Plant Defense: A GlimpsePlant Defense: A Glimpse

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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!