• NH4+ (GS-GOGAC cycle) amino acid.

Concentration of ammonia below 1 mM

These pathway are highly
regulate as the concentration
of ammonia

High concentration of ammonia : Direct incorporation of ammonia

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 The biology of symbiotic development

How does the plant allow bacteria to infect and

initiate the program?

How does the plant response to the Nod factors?

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 Rhizobia

• Nod factors Symbiosis signaling

• Receptors
• Calcium spiking
Symbiosis island transfer
• Type III and IV secretion system

Summary

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Model Legumes

Medicago truncatula

Lotus japonicus

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A proposed model of Nod factor
signaling pathway

Modified from Rie et al. (2004)

and Umehara et al. (2004)

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Secretion
system

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 Nod Factor

-1,4-linked N-acyl-D-
glucosamine backbone
containing substitution of
Reducing End acyl chain at non-
reducing terminal sugar

Acyl side-chain

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 The structure of Nod factor is the key to host specificity.

(Sulfotransferase)
NodL NodH
NodC
• The length of
glucosamine backbone
NodA • The structure of acyl chain
(N-acyltransferase)

• The specific substitution

NodF

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Sinorhizobium meliloti
(Medicago Sativa)

Rhizobium leguminosarum
(Pisum sativam)

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Secretion
system

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 Secretion system

An common theme of bacteria-eukaryotic interactions is the ability

to transfer proteins into host cells via specialised secretion systems:

Type III Secretion Systems Type IV Secretion Systems

• Pseudomonas syringae • Agrobacterium tumefaciens
• Yersinia pestis • Bordetella pertussis
• Salmonella enterica • Brucella suis

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 Secretion system

• The secretion systems are composed of several proteins

that are associated with the inner and outer bacterial membranes.
• Upon contact with host cells, proteins are secreted
via TTSS membrane channels into eukaryotic cytoplasm.

• The secretion systems are required for virulence

and used deliver effector proteins into cells of eukaryotic hosts.

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 Secretion system

Type III secretion system (T3SS)

• symbiotic plasmid of NGR234; NGR234a (1997)
• B. japonicum USDA110 (2001)
• M. loti strain MAFF303099 (2000)
• Sinorhizobium fredii, USDA257 (2003)

Type IV secretion system (T4SS)

• M. loti strain MAFF303099

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 Secretion system

T4SS
• Highly similar to the VirB/D4 T4SS
of A. tumefaciens (Sullivan et al., 2002).

• T4SS is required for T-DNA transfer to plants.

T3SS
• Rhc proteins are similar to Ysc and Hrc protei
nsfrom Yersinia sp. and plant pathogens, respec
tively.
• Rhc is required for the secretion of virulence f
actors

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The vir T4SS genes of M. loti strain R7A

vir-box 1 vir-box 2
nod-box virB
virA 11 10 9 8 7 6 5 4 32 1 virG msi059 virD4 msi061

ISfr

AH67 AH73AH58 AH60 AH48 AH47 AH50 AH49 AH51

AH54

Protein % amino acid ID to A. tumefaciens Vir proteins

VirA 45
VirG 76
VirB1-11 52, 66 ,82, 82, 57, 69, 54, 76, 68, 59, 82
VirD4 79
Msi061 26 (VirF)
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 NFR

Secretion
system

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 Nod factor receptor

NFR1 and NFR5

• NFR1 / NFR5 from L. japonicus NFR1

• Transmembrane receptor-like kinase NFR5

with extracellular LysM domain

• NFR1 regulate kinase activity by activation loop.
Kinase domain
• NFR5 lacks activation loop Activation loop
another way for activation? LysM domain
associate to NFR1?

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 • deficient in membrane polarization Nod factor receptor
• could not response to Nod factors

nfr1 and nfr5 mutants

• Root hair curling occurred in wild type L. japonicus. NFR1

• The curling response was not observed in nfr1 NFR5

and nfr5 mutants.

• Root hair of the wild type occurred depolarization, Kinase domain
• Whereas nfr5 and nfr1 mutants had no depolarization Activation loop
response. LysM domain

Simona et al. (2003)

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Secretion
system

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Ion flux and plasma membrane depolarization

Ca2+ This event lead to;

• Accumulation of Ca2+ in
R root hair tip

• Root hair deformation

Cl K+
-

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Secretion
system

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Nodulation receptors required for calcium spiking

DMI1
• Novel transmembrane protein
(homology with prokaryotic ion channel)

• acts as a complex with DMI2???

DMI2
• DMI2 from M. truncatula or SYMRK from L. japonicus Kinase domain
• Receptor-like kinase with extracellular LRR domain Activation loop
(3 Leucine-rich repeat) LRR domain

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 DMI2 is also required for
DMI2 mutant Wild type infection thread
development and switch
to the release of rhizobia
DMI2 mutant DMI2-transformed nodule Wild type in the infection zone.
M. truncatula

Limpens et al. (2005)

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Secretion
system

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Nodulation receptors required for calcium spiking

DMI3
• Calcium-calmodulin dependent protein kinase
(CCaMK)
• induce nodulation by binding both free calcium
and calmodulin-bound calcium

• regulate negative feedback of calcium spiking Kinase domain

Activation loop
Oldroyd et al., 2001 Ca2+ ion
EF hand
Calmodulin
CaM binding domain

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 Calcium spiking

Pharmacological experiments
• Type-IIA Ca2+ pump and PLC are involved in
Nod factor- calcium process.

• Trimeric G-protein is involved in Nod factor

signaling pathway.
Kinase domain
Activation loop
Ca2+ ion
EF hand
Calmodulin
CaM binding domain

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 Pharmacological analysis

• CPA; inhibitor of plant

type IIA calcium pumps
• U-73122; inhibitor of
phospholipase C
CAP application • CAP and U-73122
blocked ENOD12
expression and root hair
U73122 application
deformation
(Antagonist activity)

(Limpens and Bisseling, 2003)

Engstom et al. (2002)44

Conclusion

Molecular key Phenomenon Plant response

● Nod factors – ● Ion flux and ● Swelling and curling
NFR1 and NFR5 Membrane depolarization of root hair

● DMI1, DMI2 and DMI3 ● Ca2+ spiking ● ENOD gene expression,

PLC and G-protein cortical cell division and
nodule formation

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Symbiosis Island
Transfer

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 Rhizobia

Horizontal transfer; Symbiosis Island

• Discovery of Symbiosis Island Symbiosis signaling

• Symbiosis Island transfer

Symbiotic island transfer

Easy to do engineering

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 Mesorhizobium loti

• M. loti nodulates legumes of the genus

Lotus including L. corniculatus and
L. japonicus

• Symbiosis genes located on

a transmissible symbiosis island

Birdsfoot trefoil

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Discovery of the Symbiosis Island:

Field site established in

1986 with L. corniculatus
seed inoculated with a
single M. loti strain, R7A,
in a tussock grassland
environment lacking
indigenous rhizobia.

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Discovery of the Symbiosis Island:

Seedlings inoculated with R7A Uninoculated seedlings

are healthy 3 months 3 months post-planting are
post-inoculation suffering from lack of nitrogen Site lacks indigenous
rhizobia capable of
nodulating the plant.

Uninoculated seedlings
die from nitrogen
deficiency within a year.

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Sampling 7 years after field site established:

A B

- Diverse strains found in

nodules.

- Strains found to contain

symbiotic DNA identical to
that of R7A.

Genome fingerprints Nod gene probe

Diversity Conservation

A) Pulse-field agarose gel of SpeI digests of NZP2238, R7A and 6 nodule isolates.
B) Southern blots hybridized to R. loti nod cosmid pPN377.

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Sampling 7 years after field site established:

A B
- Diverse strains found in
nodules.

- Strains found to contain

symbiotic DNA identical to
that of R7A.

A) Southern blot of EcoR1 digests of NZP2238, R7A, and 6 nodule isolates hybridized to
a collection of 5 HindIII fragments randomly cloned from NZP2238.
B) Southern blots hybridized to R. loti nod cosmid pPN377.

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Sampling 7 years after field site established:

A B
The diverse strains arose
by transfer of chromosomal
symbiotic genes from M. loti
NZP2238 to nonsymbiotic
rhizobia in the environment.

A) Southern blots hybridized to R. loti nod cosmid pPN377 (NZP2238, R7A, and 5 nodule isolates).

B) Southern blots hybridized to R. loti nod cosmid pPN377

(R. loti strains of diverse geographical origin ).
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Transfer of the Symbiosis Island in the laboratory:

A B
Southern blot analysis
demonstrating transfer of
the symbiotic element to
4 species of nonsymbiotic
mesorhizobia.

Genomic DNA from the donor strain ICMP3153 and pairings of nonsymbiotic and symbiotic strains
was digested with EcoRI and hybridized with;
A) three random genomic HindIII fragments of ICMP3153
B) pRLnodAC containing M. loti nodAC genes 54
The M. loti R7A Symbiosis Island:

• 501,801-bp mobile, chromosomally-

integrated element; encodes ~414 genes
• integrates into a phe-tRNA gene in a
process mediated by a phage-type
integrase.

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 Genomic Island Mobility:

• A large chromosomal DNA fragment.

• G + C content is usually different from the rest of
the host chromosome, suggesting that they originate
from horizontal transfer between different bacterial
genera.
• carries an integrase gene or homologous to the
integrase gene.
• It is integrated into tRNA loci that is a preferential
sites for phage integration.

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Pathogenicity Island

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Nucleotide sequence analysis:

The 501,801-bp island contains genes for:

 Nodulation and nitrogen fixation
 Biotin, thiamin and nicotinate biosynthesis
 Several other metabolic functions including
C4-dicarboxylate transport

 Integration/excision and transfer by

conjugation
 Transfer of protein effector molecules to the
plant cell.
 Many unknown functions

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Symbiotic Island

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 Genomic Island Mobility: Msi109 ; small basic
protein excisionase

• A large chromosomal DNA fragment. 230 bp

• G + C content is usually different from the rest of

the host chromosome, suggesting that they originate
from horizontal transfer between different bacterial
genera.

traF

msi110
Msi106: similarity to
• carries an integrase gene or homologous to the Agrobacterium VirD2
integrase gene. Msi107: similar to lytic
transglycosylases
• It is integrated into tRNA loci that is a preferential
sites for phage integration.

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How does the island transfer?

• P4-phage subfamily integrase gene intS

required for island integration and excision

• Nucleotide sequence also revealed:

– trb operon likely involved in mating pore
formation, and traG gene
– operon potentially involved in DNA
transactions required for conjugation
– vir T4SS genes

Conjugative transposition
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 Conjugative transposition
Excision

ints = P4-phage integrase gene

 for integration & excision
Trb operon involved in mating pore formation
Transfer
Trb operon & traG,F
involve plasmid conjugative transfer
Tra = conjugate the conjugal transfer
Replication rep = vegetative replication
Dtr = DNA transfer and replication
OriT = origin of transfer
Mpf = mating pair formation gene
Integration vir = Virulence gene

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 Model for island transfer

Excision forms a transient circular intermediate. Single strand transferred through

mating pore by rolling circle replication. Second strand synthesis in recipient and
replacement synthesis in donor are followed by (re)integration.
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 Thank you
for your attention

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65
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Conservation of putative island transfer genes: a new island-based Mpr/Dtr
family

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Significant
• Assimilation
Plant SM
PBS BM Bacteroid
N-assimilation / on N-fixation / on
N-assimilation / off

H+ ATPase H+
+ ATP
N-compound NH3 NH3 NTase N2
e-
GLN
NH4+ EXCR NH4+
GS
+
GLT GLT
+
GS
NH4+ AMT AMTB
GLN

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Transfer of the Symbiosis Island in the Field
% occupancy of nodules

100
90 In new field experiment,
80
70 after 4 years only 25% of
60
50
nodules were occupied by
40
30
R7A; 75% of nodules were
20 formed by diverse
10
0 mesorhizobia that had
AUTUMN '97
AUTUMN '95

AUTUMN '96

AUTUMN '98

AUTUMN '99
SPRING '95

SPRING '96

SPRING '97

SPRING '98
received the symbiosis
island from R7A

Strains Present in Nodules

R7A
NEW DIVERSE STRAINS

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 Table 7 Activation of NodD protein by flavonoid compounds
Bacteria NodD Flavones Flavanones Isoflavones Others
5,7,3',4'-Tetra hydroxy,
R.l.eguminosarum . bv.
D 5,7,3'-Trihydroxy, 5,7,4'-Trihydroxy    
trifoli
7,4'- Dihydroxy

5,7,4'-Trihydroxy,
5,7,3',4'-Tetra hydroxy,
R. leguminosarum 5,7,3',4'-Tetra hydroxy,
D 5,7,3'-Trihydroxy,    
bv..viciae 5,7,3'-Trihydroxy-4'-
7,3',4'-Trihydroxy
methoxy

5,7,3',4'-Tetra hydroxy,
4,4'-Dihydroxy-2'-
R. meliloti D1 7,3',4'-Trihydroxy,    
methoxychalcone
5,7,4'-Trihydroxy -3'- methoxy

4,4'-Dihydroxy-2'-
R. meliloti D2      
methoxychalcone
R.leguminosarum
D2 5,7,3'-Trihydroxy 5,7,4'-Trihydroxy 5,7,4'-Trihydroxy  
bv.phaseoli
5,7,3' Trihydroxy,
R.tropici D1 7,4'-Dihydroxy, 5,7,4'-Trihydroxy,    
5,7- Dihydroxy

5,7,4'-Trihydroxy
Br. japonicum D      
7,4'-Dihydroxy

5,7,3',4'-Tetra hydroxy,
5,7,4'-Trihydroxy 5,7,3'-Trihydroxy-
5,7,3'-Trihydroxy, 5,7,4'-Trihydroxy
R.sp NGR234 D 5,7,3'-Trihydroxy-4'- flavonol,
7,4'-Dihydroxy, 7,4'-Dihydroxy
methoxy 5,7,4'-Trihydroxy-flavonol
5,7-Dihydroxy
Az. caulinodans D   7,4'-Dihydroxy    

Rhijn et al (1995)
70
 Table 8 Nod Factors of rhizobia
rhizobia Q R1 R2 R3 R4, 5
R.leguminosarum bv. viciae- C18ข2 4 6 11,
CH3CO H H H
RBL5560 C18ข11

C16ข2 9, H,
R.meliloti AK41 HSO3 H H
C16ข2 4 9 CH3CO

C16ข9,
R.meliloti 2011 C16ข2 9, C16ข2 4 9, H,CH3CO HSO3 H H
(ƒถ-1)-OH C18-26

R.tropici CFN299 C18ข11 H HSO3 CH3 H

B. japonicum USDA110 C18ข9 H 2-O-Me-Fucosyl H H

B. japonicum USDA135 C18ข9, C16 H, CH3CO 2-O-Me-Fucosyl H H

Fucosyl-,
B. elkani USDA61 C18ข9 H, CH3CO H, CH3 H, NH2CO
2-O-Me-Fucosyl

R.fredii USDA234 C18ข11 H Fucosyl, 2-O-Me-Fucosyl H H

2-O-Me-Fucosyl,
R.sp. NGR234 C18ข11,C16 H 2-O-Me-4-O-SO3H-Fucosyl, H H, NH2CO
2-O-Me-3-O-CO-CH3-Fucosyl

H<TDO-AC-
R. etli C18ข1, C18 CH3 NH2CO
FUCOSYL<TD>

R.loti NZP2037 C18ข11, C18 NH2CO 4-O-Ac-Fucosyl CH3 NH2CO

H,
A. caulinodans ORS57 C18ข11, C18 H, NH2CO CH3 H
D-Arabinosyl

Rhijn et al (1995)
71
The symbiosis island Dtr operon and oriT:
Msi109 ; small
230 bp
(89 aa) basic protein
excisionase

msi110
traF
Msi106: weak similarity
to Agrobacterium VirD2
Msi107: similar to lytic
transglycosylases

230-bp region enables transfer of pLAFR1 from R7A to E. coli.

72
Transfer dependent on the island trb operon, traG, msi106,
 The vir T4SS genes of M. loti strain R7A

vir-box 1 vir-box 2
nod-box virB
virA 11 10 9 8 7 6 5 4 32 1 virG msi059 virD4 msi061

ISfr

AH67 AH73AH58 AH60 AH48 AH47 AH50 AH49 AH51

AH54

Protein % amino acid ID to A. tumefaciens Vir proteins

VirA 45
VirG 76
VirB1-11 52, 66 ,82, 82, 57, 69, 54, 76, 68, 59, 82
VirD4 79
Msi061 26 (VirF)
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A proposed model of
Nod factor signaling pathway

74
 DMI2
Nod factor DMI1
Calcium Influx

R
5 PLC
NF
P?
3/ PM
K
LY FR 1
N
Kinase domain

Depolarization Activation loop

LysM motif

Root hair swelling Ca2+ ions

P IP3
LRR domain
Root hair curling
EF hands

Calmodulin
Cortical cell divisions
CaM Calcium spiking CaM binding
domain
ENOD expression

Nodule formation
DMI3

IP3
Type-IIA Ca2+ pump
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Perinuclear region
Rhizobium – Legume Symbiosis

Flavonoid

NodD

Symbiosis

Nod factor

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 Conjugative transposition
Excision

ints = P4-phage integrase gene

 for integration & excision
Trb operon = involved in mating pore formation
Transfer
Trb operon & traG gene involve plasmid conjugativ
Tra = conjugate the conjugal transfer
rep = vegetative replication
Replication Pi = tumor inducing
Dtr = DNA transfer and replication
OriT = origin of transfer
Mpf = mating pair formation gene
Integration
Vir = Virulence

77