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

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 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

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 Col-0 detached leaves @ 25°C in light - 3 dpi

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

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 HR-like 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) L S Col-0 - + PR1 UBQ5 ein2-1

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 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? C2H4 JA SA ROS NahG PAD3 EIN2 COI1 catechol camalexin NPR1 ? ? EIN3 EIL1? LOCAL RESISTANCE 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