1. Simone Ferrari Lab meeting 12/19/00
Local and systemic resistance to Botrytis cinerea in Arabidopsis thaliana mutants
Simone Ferrari
Lab meeting 12/19/00

2. 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

3. B. cinerea infection
germination
penetration
lesion initiation
(HR-like necrosis)
lesion spreading
(maceration)

4. Col-0

5. 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

6. ein2-1

7. 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?

8. 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

9. 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

10. Ethylene transduction and Botrytis resistance?
EIN5
EIN6
EIN7 ? ? ?
Botrytis resistance
Chang and Shockey, 1999

13. 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

14. 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)

15. Local resistance to Botrytis
Mutations that reduce growth rate
Mutations that increase growth rate
Factors involved in secondary lesion spreading

16. 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?)

17. Col-0

18. edr5-1

19. edr5-1

22. edr5 resistance to B. cinerea
Col-0
detached 25°C in light - 3 dpi

23. B. cinerea infection at 4ºC in darkness
Col-0
edr5-1
edr1-1
Mock
(10 dpi)
Infected
(10 dpi)

27. 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

28. 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)

29. Role of SA and camalexin
Necrotic lesions are initiated through HR-like localized cell death (Govrin EM and Levine A. Curr Biol 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

30. PR1 induction by Botrytis is localized at the lesion site
PR1-GUS (3dpi) L S
Col-0 - +
PR1
UBQ5
ein2-1

32. 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

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

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

37. 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