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Local and systemic resistance to
Botrytis cinerea in Arabidopsis
thaliana mutants
Simone Ferrari
Lab meeting 12/19/00
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
B. cinerea infection
germination
penetration
lesion initiation
(HR-like necrosis)
lesion spreading
(maceration)
Col-0
Botrytis cinerea resistance in defenserelated 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
ein2-1
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?
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
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
Ethylene transduction and Botrytis resistance
?
EIN5
EIN6
EIN7
?
?
Botrytis resistance
Chang and Shockey, 1999
?
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
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)
Local resistance to Botrytis
• Mutations that reduce growth rate
• Mutations that increase growth rate
• Factors involved in secondary lesion
spreading
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?)
Col-0
edr5-1
edr5-1
edr5 resistance to B. cinerea
edr5
Col-0
detached leaves @ 25°C in light - 3 dpi
B. cinerea infection at 4ºC in darkness
Col-0
Mock
(10 dpi)
Infected
(10 dpi)
edr5-1
edr1-1
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
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)
Role of SA and camalexin
• Necrotic lesions are initiated through HRlike localized cell death (Govrin EM and Levine A.
Curr Biol. 2000 Jun 29;10(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
PR1 induction by Botrytis is localized
at the lesion site
PR1-GUS (3dpi)
-
Col-0
+
-
L S L S
PR1
UBQ5
+
L S L S
ein2-1
+
L S L S
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
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
Defense genes induction in arabidopsis
by different fungal pathogens
B. cinerea
EDS16?
PAD4
ETR1?
ROS
SA
C2H4
JA
EIN2
COI1
NahG
PAD3
camalexin
catechol
NPR1
?
LOCAL RESISTANCE
?
EIN3
EIL1?
SYSTEMIC RESISTANCE
Future plans
• Compare lesion size and gene expression
in wt and mutants at RT and 4°C
• Test more eds mutants for systemic
resistance
• Determine lesion formation in coi1, eds16,
cpr mutants, edr5 NahG plants, ethylene
mutants