plant pathology 602 plant-microbe interactions...plant pathology 602 plant-microbe interactions...

Plant Pathology 602Plant-Microbe Interactions

Sophien [email protected] Ohio State UniversityOhio Agricultural Research & Development Center

Fungal PathogenesisInfection processes

Pathogen must: Find the host and the appropriate entry site

Develop specialized infection structures

Overcome preformed and induced host defenses

Take up nutrients, grow, and colonize host tissue


Infection processes

Finding the host Development of infection structures

Overcoming plant defenses


Outline - infection processes





Finding the host

Passive vs. active

Wait in favorable site for host

Dispersal by animal vectors

Use dispersal stages (spores, zoospores…)

Stages leading to infection by oomycetezoospores

http://helios.bto.ed.ac.uk/bto/microbes/zoospore.htm

Chemotaxis of Phytophthora zoospores

Tactic response allows zoospores to reachinfection sites in roots of host plants

Positive chemotaxis towards plantcompounds (ex: P. sojae to isoflavones)

Electrotactic responses also noted (weakelectric fields generated by roots)

Phytophthora and Pythium zoospores swimtowards amino acids and sugars

http://helios.bto.ed.ac.uk/bto/microbes/zoospore.htm

Zoospore-root interactions(from P. van West)

Phytophthora palmivoramarked with GFP isattarcted to the root tip(electrotaxis?)

Pythium aphanidermatumis attracted to wounds(chemotaxis to rootexudates?)





Morphological changes are essential topenetration and adaptation to plant tissue

Many fungal/oomycete pathogens develophighly specialized infection structures inresponse to signals such as contact withthe plant surface and plant tissueenvironment

Most important structures are appressoriaand haustoria

Developmental processes and infectionDevelopmental processes

and infection: rust fungi

Not all fungal plant pathogens producespecialized infections structures:

Cladosporium fulvum-tomato

Pathogen: virulent, plant: susceptible

Forcible entry: rice blast fungus

A spore from Magnaporthegrisea has germinated on thesurface of a rice leaf andformed a dome-shapedappressorium. Theappressorium has to breachthe thin but tough rice leafcuticle to invade the leaf andcause disease.Talbot, NJ. 1999. Science 285:1860

Penetration of plant epidermis

Infection process by appressoria caninvolve enzymatic action

External matrix around appressoriacontains cutinase, cellulases, and otherenzymes to help soften the cuticle, therebyaiding adhesion and penetration

However, some fungi can physically forcetheir way through plant cuticles

Penetration of plant epidermis Fungi such as Colletotrichum and

Magnaporthe produce appressoria withtough melanin-pigmented cell walls

Appressoria generate pressures between 6-8 Mega Pa (30-40x pressure of a car tire)

Produce hydrostatic turgor by accumulatingmolar concentrations of glycerol

Can puncture artificial plastic membranes Melanin mutants are nonpathogenic and

cannot accumulate turgor

Haustoria: feeding and delivery ofvirulence factors (effectors) Numerous fungi, oomycetes and parasitic

plants produce feeding structures (haustoria) Formed within living cells of host organism Assumed to play a major role in the

absorption of plant metabolites and thusinfection

Difficult to study because of intimateassociation with plant cells

Haustoria: feeding and delivery ofvirulence factors (effectors)

Haustoria of rust fungi can be purifiedbiochemically

Hahn et al. Identified several genes that areexpressed in haustoria

An Amino Acid Transporter protein of theRust Fungus Uromyces fabae (host: broadbean) is specifically localized in haustoria

Confirms role in nutrient assimilation

Haustoria: feeding structuresAmino Acid Transporter in Haustoria of the Rust FungusUromyces fabae (host: broad bean)

Detection of the amino-acidtransporter protein PIG2 inrust-infected leaves byimmunofluorescencemicroscopy. Only the surfaceof the haustorial bodies (h) isshowing fluorescencelabeling. Haustorial neck(arrow), haustorial mother cells(m), and inter-cellularhyphae (i) exhibit nofluorescence.Hahn et al. 1997. MPMI 10:438

A, Early stage of haustorium development. B, Fully developed haustorium.

Susceptible Resistant

Haustoria: effector delivery into plant cellsAvirulence protein recognition by Resistance protein -Flax-rust interaction

HR

cell death

A.M. Catanzariti, P. Doods, and J. Ellis

Signal transduction pathways allowpathogen to recognize and respond to theplant surface/plant tissue environment

Proteins involved in these signaltransduction pathways can then bepredicted to be esssential for pathogenicity

These signal transduction pathways areoften conserved between pathogenic andnonpathogenic organisms

Developmental processes and infection

Pathways involving the second messengercyclic AMP (cAMP) have been implicated insignal perception and transduction duringinfection by:

Rice blast (Magnaporthe grisea) Corn and barley smut (Ustilago maydis/U.

hordei) Chestnut blight (Cryphonectria parasitica) Sclerotinia (Sclerotinia sclerotiorum)

Signal transduction pathways: cAMP Signal transduction pathways: cAMPExample of pathways in pathogenic fungi

AppressoriumPlant surface?Rice blast

MorphogenesisUnknown?Corn/barleysmut

GrowthUnknown?Chestnut blight

Signal Response(s)

SclerotiaUnknown?Sclerotinia

Signal transduction pathways: cAMPExample of a generic pathway

Signal ReceptorsConversion of ATP into cAMP

Activation of Protein kinases

Downstreamresponses

Signal transduction pathways: cAMPExample of a generic pathway

GTP-bindingProteins

(G-proteins)

AdenylylcyclaseS

ATP

cAMP ProteinKinase

Downstream responses

Signal transduction pathways: cAMPMutagenesis approach in Magnaporthe

GTP-bindingProteins

(G-proteins)

AdenylylcyclaseS

ATP

cAMP ProteinKinase

Downstream responses

Signal transduction pathways: cAMPMutagenesis approach in Magnaporthe

magB mutants are deficient in G protein mac1 mutants are deficient in adenylate

cyclase Both mutants are deficient in appressoria

formation and pathogenicity Kinase mutants?

Signal transduction pathways: cAMPKinase identification in Magnaporthe

Appressoria pressure produced byhydrostatic turgor by accumulating molarconcentrations of glycerol

Hog1 (high osmolarity turgor): kinase thatregulate cellular turgor in yeast

Magnaporthe homolog isolated by PCR


Magnaporthe griseaOSM1 gene isolated

Encodes a proteinkinase with highsimilarity to yeastHOG1

Gene knockout

Dixon KP et al. 1999 Plant Cell 11:2045


Wild-type

Gene replacement plasmid

Mutant: deleted for osm1


DNA (Southern) blot of: wild-type (1) nontransformed strain (3) mutants (2, 4-6)


Probe:OSM1 gene

Probe:HPH gene


Osm1 mutant showsaberrant hyphalmorphology andosmotic sensitivity atthe mycelium stage


medium

Medium+

0.4M NaCl

Wild-type Osm1 mutant


Osm1 mutant produceappressoria with high cellularturgor

Osm1 mutants can infect ricesimilarly to the wild-type strain

Osm1 is not directly involved inappressoria formationpathway!!!



Osm1 mutant produces multipleappressoria duringhyperosmotic stress

Osm1 may prevent formation ofappressoria during stressfulenvironmental conditions


Signal transduction pathways: cAMPMultiple kinase signaling pathways in therice blast pathogen Magnaporthe grisea

input

kinase

output





Overcoming plant resistance

Plants are resistant to most pathogens.Thus, most pathogens are to a greater orlesser extent host-specific, beingspecialized for infection of a particular setof host plants

Plants are resistant to most pathogens

Compendium of soybean diseases (APSPress) lists about 100 pathogens known toaffect soybeans, about 35 of which areimportant economically

Example: soybean

Plants are resistant to most pathogensDisease is the exception!

Species A Species B

Species C Species D

Susceptibility or infection = How hostresistance and defenses are defeated!

Plants can rapidly detect physical and molecularperturbations caused by invading microbes

Successful pathogens must suppress, evade, ortolerate resistance and defenses responses

Plants can be very sensitive:Touch of Phytophthora

Parsley cells were embedded in agarose. After the cell was stimulated for a fewminutes with a fine tungsten needle the generation of intracellular reactiveoxygen intermediates was monitored with a dye, which becomes fluorescentupon oxidation by ROI. (A = bright field picture, B= flourescence picture; n =nucleus, cs cytoplasmic strands). http://www.eurekalert.org/releases/mpg-tto071498.html

Titel corps 28 News Gothicregular

Broodtekst diapositief corps 18 NewsGothic

No response of susceptible potato toPhytophthora infestans 1 day after inoculation Coevolutionary battle between plants

and pathogens - arms race model

Plant is resistant

Pathogen defeats resistance/defense barriers Host faces selection pressure to develop new

resistance/defense barriers

Pathogen evolves alternative mechanism forvirulence etc...

Coevolutionary battle between plantsand pathogens - arms race model

Not every disease/interaction is explained bythe arms race model. Opportunisticdiseases/interactions do occur!

More on this later!!!

Caution!!!! Preformed vs. induced

Active vs. passive

Specific vs. general

Resistance mechanisms -Lets review some of the mechanisms and find outhow pathogens overcome them

Physical barriers (cutin, cell wall…)

Preformed antimicrobial compounds

Preformed resistance mechanisms

Degradative enzymes (cutinase, pectinaseetc...)

Force penetration through specializedstructures (appressoria)

Evasion (stomata, wounds...)

How to overcome physical barriers?

Plants produce a diverse array ofsecondary metabolites

Some of these have antimicrobial activity Phytoanticipins distinguish preformed

compounds from those that are synthesizedfrom remote precursors following pathogeninfection (phytoalexins)

Preformed antimicrobial compounds-Phytoanticipins

Constitutive: saponins (glycosylatedcompounds)

Inactive precursors activated by tissuedamage or pathogen attack: cyanogenicglycosides and glucosinolates

Preformed antimicrobial compounds-Phytoanticipins

Preformed antimicrobial compounds-Saponins: oat avenacin

Preformed antifungalcompound ‘‘anticipating’’fungal attack:Autofluorescent avenacinsin the epidermal cell layer ofa young oat root.

Osbourn A. 1999. Fun. Genet. Biol. 26:163

Membrane composition: saponins act onmembrane sterols and cause pore formation.

The oomycetes Pythium and Phytophthoracontain little sterol in their membranes makingthem resistant to saponins.

Alternaria alternata lowers the pH at theinfection site to levels at which tomatine isineffective.

How to overcome preformedantimicrobial compounds?

Enzymatic detoxification: saponin-detoxifyingenzymes are produced by several fungalpathogens.

Avenacinase produced by Gaeumannomycesgraminis var. avenae is essential for avenacinresistance and infection of oats .

Several fungal pathogens of tomato producetomatinase (glycosyl hydrolases) that detoxifyalpha-tomatine.

How to overcome preformedantimicrobial compounds?

Avenacinase gene cloned from G. graminisavenae by complementation into the fungusNeurospora

Gene disruption was used to generate anavenacinase mutant in Gga

Gga avenacinase mutant can still infect wheatbut cannot infect oat

Saponin detoxificationBowyer P et al. 1995. Science 267:371

Physical barriers (cell wall thickening,callose, lignification…)

Oxidative burst (cross-linking of cell wall) Induced antimicrobial compounds Antimicrobial proteins (PR proteins,

defensins …) Hypersensitive response (cell death)

Induced resistance mechanisms

Extracellular deposits lignification-like

Lignification-like response Hypersensitive response

Hypersensitive responseVleeshouwers et al. Planta (2000) 210:853

Trypan blue staining Autofluorescense

An effective defense reaction that functionsagainst all classes of biotrophic pathogens

HR is a pattern of localized cell death within theplant tissue at the site of infection

This local cell death blocks pathogen growth bydepriving biotrophic pathogens of nutrition andby creating a highly oxidizing environment thatdamages proteins and cell structures

The hypersensitive response (HR)

Pathogens do not induce plant cell death

Pathogens suppress plant cell death

Both strategies involve pathogen effectors

More on this whgen we talk about gene-for-gene

interactions

How do pathogens overcome the HR?

Pathogen

Plant

DiseaseResistance

Strain A Strain B

A XA


X

Pathogen

Plant

DiseaseResistance

Strain A Strain B

A AS


Secreted proteins of diverse structures thatare induced by pathogens and by salicylicacid; marker for SAR

Some PR proteins are enzymes PR2 = glucanases PR3 = chitinases PR7 = proteases

Induced antimicrobial compounds-Pathogenesis-related (PR) proteins

Phytophthora spp. evolved secreted enzymeinhibitors, some of which target PR proteins

Inhibitors of glucanases (PR2)

Inhibitors serine proteases (PR7) Inhibitors of cysteine proteases (PR7)

Inhibition of enzymatic activity of PR proteins isa counterdefense mechanism in Phytophthora

Inhibition of soybean PR2 glucanase is acounterdefense mechanism in P. sojae

Soybean secretes EGaseAthat degrades pathogen cellwall and releases glucanfragments that elicit defenseresponses

P. sojae secretes GIP1 thatinhibit EGaseA activity

Rose et al. 2002. Plant Cell. 14:1329

Tomato

P. infestans

I

P

EPI1EPIC2

P69B PIP1

defense

EPIC1RCR3

Inhibition cascade of tomato proteases leading to suppression of host defenses?

Different sites of interaction:apoplast and host cytoplasm

Low molecular weight antimicrobial compoundsthat accumulate following pathogen attack

Generically called phytoalexins

Belong to diverse chemical families

Legume isoflavanoids are best characterized

Induced antimicrobial compounds-Phytoalexins

Induced antimicrobial compounds-Phytoalexins

Formation and mobilization ofphytoalexin-producing vesiclestoward the site of fungal invasionin sorghum resisting infectionby Colletotrichum graminicola.The dark structure in the centerof the photograph is a fungalappressorium. The red3-deoxyanthocyanidinphytoalexins are visualized bylight microscopy.Snyder BA et al. 1990 Science 248:1637

Legume isoflavanoids

Genes encoding enzymes in thebiosynthetic pathway ofisoflavanoids, are induced uponpathogen attack thus resulting inincreased isoflavanoidaccumulation

Detoxification or tolerance of phytoalexins Fungal pathogens of pea are tolerant to pisatin

and/or can metabolize pisatin to less toxicproducts

Nectria hematococca encodes an enzyme pisatindemethylase or pda (cytochrome P-450monoxygenase family) that detoxifies pisatin

How to overcome phytoalexins?

ATP-binding cassette (ABC) membrane transportproteins are involved in resistance to a wide rangeof unrelated antifungal compounds (multidrugresistance)

ABC transporters are involved in the resistance ofbacterial pathogens of animals and plants to host-produced antimicrobial peptides

ABC transporters play a role in tolerance tophytoalexin in fungi

How to overcome phytoalexins?Role of ABC transporters?

Pathogen must: Find the host and the appropriate entry site (zoospore

chemotaxis)

Develop specialized infection structures (appressoria andhaustoria)

Overcome preformed and induced host defenses (variousstrategies for suppressing or evading defenses)

Take up nutrients, grow, and colonize host tissue (role ofhaustoria)

Mechanisms of infection in fungi andoomycetes - Summary

plant pathology 602 plant-microbe interactions...plant pathology 602 plant-microbe interactions...

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