Local and systemic resistance to Botrytis cinerea in Arabidopsis thaliana mutants

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Local and systemic resistance to Botrytis cinerea
in Arabidopsis thaliana mutants

• Simone Ferrari
• Lab meeting 12/19/00

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Botrytis cinerea

• Necrotrophic fungal pathogen
• Causal agent of gray mold
• Over 200 species infected
• Disease down to 2ºC (post-harvest problem)
• Weak pathogen on healthy tissue

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B. cinerea infection
germination
penetration
lesion initiation (HR-like necrosis)
lesion spreading (maceration)
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Col-0
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Botrytis cinerea resistance in defense-related
arabidopsis mutants

• ein2 and coi1 are highly susceptible (systemic
  infection)
• npr1 and NahG transgenic plants show no systemic
  infection
• pad1 and pad3 show no systemic infection

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ein2-1
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Questions

• What is the role of SA, ethylene and jasmonic
  acid in local vs systemic infection?
• What is the effect of mutations affecting
  Erysiphe resistance?
• What defense responses are important for Botrytis
  resistance?

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Analysis of known mutants systemic infection

• enhanced desease susceptibility eds1, eds4,
  eds5, eds9, eds16
• SA-defective/insensitive NahG, npr1, eds5 npr1,
  pad4
• constitutive PR1 cpr1, cpr5, cpr6
• camalexin-defective pad1, pad2, pad3
• ethylene insensitive etr1, ein2, ein3, eil1,
  ein3 eil1, ein5, ein6
• other mutants jar1, acd2, edr1, edr5

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Analysis of known mutants systemic infection

• enhanced desease susceptibility eds1, eds4,
  eds5, eds9, eds16
• SA-defective/insensitive NahG, npr1, eds5 npr1,
  pad4
• constitutive PR1 cpr1, cpr5, cpr6
• camalexin-defective pad1, pad2, pad3
• ethylene insensitive etr1, ein2, ein3, eil1,
  ein3 eil1, ein5, ein6
• other mutants jar1, acd2, edr1, edr5

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Ethylene transduction and Botrytis resistance
?
EIN5 EIN6 EIN7
?
?
?
Botrytis resistance
Chang and Shockey, 1999
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Ethylene mutants - summary

• EIN3, but not EIL1, is required for full systemic
  resistance
• EIL1 may partially compensate for loss of EIN3
  function
• EIN5, and maybe EIN6, are not required for
  systemic resistance

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Ethylene mutants in progress

• Resistance of ERF1 overexpression lines
• Resistance in etr1 alleles role of other
  receptors (ein4 loss of function mutants?)
• Search for suppressors of ein2 (activation
  tagging mutagenesis)

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Local resistance to Botrytis

• Mutations that reduce growth rate
• Mutations that increase growth rate
• Factors involved in secondary lesion spreading

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enhanced disease resistance mutants

• edr1 and edr5
• isolated for virulent P.syringae resistance
• enhanced resistance to Erysiphe orontii
• no constitutive PR gene expression
• PR1,BGL2, PR5 induction is more rapid
• HR-like lesions (spontaneous or induced?)

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Col-0
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edr5-1
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edr5-1
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edr5 resistance to B. cinerea
edr5
Col-0
detached leaves _at_ 25C in light - 3 dpi
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B. cinerea infection at 4ºC in darkness
Col-0
edr5-1
edr1-1
Mock (10 dpi)
Infected (10 dpi)
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edr5 summary

• edr5 is more resistant to different types of
  pathogens (virulent bacteria, biotrophic and
  necrotrophic fungi)
• B.cinerea can initiate necrotic lesions in edr5,
  but fungal growth is restricted, both in light
  and darkness
• edr5 seedlings are as susceptible as the wild-type

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edr5 in progress

• Kinetic of defense responses after B. cinerea
  infection (PR proteins, defensins)
• Generation of ein2 edr5 double mutant
  (epistatic?)
• Cloning of EDR5 (cosmid library complementation)

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Role of SA and camalexin

• Necrotic lesions are initiated through HR-like
  localized cell death (Govrin EM and Levine A.
  Curr Biol. 2000 Jun 2910(13)751-7).
• B. cinerea cannot efficiently grow on established
  HR lesions
• The rate of colonization must depend on the
  balance between localized cell death and
  induction of defense responses

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PR1 induction by Botrytis is localized at the
lesion site
PR1-GUS (3dpi)
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camalexin mutants

• pad1 30 camalexin induction with P.syringae
  pleiotropic susceptible to virulent Psm
• pad2 10 susceptible to virulent Psm
• pad3 undetectable (cyt P450) with both Psm and
  A.alternata susceptible to A. alternata
• pad4 10, low SA susceptible to virulent Psm
  and E. orontii

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B. cinerea and A. brassicicola induce different
responses in Arabiopsis

• B. cinerea
• Tissue maceration
• Localized PR1 induction
• COI1 and EIN2 are required for systemic
  resistance
• SA and camalexin seem required for local
  resistance

• A. brassicicola
• Small necrotic lesions
• Local and systemic PR1 induction
• COI1 and camalexin, but not EIN2 and SA, are
  required for local resistance

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Defense genes induction in arabidopsis by
different fungal pathogens
B. cinerea
EDS16? PAD4
ETR1?
JA
C2H4
ROS
SA
NahG
PAD3
COI1
EIN2
camalexin
catechol
NPR1
?
?
EIN3 EIL1?
LOCAL RESISTANCE
SYSTEMIC RESISTANCE
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Future plans

• Compare lesion size and gene expression in wt and
  mutants at RT and 4C
• Test more eds mutants for systemic resistance
• Determine lesion formation in coi1, eds16, cpr
  mutants, edr5 NahG plants, ethylene mutants