Plant MAPK

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BIO 567 Signal Transduction
Plant MAPK cascadescomplexity diversity
by Burcu Kaplan
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Mammalian MAPK cascades
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MAPK cascades

• MAPK,the last kinase in the cascade, is activated
  by dual phosphorylation of the Thr and Tyr
  residues in a tripeptide motif (ThrXaaTyr,
  where Xaa could be Glu, Gly, Pro or Asp) located
  in the activation loop (T-loop) between
  subdomains VII and VIII of the kinase catalytic
  domain.
• This phosphorylation is mediated by a MAPK kinase
  (MAPKK or MEK),
• which, in turn, is activated by a MAPKK
  kinase (MAPKKK or MEKK) through phosphorylation
  of conserved serine and/or threonine residues in
  their T-loop

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TRENDS in Plant Science Vol.7 No.7 July 2002
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Convergence of various stress stimuli onto MAPK
pathways.
Trends Plant Sci 2001. 6, 520527
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A. Thaliana MAPK modules
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Plant MAPKs

• Phosphorylation site
• TEY (A,B,C)
• TDY (D)
• CD domain, putative MAPKK docking site at
  C-terminus
• L/H-L/H/Y-D-X-X-D/E-X-X-D/E-E-P-X-C
• MHK(Mak-homologous kinase) lacks the CD domain
• No biochemical evidence that they are MAPKs
• Comparable sequence similarity to CDC2-like
  kinases

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http//genetics.mgh.harvard.edu/sheenweb/mapk_proj
ect.htmlmapk_illustrations
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Plant MAPKs

• Group A
• WIPK
• SAMK Subfamily I
• AtMPK3
• SIPK
  stress responses
• SIMK Subfamily II
• AtMPK6
• Group B
• MMK2
• MMK3 Subfamily III
  stress responses
• AtMPK4
  cell cycle
• Ntf6

Red Arabidopsis thaliana Blue
Nicotiana tabacum ( tobacco) Orange Medicago
sativa (alfalfa)
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Transcriptional activation of genes for plant
MAPKs by stress

• Some of the early evidence for the involvement of
  plant MAPKs in stress signaling was the
  transcriptional activation of gene WIPK by
  wounding.
• Later, WIPK transcript was shown to be induced by
  various elicitors and pathogens.
• Other members in the same subfamily are also
  induced at the mRNA level such as
• SAMK
• MPK3
• ERMK from parsley (Petroselinum crispum)
• TaWCK1 from wheat
• These results suggest that stress induced
  transcription of genes for MAPKs evolved early in
  plants, before the divergence of dicots and
  monocots, and might play an important role in
  plant defense responses.

Trends Plant Sci. 2001 6, 520527
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Biochemical evidence of stress-induced MAPK
activation
member-specific antibodies. immunecomplex
kinase assay

• Wounding, high salinity and high or low
  osmolarity, as well as pathogen-derived elicitors
  that do not induce hypersensitive response
  (HR)-like cell death, transiently activate
  SIPK/SIMK/MPK6 and sometimes also WIPK/SAMK/MPK3.
• By contrast, long-lasting activation of SIPK and
  delayed activation of WIPK are associated with HR
  cell death in resistant tobacco (NN) plants
  challenged by tobacco mosaic virus (TMV) or
  cultured cells treated with fungal elicitins.
• Ozone-induced cell death is also accompanied by
  the activation of two MAPKs one of which was
  identified as SIPK.
• Recently, MMK2 and MMK3 from alfalfa, and MPK4
  from Arabidopsis, were shown to be activated by
  fungal elicitors and several abiotic stresses.

Trends in Plant Science 2001 6 (11) 520-527
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http//genetics.mgh.harvard.edu/sheenweb/mapk_proj
ect.htmlmapk_illustrations
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MAPKK

• Putative MAPK docking motif at N-terminus
• K/R-K/R-K/R-X1-5-L/I-X-L/V/I
• Phosphorylation site
• S/TXXXXXS/T
• Group A
• AtMKK1abiotic stress, leaf
• PRKK pathogen response
• NtMEK1cell division
• Group C
• AtMMK4 pathogen response , leaf
• AtMMK5 pathogen response , leaf
• SIMKK salinity stress pathogen response
• NtMEK2 pathogen response

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http//genetics.mgh.harvard.edu/sheenweb/mapk_proj
ect.htmlmapk_illustrations
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MEKK4

• MEKK2-4 were identified by low stringency
  hybridization ,probe MEKK1
• MEKK4 has several N-terminal functional domains
• WRKY domain---direct DNA binding?
• WRKYs are plant specific zinc-finger
  tfs.activated during plant defense responses.

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MAPKKK

• Similar to animal MEKKs yeast MAPKKKs
• AtMEKK1abiotic biotic (??) , leaf
• AtMEKK4a disease resitance gene a MAPKKK?
• NPK1 cytokinesis, oxidative stress,
  auxin-response
• ANP1-3 cytokinesis, oxidative stress,
  auxin-response , leaf
• Raf-like protein kinases, extended N-terminal
  domains
• CTR1ethylene signalling , leaf
• EDR1disease resistance signalling , leaf

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Function of MAPKs cascades

• Pathogen Response
• Negative regulation of plant defense responses
• Plant defense gene activation(flg22 cascade)
• HR-like cell death
• Osmotic Stress
• Cytokinesis
• Hormone Signalling
• Oxidative Stress
• Abiotic Stresses

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Pathogen response

• ROS
• Strengthening of cell walls
• HR-response
• Increased SA levels
• PR-proteins
• Expression of JA-dependent genes

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MAPKs Pathogen Response
Tobacco
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MPK4
EDR1
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flg22 cascade

• Flagellin bacterial elicitor
• FLS2
• extracellular LRR domain binding to flagellin
• Kinase activity of the intracellular
  serine/threonine kinase domain signal
  transduction
• RESULTS
• Flg22 induces WRKY29 as an early defence
  response( RT-PCR)
• Flg22 can not induce WRKY29 in fsl2 mutant.
  (luciferase reporter)
• U0126 MPK1 reduces WRKY activation via flg22.
  (luciferase reporter)
• Transient protoplast expression assays of a
  variety of MAPKs, MAPKKs, and MAPKKKs to examine
  the roles of MAPK signalling components in the
  defence response.
• AtMPK3 and AtMPK6 are activated via flg22 ( in
  gel kinase assay, MBP), not in fls2 mutant.
• AtMPK3 and AtMPK6 are downstream of AtMKK4 and
  AtMKK5. ( in gel kinase assay, MBP)
• c.a AtMKK4 and AtMKK5 activate WRKY29, also in
  fls2 mutant . ( luciferase reporter)
• c.a AtMEKK1 activates AtMKK5 ( in gel kinase
  assay, MBP), WRKY29 ( luciferase reporter) also
  in fls2 mutant AtMEKK1 is downstream of FLS2

Nature 2002 415 977-983
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• The targets of the MAPK pathway are suggested to
  be two plant-specific transcription factors of
  the WRKY family (i.e. WRKY22 and WRKY29), because
  they are flg22-activated encode a WRKY tf.
• How the receptor kinase activates AtMEKK1?
• How AtMPK3 and AtMPK6 are connected to the WRKY22
  and WRKY29 transcription factors?
• There is no invo evidence of the cascade!
• But there is in vivo evidence for individual
  components
• Agrobacterium-mediated transient overexpression
  of truncated AtMEKK1, constitutively active
  AtMKK4 and AtMKK5, or WRKY29 conferred resistance
  of Arabidopsis leaves to infection by the
  bacterial pathogen Pseudomonas syringae or the
  fungal pathogen Botrytis cinerea.

Nature 2002 415 977-983
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Nature 2002 415 977-983
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MPK4

• An Arabidopsis line that carried a modified maize
  Ds transposon element in the AtMPK4 gene was
  identified.
• The mpk4 mutant line has a dwarf phenotype,
  showed enhanced resistance to virulent bacterial
  (P. syringae) and fungal (P. parasitica)
  pathogens, exhibited constitutive SAR, elevated
  SA levels and defence-related gene expression,
  but lacked jasmonic acid-dependent gene
  induction.
• These responses were dependent on MPK4 kinase
  activity, as a kinase inactive mutant allele
  failed to complement the mpk4 mutation.
• Thus, the potential kinase cascade utilising MPK4
  apparently has a negative regulatory role in the
  plant defence.

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MPK4 abiotic stress
Touch, cold, salinity,drought
AtMEKK1
YIIH Y.C
Pathogen response
AtMEK1
P
AtMPK4
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EDR1

• Arabidopsis edr1 mutant, isolated from a genetic
  screen, displayed enhanced resistance against the
  usually virulent bacterial strain P. syringae and
  the fungal powdery mildew pathogen Erysiphe
  cichoracearum.
• EDR1 codes for a MAPKK kinase. The recessive
  nature of the mutation suggests that EDR1, like
  MPK4, may function at the top of a MAP kinase
  cascade that negatively regulates defence
  responses.
• Plant mitogen-activated protein kinase
  cascadesNegative regulatory roles turn out
  positive PNAS 2001 vol. 98 (3)

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Tobacco

• Various pathogenic signals activate WIPK and SIPK
  in tobacco.
• High levels of WIPK and SIPK activity also result
  from wounding and various abiotic stresses.
• Overexpression of constitutively active NtMEK2 in
  tobacco activates a subset of defence genes and
  induces a hypersensitive response (HR)-like cell
  death, which is typical of the pathogen defence
  response. The HR is preceded by the activation of
  endogenous SIPK and WIPK.

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SA JA

• WIPK silencing
• Increased SA prodn
• Abrogation of JA induced gene expression upon
  wounding.
• WIPK overexpression
• Increased JA levels
• Constitutive exp. of JA-responsive genes.

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MAPKs Osmotic Stress
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MAPKs Cytokinesis
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MAPK cascade

• Ntf6 MMK3
• localization to the cell plate
• Activation during cytokinesis
• NtMEK1
• Yeast two hybrid screen, bait Ntf6
• NPK1
• Activates NtMEK1
• Localized to the cell-division plane
• Activated during cytokinesis
• N-terminus kinase domain
• C-terminus - regulatory domain, interaction with
  kinesin microtubule motor proteins NACK1/2
• NACK1/2
• Dominant negative mutant forms leads to
  incomplete cytokinesis

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MAPKs Hormone Signalling

• ctr1 cons. exp. of ethylene inducible genes
• ACC treatmentMMK3, SIMK AtMPK6
• Protoplast transient exp
• SIMKK activates SIMK MMK3 , ACC inhibits
  activation.

TRENDS in Plant Science 2003 Vol.8 (8) 365-368
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Ethylene signalling molecule involved in
plant defence
Current Opinion in Plant Biology 2004 vol 7 4049
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Auxin

• Auxin activates a MAPK-like kinase in Arabidopsis
  roots, and that auxin- but not salt-induced
  MAPK-like activation was inhibited in
  auxin-resistant4 (axr4) mutants.
• On the other hand, there is good evidence that
  an H2O2-induced MAPK pathway blocks
  auxin-responsive genes while inducing genes that
  respond to oxidative stress.
• These data indicate that there is crosstalk
  between the oxidative-stress and auxin pathways.
• H2O2 is a signalling molecule that is implicated
  not only in various cellular processes, such as
  the response to wounding and pathogen defence,
  but also in the regulation of the cell cycle and
  in cell death.
• The H2O2-induced MAPKs were identified as AtMPK3
  and AtMPK6, and only truncated Arabidopsis ANPs
  and tobacco NPK1 could induce these MAPKs.
• Truncated NPK1 is known to repress several auxin
  inducible promoters and to result in defective
  embryo and seed development.

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MAPK Oxidative-stress
Stress wounding Pathogens Cell cycle death
NACK12
H2O2
NPK1
ANPs
Cell plate formation
Biotic stress
?
Abiotic stress

• Regulation
• of auxin

MPK3

Ethylene Prodn
MPK6
Regulation of H2O2
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Antagonistic MAPK signaling pathways.
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Conclusion

• Still no in vivo MAPK module is defined.
• Methods used in identification of MAPKs /
  cascades are not fully reliable
• Sequence similarity
• Yeast 2-hybrid screens
• SIPKK was identified by a yeast two-hybrid
  screening using SIPK as bait, but SIPKK cannot
  phosphorylate SIPK.
• Yeast complementation assays
• Mutant studies
• Transient protoplast assays

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Transient protoplast transformation

• Transient transformation of cell or leaf
  protoplasts has been established enabling the
  expression of epitope tagged kinase variants with
  a reporter construct to study their effect on
  transcription. This elegant system allows the
  cell-based reconstitution of entire signalling
  cascades
• The generation of protoplasts from leaves or cell
  cultures interferes with some gene-for-gene
  interaction systems, and defence responses are
  already activated due to protoplasting and/or
  protoplasts do not respond to elicitation.

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• Before we can reconcile all the data generated at
  different levels of a MAPK pathway, it is crucial
  to identify the three kinases in a cascade and
  the substrates of the MAPK based on in vivo
  evidence.
• The loss of a functional gene product might not
  reveal the exact function of a MAPK cascade
• MAPKs are regulated post-translationally by
  phosphorylation
• Their function depends on the kinetics and
  amplitude of their activation
• The function of a MAPK cascade in cells is
  frequently pleiotropic,
• The disruption of a MAPK gene will generate
  nonspecific effects.
• The constitutive activation of a MAPK might also
  result in nonspecific effects

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