A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis
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A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis. From: 2007 Jan SCIENCE Vol315 5. Now We Known. Sample. Screen Lotus japonicus har1-1 hypernodulation. Hyperinfected 1(HIT1) low nodulation, excessive formation of IT.

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A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis

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A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis

From: 2007 Jan SCIENCE Vol315 5


Now we known

Now We Known


Sample

Sample

  • Screen Lotus japonicus har1-1 hypernodulation.

  • Hyperinfected 1(HIT1)

    low nodulation, excessive formation of IT.

  • (hit1-1 har1-1), (hit1-1)

  • Mesorhizobiumloti carry reporter gene.


Hit1 1 har1 1

hit1-1 har1-1

  • 10dai

J. Murray, 07


Hit1 1 har1 11

hit1-1 har1-1

  • 14dai


Hit1 1

hit1-1

J. Murray, 07


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

hit

Cortex Epidermis

  • Lots IT, but less or no nodule organogenesis.

J. Murray, 07


Role of hit1 in np organogenesis

Role of HIT1 in NP organogenesis

  • ENOD40(cortical cell divison, mRNA)

  • Nin(IT formation, initiation of primordia)


Role of hit1 in np organogenesis1

Role of HIT1 in NP organogenesis

inoculate

J. Murray, 07


Role of hit1 in np organogenesis2

Role of HIT1 in NP organogenesis

J. Murray, 07


Role of hit1 in np organogenesis3

Role of HIT1 in NP organogenesis

  • L. japonicus root hairless( Ljrhl1-1 )

    - lots uncolonized NPs.

    - no root hairs and ITs.

  • ( Ljrhl1-1 ) and (hit1-1) double mutant.


Role of hit1 in np organogenesis4

Role of HIT1 in NP organogenesis

J. Murray, 07


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • Jens Stougaard’s group

    - L. japonicus Spontaneous nodule formation (Lotus hitidine kinase - Lhk)


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

har + AR

hit har + AR

hit har + Lhk1


Expression of lhk1 in various l japonicus tissues as assayed by rt pcr

Expression of Lhk1 in various L. japonicus tissues as assayed by RT-PCR.


Phylogenetic analysis of lhk1 and lhk1 like proteins

Phylogenetic analysis of LHK1 and LHK1-like proteins.

Analysis of the full-length Lhk1 cDNA revealed

a 2979–base pair open reading frame

encoding a predicted protein of 993 amino acids

The LHK1 protein had 64% identity

with the Arabidopsis cytokinin histidine kinase

receptor AHK4, and 49 and 45% identity with

AHK2 and AHK3, respectively. Similar to

LHK1, LHK2 was more closely related to

AHK4 than to other Arabidopsis cytokinin

receptors


Lhk1 cdna confers cytokinin responsiveness to yeast cells

Lhk1 cDNA confers cytokinin responsiveness to yeast cells.

cells were transformed with either p415CYC carrying the Lhk1 cDNA, the hit1-2 cDNA or the vector alone


Hit1 1 roots are insensitive to exogenously applied cytokinin

hit1-1 roots are insensitive to exogenously applied cytokinin.


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • We further investigated a role for the HIT1

    locus in NP organogenesis by studying Early

    Nodulin 40 (ENOD40) and Nin expression,

    two markers for NP initiation


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

50 nM benzyl adenine (BA) increased the steady-state level of Nin transcript

qPCR showing significant attenuation of Nin mRNA in hit1-1 versus wild-type roots upon exogenous application of 50nM BA. BA-stimulated accumulation of Nin transcripts in hit1 roots was significantly higher in comparison to water treatments


Nodule organogenesis

Nodule organogenesis


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • The LHK1 function as cytokinin receptors

  • hit1-1 roots are insensitive to exogenously applied cytokinin

  • 50 nM benzyl adenine (BA) increased the steady-state level of Nin transcript

  • Nin mRNA in hit1-1 less then in wild-type


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

The LHK1 function as cytokinin receptors, which may explain a leaky (formation of some nodules) symbiotic phenotype and lack of more general developmental abnormalities in mutants carrying hit1 alleles.

Nin expression, supporting IT formation, may be regulated by an Lhk1-independent mechanism in the root epidermis, possibly involving another cytokinin receptor.

cytokinin biosynthesis and/or action are crucial downstream targets of NF perception

recruitment of a cytokinin receptor could have been an essential event during the evolution of nitrogen-fixing nodule symbiosis


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

祥霖


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • Arabidopsis ahk2 ahk3 ahk4 triple mutants display a reduced number of root vascular cell files→ impaired periclinal procambial cell divisions

  • snf2 mutant?


Phenotypic characterization of the snf2 mutant

Phenotypic characterization of the snf2 mutant

5 week

time 0

6 day

3 week

wt

snf2

On hormone free medium


Phenotypic characterization of the snf2 mutant1

Phenotypic characterization of the snf2 mutant

auxin

cytokinin


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • ARR(type-A response regulator) gene was Cytokinin-induced cellular processes in plants

  • ARR5 promoter-GUS was expressed during nodulation

  • L266F receptor protein→cytokinin- independent activity in E. coli

  • Lotus Lrr5?


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

Expression of Lrr5, Lhk1, and Nin in intact wild-type and snf2 roots


Expression of lrr5 in wild type and snf2 root explants

Expression of Lrr5 in wild-type and snf2 root explants

Medium with or without 0.5 mg/ml of BAP(cytokinin) for 10 days


Expression of nin in wild type and snf2 root explants

Expression of Nin in wild-type and snf2 rootexplants

Medium with or without 0.5 mg/ml of BAP(cytokinin) for 10 days


Root length of wt and snf2 plants in the presence of increasing concentration of bap

Root length of wt and snf2 plants in the presence of increasing concentration of BAP

BAP: 0 3 6 10 ng/ml

Wt/snf2

  • snf2 mutants growth were strongly affected by external cytokinin

BAP: 30 50 100 500 ng/ml


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

signal

Nod-factor

snf2

nod formation

  • The phenotype of snf2 mutants suggests that cytokinin signaling acts downstream of Nod-factor signal transduction


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

symRK

snf2

nod formation

ccamk

  • Signal transduction:

  • symRK mutants lacking Ca2+ spiking

  • ccamk (Ca2+- and calmodulin-dependent protein kinase)

  • develop spontaneous nodules in snf2 transgenic roots


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • nfr1, nfr5, symrk, ccamk mutants→unable to form nodules in response to rhizobia inoculation

  • nin, nsp2 mutants→arrested before initiation of cell division induced by Nod-factor signaling, no spontaneous nodules were observed in snf2 transgenic roots

symRK

nin

snf2

nod formation

nsp2

ccamk

Cytokinin signal perception acts upstream of cell division initiation


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • snf1-1 mutants(CCaMK protein impaired in autophosphorylation)→ 7±0.9

  • snf2 mutants→3±0.5

  • snf1-1+snf2→17±0.9

  • Parallel signaling

  • Deregulated signaling in snf1 results in a local increase in cytokinin levels transcriptionally up-regulating snf2


A cytokinin perception mutant colonized by rhizobium in the absence of nodule organogenesis

  • har1-1(hypernodulating)→no nodule

  • snf2 mutant→3±0.5

  • snf2+har1-1 double mutants→14±1.4

  • Only a few cells dedifferentiate

  • Only a few dedifferentiated cells sustain cell divisions during the snf2 nodule-initiation process


Model for functional role of lhk1 in nodulation

Model for functional role of Lhk1 in nodulation


Discuss

Discuss

  • Applications of auxin transport inhibitors resulted in empty nodule-like structures, which suggested that local inhibition of auxin transport sensitizes cells for division

  • Externally supplied cytokinin induces cortical cell division and activation genes, and expression of cytokinin biosynthesis gene in a nodulation-deficient Sinorhizobium meliloti resulted in nodule-like structures


Conclude

Conclude

  • cytokinin signaling plays an important role in plant meristem formation and is directly involved in initiating root nodule organogenesis


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