Pat Okubara

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Plant Innate Immunity Host Responses to
Necrotrophic Fungi
Pat Okubara
PLP535 January 22, 2008
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Necro- vs. Bio-
Mode of pathogenesis Nutrient acquisition Resist
ance to necrotrophs is rare
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Defense Signaling against Biotrophs
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Defense Signaling against Necrotrophs
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What Is Innate Immunity?

• Partially preformed, partially inducible
• Part of normal plant growth, development
• Triggered by host pathogen elicitors
• Distinct from gene-for-gene resistance
• Not as strong as gene-for-gene resistance
• Durability unknown, but can lose via mutation

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Model Systems for Dissecting Molecular Responses

• Arabidopsis thaliana preferred dicot host
• Small genome size (125 Mb)
• Selfing genetic crosses give lots of progeny
• 3-month generation time
• Genetic stocks harboring gene knock-outs
• Transformable with Agrobacterium
• Detailed genetic map
• Complete, annotated genome sequence
• Numerous model pathogens
• Pseudomonas syringae pvs.
• Magnaporthe oryzae (formerly grisea)
• Phytophthora infestans

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Shortcomings of Model Systems

• Not representative of all pathosystems
• Usually not crop plants (exception rice)
• Avoid problems that need to be addressed in
• crop hosts repetitive DNA, transformability

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Conventions in Molecular Biology

• Gene, mRNA and mutant names italicized
• PROPEP1
• Protein names not italicized
• AtPEP1
• Mutant names historical, indicate phenotype
• ein1 ethylene insensitive1 (first locus
  mutated)
• sid2-2 second mutant allele
• Generally, small letters indicate mutant (ein1)
  if mutation is
• recessive wild type indicated by capital
  letters (Ein1)
• Capital vs. small letters indicate
  genetic dominance and
• recessiveness, resp.

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Jasmonic Acid - Key Molecule in Innate Immunity
Methyl jasmonate
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JA Biosynthesis and fad Mutants
Octadecanoid pathway
Membrane
Fatty acid desaturases
FAD3, FAD7, FAD8
a-Linolenic acid precursor
Lipoxygenase (LOX)
Allene oxide synthase (AOS)
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Involvement of JA in Pythium Immunity
Wild type Arabidopsis vs. fad3, fad7, fad8
Pythium mastophorum
Observations fad triple mutant did not thrive
in the greenhouse. Pythium was isolated from
symptomatic roots.
Vijayan et al. (1998) PNAS 95 7209-7214
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fad Mutant Showed Enhanced Susceptibility
Vijayan et al. (1998) PNAS 95 7209-7214
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fad Mutant Harbored More Oospores
Applied MeJ Partially Reversed the Infection
Vijayan et al. (1998) PNAS 95 7209-7214
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JA Signaling - Key Pathway in Innate Immunity
Fad3 Fad7 Fad8
JA
JAR1
Metabolic protein
COI1
Regulatory protein
Regulatory protein
JAZ1
Thi2.1 PR-4 PDF1.2
Defense (Pr) proteins
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Identification of COI1 As a JA Pathway Regulator
Arabidopsis - Pseudomonas syringae vir
interaction

• Mutant with insensitivity to the toxin
  coronatine
• coi1 coronatine insensitive1

• coi1 phenotype not restored to wild type by
  applied JA

Xie et al. (1998) Science 280 1091-1094
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Thionin2.1-Based JA Signaling Reporter System
Agrobacterium
Loss of GUS expression in coi1
wild type
coi1
GUS conversion of colorless substrate to blue
product
Xie et al. (1998) Science 280 1091-1094
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COI1 Independently Indentified in Other Mutant
Screens

• Enhanced resistance to P. syringae (biotroph)
• Non-expression of Vegetative storage protein
  gene
• (VSP1), a JA-induced gene

• Jasmonate insensitive mutant (jai1) of tomato
  shows similar phenotype(s) as Arabidopsis coi1

• Connection between COI1 and JA action

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Cloning of COI1 Gene

• coi1 mutation between markers C83 and B23

genetic map
600 kb clone

• coi1 mutation on either of 2 ORFs

Clone T-36 (8ks)
Xie et al. (1998) Science 280 1091-1094
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Clone T-36 complemented coi1 phenotype
-- Restored sensitivity (stunting) to JA
Xie et al. (1998) Science 280 1091-1094
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ORF1 (COI1) Restored Expression of Thionin-2.1
Particle gun bombardment using gold beads coated
with clone DNA
GUS expression in bombarded leaves
Xie et al. (1998) Science 280 1091-1094
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Deduced Amino Acid Sequence of COI1

• COI1 encodes an F-box protein
• -- F-box conserved motif - SKP1 binding
  function
• -- leucine-rich repeats (LRR)
  protein-protein interactions

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F-box Proteins Function in the Ubiquitylation
Complex

• Protein degradation
• Altered protein function

Adapted from diagram in Zheng et al. 2002
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COI1 Function Verified Using Biochemical
Approaches

• Soybean COI1 protein binds cullin, ASK1 and
  RBX1 in co-immunoprecipitation experiments
• Binds ASK1 and ASK2 in yeast two-hybrid screen

Wang et al. (2005) Mol Plant-Microbe Interact 18
1285-1295
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Current Hypothesis of JA Signaling
JA-induced gene before COI1-mediated degradation
After COI1-mediated degradation
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(No Transcript)
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Ethylene Another Key Molecule in Innate Immunity
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Ethylene Signaling Pathway
etr1 phenotype?
ein2 phenotype?
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Evidence for Involvement of E Signaling
etr1 ein2
Arabidopsis plants
etr1 ein2 cloned genes
Agrobacterium
Transgenic tobacco expressing etr1 ein2

• Tobacco expressing Arabidopsis etr1 and ein2
  were more susceptible to diseases in greenhouse.
• Geraats et al. (2003) Phytopathology 93
  1078-1085
• etr1 and ein2 tobacco sustained more damage by
  Pythium spp. Geraats et al. (2002) Mol
  Plant-Microbe Interact 15 1078-1085

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ERF1 Overexpression Confers Enhanced Resistance
Wild type Arabidopsis-ERF1 necrotrophic foliar
pathogens
Plectosphaerella cucumerina spore
suspension Symptoms evaluated at 8 dpi
Berrocal-Lobo et al. (2002) Plant J 29 23-32
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ERF1 Overexpression P. cucumerina
Berrocal-Lobo et al. (2002) Plant J 29 23-32
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ERF1 Overexpression Botrytis cinerea
Berrocal-Lobo et al. (2002) Plant J 29 23-32
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ERF1 Overexpression Altered Defense Gene
Expression
Berrocal-Lobo et al. (2002) Plant J 29 23-32
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Innate Immunity Protects against Necrotrophic
Fungi and Wounding Insects

• fad triple mutant sustained more damage by the
  wounding
• insect Bradysia impatiens (fungal gnat).
• McConn et al. (1997) PNAS 95 5473-5477
• Tomato mutant deficient in JA signaling was more
  
• susceptible to the two-spotted spider mite.
• Li et al. (2002) Plant Physiol 130 494-503

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necrotrophic pathogens herbivory, wounding,
elicitors
CEV1
Fad 3,7,8
CTR1
JA E
ABA
JAR1
ETR1
JA-ile
COI1 EIN2
ERF1
JAZ1
EDS8 PAD1
MYC2
Thi2.1 PR-4 PDF1.2
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Defense Signaling against Necrotrophs
Innate immunity constitutive defense
Reduced disease symptoms
Necrotrophs
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Novel Sources of Tolerance to Rhizoctonia solani
AG-8 in Wheat

• EMS mutant of cv. Scarlet K. Kidwell C.
  Steber
• cv. Chinese Spring perennial Chr 4 S. Jones
• ABD synthetic wheat CIMMYT-Arthur Klatt

Resistance is not futile!!
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Small Peptide Triggers of Innate Immunity
Derived from previous work on systemin, a
wound-inducible peptide causing JA-mediated
defense gene induction and anti-feeding activity
in insects. Reviewed in Ryan (2000) Biochim
Biophys Acta 1477 112-121.

• Does Arabidopsis encode peptide elicitors of
  defense?
• What are they?
• How do they act?

Huffaker et al. (2006) PNAS 103 10098-10103
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Biochemical Approach to Identifying Small Peptide
Triggers of Innate Immunity
whole Arabidopsis plants
ground to fine powder
HPLC
collected fractions
screened for ability to cause alkalinization (inte
ract with cell surface receptors)
Huffaker et al. (2006) PNAS 103 10098-10103
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AtPep1 A Small Peptide Candidate
AtPep1
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Obtaining the AtPep1 Gene from Its Peptide
Sequence
N-term
C-term
Searched NCBI Arabidopsis Genome Database
tblastn
Hypothetical gene At5g64900 PROPEP
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PROPEP1 Expression Signaling Is JA- and
E-Dependent
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PROPEP Gene Family and Orthologs in Other Plants
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PROPEP1 Expression Is Induced by Pathogens
1Determined using RT-PCR
PROPEP1 also induced by elicitors (PAMPs).
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Thurs. Discussion Paper Chuntao Yin Huffaker
and Ryan (2007) Endogenous peptide defense
signals in Arabidopsis differentially amplify
signaling for the innate immune response. Proc
Natl Acad Sci 104 10732-10736.
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For Thurs. Discussion Paper

• sid2-2 mutation in isochorismate mutase gene
  for
• salicylic acid biosynthesis
• npr1 mutation in SA-dependent signal regulator
  gene
• What data support the authors hypothesis and
  what
• anomalies did they observe?

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pokubara_at_wsu.edu 335-7824 333 Johnson Hall