A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis From: 2007 Jan SCIENCE Vol315 5
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 • 10dai J. Murray, 07
hit1-1 har1-1 • 14dai
hit1-1 J. Murray, 07
hit Cortex Epidermis • Lots IT, but less or no nodule organogenesis. J. Murray, 07
Role of HIT1 in NP organogenesis • ENOD40(cortical cell divison, mRNA) • Nin(IT formation, initiation of primordia)
Role of HIT1 in NP organogenesis inoculate J. Murray, 07
Role of HIT1 in NP organogenesis J. Murray, 07
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 organogenesis J. Murray, 07
Jens Stougaard’s group - L. japonicus Spontaneous nodule formation (Lotus hitidine kinase - Lhk)
har + AR hit har + AR hit har + Lhk1
Expression of Lhk1 in various L. japonicus tissues as assayed by RT-PCR.
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. cells were transformed with either p415CYC carrying the Lhk1 cDNA, the hit1-2 cDNA or the vector alone
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
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
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
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
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 5 week time 0 6 day 3 week wt snf2 On hormone free medium
Phenotypic characterization of the snf2 mutant auxin cytokinin
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?
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 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 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
signal Nod-factor snf2 nod formation • The phenotype of snf2 mutants suggests that cytokinin signaling acts downstream of Nod-factor signal transduction
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
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
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
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
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 • cytokinin signaling plays an important role in plant meristem formation and is directly involved in initiating root nodule organogenesis