MOLECULAR CHARACTERIZATION OF GENE FOR GENE

SYSTEMS IN PLANT- FUNGUS INTERACTION AND THE

APPLICATIONS OF AVIRULENCE GENES IN CONTROL OF

PLANT PATHOGENS

DOCTORAL SEMINAR-II

VINOD UPADHYAY

ID: 440561

INTRODUCTION

NON HOST RESISTANCE :

• Apple trees and tomato pathogens

• Powdery mildew on wheat (Blumeria graminis f. sp. tritici)

and barley

Disease is exception rather than rule

HORIZONTAL RESISTANCE :

• General resistance, quantitative resistance

• Incomplete resistance but durable

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VERTICAL RESISTANCE :

• Race specific , qualitative resistance, differential resistance

• Complete resistance, not durable – high selection presssure

• Follow gene for gene system

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PATHOGEN RACE

PATHOGEN RACE

PLANT VARIETY 1 2 PLANT VARIETY 1 2 3 4

A _ + A _ + + +

B + _ B + _ _ +

C _ + _ +

D + _ + _

Reaction of plants to attacks by various pathogens in relation to resistance of the plant

4Agrios (2005)

GENE FOR GENE HYPOTHESIS

Each gene that confers avirulence (Avr) to the pathogen there is a

corresponding gene in the host that confers resistance (R) to the host

and vice versa – H.H.Flor (1946)5

RESISTANT OR SUSCEPTIBILITY GENES IN THE PLANT

VIRULENCE OR AVIRULENCE GENE IN

PATHOGEN

R (resistant)dominant

r ( susceptibility) recessive

A (avirulent) dominant AR (-) Ar (+)

a ( virulent) recessive Ar (+) ar (+)

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RESISTANCE (R) OR SUSCEPTIBILITY ( r) GENES IN THE PLANT

R1 R2 R1 r2 r1 R2 r1 r2

VIRULENCE (a) OR AVIRULENCE (A)

GENES IN THE PATHOGEN

A1 A2 - - - +

A1 a2 - - + +

a1 A2 - + - +

a1 a2 + + + +

ELICITOR AND RECEPTOR CONCEPT

7Agrios (2005)

R-AVR GENE INTERACTION AS EFFECTOR TRIGGERED IMMUNITY (ETI)

8Jones & Dangl (2006)

OUTCOME OF R- AVR INTERACTION

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FUNCTIONS AND FEATURES OF AVR GENE CODED PROTEINS

Hydrophilic- lacking stretches of hydrophobic amino acids - enable them to be anchored in cell membranes

May produced and localized in pathogen cytoplasm or secreted through membrane pores

If secreted externally – directly acts as elicitors

If localized in cytoplasm of pathogen - acts indirectly as enzyme to produce elicitor molecules

Acting as avirulence factors in elicitor-receptor model (plant defense)

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Contribution towards the virulence of pathogen. eg. AvrBs2 gene of X. campestris pv. Vesicatoria

Avr proteins interact with specific plant proteins (virulence target) -enhances availability of nutrients to pathogen .

RESISTANCE GENES

Most R proteins contain amino acid leucine rich domain (LRR- leucine rich repeats),

Depending on R protein LRR reside : cytoplasmic LRRs or extracytoplasmic LRRs.

Leucine-rich repeats (LRR) region of R-genes is involved in recognizing pathogens

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MAJOR CLASSES OF R PROTEINSS. NO MAJOR R-GENE

CLASSESEXAMPLE

1 NBS-LRR-TIR N, L6, RPP5

2 NBS-LRR-CC I2, RPS2, RPM1

3 LRR-TrD Cf-9, Cf-4, Cf-2

4 LRR-TrD-Kinase Xa21

5 TrD-CC RPW8

6 TIR-NBS-LRR-NLS- WRKY RRS1R

7 LRR-TrD-PEST-ECS Ve1, Ve2

8 Enzymatic R-genes Pto, Rpg1 LRR - Leucine rich repeats; NBS - Nucleotide-binding site; TIR -Toll/Interleukin-1- receptors; CC - Coiled coil; TrD –Transmembrane domain; PEST -Amino acid domain; ECS - Endocytosis cell signaling domain; NLS - Nuclear localization signal; WRKY -Amino acid domain; HC toxin reductase - Helminthosporium carbonum toxin reductase enzyme.

12 Gururani et.al.,2012

13Agrios (2005)

TYPES OF R-CODED RECEPTOR PROTEINS

GENE PLANT PATHOGEN YEAR ISOLATED

Pto Tomato Pseudomonas syringae pv. tomato (avrPto)

1993

PBS1 Arabidopsis Pseudomonas syringae pv. phaseolicola(avrPphB)

2001

RPS2 Arabidopsis Pseudomonas syringae pv. maculicola (avrRpt2)

1994

N Tobacco Tobacco Mosaic virus 1994

Bs2 Pepper Xanthomonas campestris pv. vesicatoria (avrBs2)

1999

RRS-1 Arabidopsis Ralstonia solanacearum 2002Pi-ta Rice Magnaporthe grisea(avrPita) 2000Cf-9 Tomato Cladosporium fulvum(Avr9) 1994Ve1Ve2

Tomato Verticillium albo-atrum 2001

Xa-21 Rice Xanthomonas oryzae pv.oryzae (all races)

1995

Pi-d2 Rice Magnaporthe grisea 2006

14 Gururani et.al.,2012

Plant proteins belonging to the nucleotide-binding site–leucine-rich repeat (NBS-LRR) family are used for pathogen detection.

(R-PROTEIN)

Harmful organismRecognition by resistance protein

Signal to cell nucleus

Genetic material Defense

Response Defense protein (R-PROTEIN)

Out

side

pla

nt

cell

Insi

de p

lant

cel

l

Diagram of a plant disease resistance protein in action. A portion of the protein (MAROON) lies outside the cell and specifically recognises the harmful organism. The remaining portion of the protein (RED) resides inside the cell and communicates a signal to the plant’s genetic material, which in turn stimulates a defense response against the invading organism.

R-GENE IN ACTION

NBS-LRRPROTEIN

(R-GENE)

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MODELS FOR R- AVR GENE INTERCTION

• INDIRECT PERCEPTION OF AVR PROTEINS:

Protease dependent defense elicitation model

The co-receptor model

The guard hypothesis

The decoy hypothesis

Bait and Switch model

•DIRECT PERCEPTION OF AVR PROTEINS :Elicitor- receptor model

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Albersheim and Anderson Prouty, 1975 proposed this model.

Avirulence (Avr) gene of a pathogen encodes an elicitor (Avr) protein

that is recognized by a receptor protein encoded by the matching

resistance (R) gene of the host plant.

eg. Pi-ta R gene from rice and AvrPi-ta from Magnaporthe grisea

ELICITOR– RECEPTOR MODEL

17Staskawicz et.al.,1995

PROTEASE DEPENDENT DEFENSE ELICITATION MODEL

Kruger et al., 2002 proposed this model.

eg.Tomato leaf mold –Cladosporium fulvum interaction.

Rcr3 required for Cf-2 mediated resistance towards C.fulvum

strains carrying Avr2, encodes a tomato cysteine endoprotease.

Rcr3 might process Avr2 to generate a mature ligand, or Rcr3

might degrade Avr2 - releasing active elicitor peptides that

interact with the extracellular LRR of Cf2. 18

19Jones & Dangl (2006)

THE CO-RECEPTOR MODEL

Jones and Jones,1996 proposed this model.

RPS5 an Arabidopsis NB-LRR protein localized to a membrane

fraction - activated by the AvrPphB cysteine protease effector from P.

syringae.

AvrPphB is cleaved, acylated and delivered to the host plasma

membrane. Activated AvrPphB cleaves the Arabidopsis PBS1 serine-

threonine protein kinase, leading to RPS5 activation.

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Jones & Dangl (2006)

THE GUARD HYPOTHESIS

Van-der-biezen and Jones, 1998 proposed this model.

Interaction between guardee and Avr is recognized by the R

protein

eg. AvrPto of Pseudomonas syringae and Pto gene of tomato,

Prf gene act as guardee.

Evolutionary unstable situation

R protein is absent -evolution of the guardee to avoid binding

R protein is present - selection will favor binding22

23Zhang et.al., 2013

Van der Hoorn and Kamoun, 2008 proposed this model.

Host protein termed as “decoy” - specializes in perception of the effector by the R protein

Not contributing pathogen fitness in the absence of its cognate R protein.

Effector target monitored by the R protein is a decoy that mimicsthe operative effector target

e.g. AvrPto and AvrBs3 - some host targets of effectors act as decoys to detect pathogen effectors via R proteins

DECOY HYPOTHESIS

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25Zhang et.al., 2013

Peter Moffett , proposed this model in 2002.

The NB-LRR protein - primed (signaling competent) but autoinhibited (restrained from signaling) state.

Functional nucleotide binding pocket and multiple intramolecular interactions - fine-tuned balance between the LRR and ARC2.

Avr protein is brought into the NB-LRR system via the bait protein - direct binding or alteration to bait.

Conformational changes within the nucleotide binding pocket- allow signaling motif - downstream signaling components.

Subsequent to signaling, intramolecular interactions within the NB-LRR protein dissociated.

BAIT AND SWITCH MODEL

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27Collier and Moffett (2009)

MOLECULAR BASIS FOR PLANT- FUNGUS INTERACTIONS

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UTILIZATION OF AVIRULENCE GENE FOR CONTROL OF PLANT PATHOGENS

DIRECT UTILIZATION THROUGH TWO COMPONENT SENSOR SYSTEM (De Witt , 1992)

40Lauge and De Wit (1998)

INDIRECT UTILIZATION OF AVIRULENCE GENES

GENE DEPLOYMENT

GENE PYRAMIDING

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Virulence/ avirulence pattern

Gene Deployment

V1 / avr2, avr3 R2, R3, not R1V2 , V3 / avr1 Only R1

V1, V2, V3/ avr4, avr5 R4, R5

R1

R1 + R2

R1 + R2+ R3

eg. Oat against crown rust

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MULTILINES

R1

V1

R2

V2

R3

V3

R4

V4

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CO-EVOLUTION OF RUST AVR AND HOST R GENEFLAX RUST –

All the virulent rust strains retain intact copies of the Avr genes

(AvrL567) but have altered their sequences

Host R genes imposed selection for new variants to escape recognition.

44Jones & Dangl (2006)

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CONCLUSION

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FUTURE PROSPECTS

Functional genomic tools to disease resistance - interactions between defense signaling and other plant processes.

Structural basis of recognition will enable us - design R proteins that recognize essential virulence factors

New transgenic resistant plants by exploiting both avirulence genes and resistance genes in molecular resistance breeding

Using avirulence gene products / race-specific elicitors - events in signal transduction pathways can be studied.

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