SAR Signaling - MGH Molecular Biology - Genetics Webserver
Transcript of SAR Signaling - MGH Molecular Biology - Genetics Webserver
Plant Defense: A GlimpsePlant Defense: A Glimpse
By By
Wisuwat SongnuanWisuwat Songnuan
OutlineOutline
BackgroundBackground Systemic Acquired ResistanceSystemic Acquired Resistance NPR1-TGAsNPR1-TGAs That’s not all…That’s not all… FutureFuture
BackgroundBackground
Background OutlineBackground Outline
Why study plant resistance?Why study plant resistance?
Pathogen RecognitionPathogen Recognition
Gene-for-gene interactionsGene-for-gene interactions
Hypersensitive Response (HR)Hypersensitive Response (HR)
Systemic Acquired Resistance (SAR)Systemic Acquired Resistance (SAR)
Why study plant resistance?Why study plant resistance?
80% of total calories consumed by human 80% of total calories consumed by human population come from only six crops: wheat, population come from only six crops: wheat, rice, maize, potatoes, sweet potatoes, and rice, maize, potatoes, sweet potatoes, and manioc (Raven, P.H. et al, 1999). manioc (Raven, P.H. et al, 1999).
We lose 12% of total crop yields to pathogen We lose 12% of total crop yields to pathogen infection– equivalent to nine hundred million infection– equivalent to nine hundred million tons worldwide annually (Krimsky S. and tons worldwide annually (Krimsky S. and Wrubel R., 1996). Wrubel R., 1996).
Plants under attackPlants under attack
Microorganisms: viruses, bacteria, fungiMicroorganisms: viruses, bacteria, fungi NematodesNematodes Insects & a few othersInsects & a few others Us?Us?
What will YOU do?What will YOU do?
Lots of enemies, attacking from all sidesLots of enemies, attacking from all sides Huge bodyHuge body Cannot escapeCannot escape No “patrol”No “patrol”
(no NIH grant)(no NIH grant)
How THEY do itHow THEY do it
Right after plants are dead, they are rottenRight after plants are dead, they are rotten No wasting energy for ‘just in case’ immunityNo wasting energy for ‘just in case’ immunity All through “signaling” All through “signaling”
Pathogen recognitionPathogen recognition
Gene-for-gene hypothesisGene-for-gene hypothesis: Upon infection by a : Upon infection by a particular avirulent pathogen, a corresponding particular avirulent pathogen, a corresponding R gene recognizes the R gene recognizes the avravr product and triggers product and triggers the defense mechanism.the defense mechanism. Why do pathogens still possess Why do pathogens still possess avravr genes? genes?
Non-host resistanceNon-host resistance: Resistance of all : Resistance of all members of a host species against all members members of a host species against all members of pathogen speciesof pathogen species
Resistance (R) GenesResistance (R) Genes
DominantDominant Many ID so farMany ID so far 5 classes recognized5 classes recognized
NBS: Nucleotide binding siteNBS: Nucleotide binding site Leucine-zipper and leucine-rich repeat (LRR)Leucine-zipper and leucine-rich repeat (LRR) Toll/IL-1R (TIR)Toll/IL-1R (TIR) Protein kinase (PK), receptor-like kinase (RLKs)Protein kinase (PK), receptor-like kinase (RLKs)
The popular ones…The popular ones…
Maize Hm1 (1992): toxin reductaseMaize Hm1 (1992): toxin reductase Tomato Pto (1993): Ser/Thr kinaseTomato Pto (1993): Ser/Thr kinase Arabidopsis RPS2: Arabidopsis RPS2: Tobacco N: Tobacco N: Tomato Cf9 Tomato Cf9 Flax L6Flax L6 Rice Xa21Rice Xa21
Hypersensitive Response (HR)Hypersensitive Response (HR)
Burst of oxygen reactive species around Burst of oxygen reactive species around infection siteinfection site
Synthesis of antimicrobial phytoalexinsSynthesis of antimicrobial phytoalexins Accumulation of Salicylic Acid (SA)Accumulation of Salicylic Acid (SA) Directly kill and damage pathogensDirectly kill and damage pathogens Strengthen cell walls, and triggers apoptosisStrengthen cell walls, and triggers apoptosis Restrict pathogen from spreadingRestrict pathogen from spreading Rapid and localRapid and local
Systemic Acquire Resistance (SAR)Systemic Acquire Resistance (SAR)
Secondary responseSecondary response SystemicSystemic Broad-range resistanceBroad-range resistance Leads to Pathogenesis-Related (PR) gene Leads to Pathogenesis-Related (PR) gene
expressionexpression Signals: SA, JA, ethyleneSignals: SA, JA, ethylene
Systemic Acquired Systemic Acquired ResistanceResistance
(SAR)(SAR)
Salicylic Acid (SA)Salicylic Acid (SA)
Accumulates in both local and systemic tissues Accumulates in both local and systemic tissues (not the systemic signal)(not the systemic signal)
Removal of SA (as in Removal of SA (as in nahGnahG plants) prevents plants) prevents induction of SARinduction of SAR
Analogs: INA or BTHAnalogs: INA or BTH
COOHOH
Mutants affecting SA synthesisMutants affecting SA synthesis
Elevated SA accumulationElevated SA accumulation dnd1dnd1 ( (defense, no death 1defense, no death 1): increased SA, but ): increased SA, but
reduced HR, DND1 gene encodes cyclic-reduced HR, DND1 gene encodes cyclic-nucleotide-gated ion channelnucleotide-gated ion channel
mpk4mpk4: constitutive SA accumulation: constitutive SA accumulation edr1edr1 ( (enhanced disease resistance 1enhanced disease resistance 1): defective ): defective
MAPKKKMAPKKK
Mutants affecting SA synthesisMutants affecting SA synthesis
reduced SA accumulationreduced SA accumulation eds1eds1 ( (enhanced disease susceptibility 1enhanced disease susceptibility 1): lipase ): lipase
homologhomolog pad4pad4 ( (phytoalexin deficient 4phytoalexin deficient 4): another lipase ): another lipase
homologhomolog sid1sid1 and and sid2sid2 ( (salicylic acid induction-deficientsalicylic acid induction-deficient): ):
defects in chorismate pathwaydefects in chorismate pathway
Mutant ScreenMutant Screen
Aimed at identifying regulatory genes of SARAimed at identifying regulatory genes of SAR Strategy: Transform Arabidopsis with GUS Strategy: Transform Arabidopsis with GUS
reporter driven by SA- and INA-responsive reporter driven by SA- and INA-responsive promotor from BGL2 genepromotor from BGL2 gene npr1 (non-expresser of PR genes) mutant: reduced npr1 (non-expresser of PR genes) mutant: reduced
induction of reporter gene with or without SA, INA induction of reporter gene with or without SA, INA cpr (constitutive expresser of PR genes) mutants: cpr (constitutive expresser of PR genes) mutants:
constitutively express reporter genesconstitutively express reporter genes
NPR1: non-expresser of PR NPR1: non-expresser of PR genesgenes
Also known as NIM1 or SAI1Also known as NIM1 or SAI1 Positive regulator of SARPositive regulator of SAR Downstream of SA, upstream of PR genesDownstream of SA, upstream of PR genes npr1npr1 mutants are susceptible to various mutants are susceptible to various
pathogenspathogens Overexpression of NPR1 generates broad-Overexpression of NPR1 generates broad-
spectrum resistancespectrum resistance Unique, but similar to IUnique, but similar to Iκκ-B (negative regulator -B (negative regulator
of immunity in animals)of immunity in animals)
NPR1 overexpressionNPR1 overexpression
Pathogen-Related (PR) GenesPathogen-Related (PR) Genes
Antimicrobial propertiesAntimicrobial properties Many identifiedMany identified Categorized according to activityCategorized according to activity ExamplesExamples
PR-2 : beta-1,3-glucanasePR-2 : beta-1,3-glucanase PR-3 : chitinasePR-3 : chitinase PR-12: defensinPR-12: defensin
Avr R gene
SA
NPR1
PR-1 PR-2 PR-5
SAR
SAR SAR
Structural features of NPR1Structural features of NPR1
593 amino acids, 67 kD593 amino acids, 67 kD Two protein-protein interaction domains: Two protein-protein interaction domains:
BTB/POZ and Ankyrin repeatsBTB/POZ and Ankyrin repeats Contains NLSContains NLS Multiple phosphorylation sitesMultiple phosphorylation sites No DNA binding domainNo DNA binding domain
npr 1-1
BTB ARD
S S
NLSnpr 1-2 nim 1-2
NPR1-GFP localizes in nucleus upon SAR inductionMS MS-INA
NPR1-GFP
GFP
TGA FactorsTGA Factors
Found to interact with NPR1 through yeast-Found to interact with NPR1 through yeast-two hybridtwo hybrid
bZIP transcription factorsbZIP transcription factors Six members in Arabidopsis (TGA1-6) Six members in Arabidopsis (TGA1-6) Might be redundantMight be redundant Bind to as-1 elementBind to as-1 element
NPR1-TGA2 interactionNPR1-TGA2 interaction
Direct visualisationDirect visualisation
TGA2 C-term interacts with NPR1TGA2 C-term interacts with NPR1
PR-1 expression reduced in PR-1 expression reduced in TGA2CT linesTGA2CT lines
Figure 2A, 2B
Reduced resistance to Reduced resistance to P.parasitica and tolerance to SAP.parasitica and tolerance to SA
Figure 2C, D
DN effects depends on NPR1DN effects depends on NPR1
Figure 3A, B
SA affects NPR1-TGA2 SA affects NPR1-TGA2 interactioninteraction
Figure 3C, D
Chimera Reporter SystemChimera Reporter System
Figure 4
TGA2-GAL4 is SA-responsiveTGA2-GAL4 is SA-responsive
Figure 5A,B
TGA2-GAL4 as an activatorTGA2-GAL4 as an activator
Figure 5C
DNA binding dependent on DNA binding dependent on NPR1 and enhanced by SANPR1 and enhanced by SA
Figure 5D
Current modelCurrent model
Figure 6
Avr R gene
SA
NPR1
PR-1 PR-2 PR-5
SAR
SAR SAR
TGA2
NPR1-TGA5NPR1-TGA5
Yeast-two hybridYeast-two hybrid
Figure 1 a-d
Co-purificationCo-purification
TGA2 mRNA accumulationTGA2 mRNA accumulation
Figure 2
untreated
P.parasitica
INA
TGA5 mRNA accumulationTGA5 mRNA accumulation
untreated
P.parasitica
INA
Figure 3a
Surprising accumulation of Surprising accumulation of TGA5 in antisense linesTGA5 in antisense lines
Figure 3b
untreated
P.parasitica
INA
PR-1 induction in TGA2 PR-1 induction in TGA2 transformantstransformants
Figure 4
Reduced PR-1 expression in lines Reduced PR-1 expression in lines with high TGA5 mRNA with high TGA5 mRNA
Figure 5
TGA5-antisense lines resistant to TGA5-antisense lines resistant to infectioninfection
Figure 6
WT
AS15
AS16
TGA5-antisense lines resistant to TGA5-antisense lines resistant to infectioninfection
AS15 resistance is independent of AS15 resistance is independent of NIM1NIM1
Avr R gene
SA
NPR1
PR-1 PR-2 PR-5
SAR
SAR SAR
TGA2 TGA5
SAR independentresistance
That’s not all…That’s not all…
A few othersA few others
Ethylene-mediated responseEthylene-mediated response Jasmonic acid-mediated responseJasmonic acid-mediated response Induced systemic resistance (ISR)Induced systemic resistance (ISR) MAPK cascadesMAPK cascades
The futureThe future
Still a lot to learnStill a lot to learn 2010 project2010 project The golden eraThe golden era
Thank you!Thank you!