Basic Principles of Phytopathology

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Dept. Botany

Faculty of Science Palacký University in Olomouc

Šlechtitelů 27 78371 Olomouc Czech Republic

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PHYTOPATHOLOGY

Syllabus of 5th lecture: - Plant responses to infectious agents - Immunity, resistance (defense mechanisms), pseudoresistance, susceptibility, tolerance, sensitivity, hypersensitivity - Plant metabolic processes resulting in susceptibility, resistance and tolerance - Causes of changes in susceptibility and resistance - Field resistance In Olomouc 30.3.2017 Assoc. Prof. Dr. Michaela Sedlářová

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Reaction of host to pathogen infection can be studied on the following levels of biological organization:

Organism

Organ

Tissue

Cell

Physiological processes

Molecular structure

All these levels are more or less involved in the expression of interaction, one being closely mutually connected to

others, conditioning each other. BOT/ZFP

Plant reactions to pathogen attack

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Timing of

host defence

mechanisms

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http://slideplayer.com/slide/2484138/

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stimulatory

(obligate biotrophs – at early stages of development stimulation of growth, No. organelles) „green islands“- biotrophic bacteria, powdery mildews, rusts, viruses (unballanced kinetin aand others CKs)

Persea americana - Oncobasidium theobromae

Effect of pathogen infection to cells

green bionissia vs.

green necronissia

Erysiphe graminis f. sp. tritici BOT/ZFP

More mitochondria and ribosomes

increased synthesis of proteins, more intensive respiration

respiration + oxidative processes + degradation of storage molecules

= chemical energy release

to cover synthesis of biomolecules

Vitality of cells depends on – functional vacuole – intact vesicular transport

Effect of biotrophic pathogens to cell

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degenerative

(necrotrophs and at later stages for obligate parasites: membrane disruption)

Lycopersicon esculentum - Phytophthora infestans

Effect of pathogen infection to cells

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Strategies of parasitic pathogens

BIOTROPHS HEMIBIOTROPHS NECTROTROPHS

Host cells Alive used as source of nutrients Alive – kills later Killed

Host range Very narrow Narrow Wide

Host cell attack

Do not affect cytoplasmic membrane

Do not affect cell wall

Affect cytoplasmic membrane

and cell wall

Toxin production Rare

At late stages of life cycle

High

HR Often Rare - BOT/ZFP

1/ Structural (passive) physical barriers against pathogen ingress Constitutive (preformed): Trichomes, leaf hairs – reduce pathogen deposition Cell wall – prevent the pathogen from entering the cell Waxy epidermal cuticle – prevents tissues from water loos +

reduces surface moisture required for spore germination Inducible: lignin, suberin, callose deposition, sticky gums, resins x Pathogens produce - cutinases, cellulases

Plant defense mechanisms

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Plant defense mechanisms

2/ Biochemical (active) production of toxic compounds, pathogen-degrading

enzymes, deliberate cell suicide secondary metabolites (specific to host-pathogen interaction): alkaloids, saponins, antokyans phenolics, tanins, melanines aromatic aminoacids proteins extensins (rich in hydroxyprolin) PR-proteins (glucanases, chitinases, peroxidases) BOT/ZFP

Induced structural defense

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Lignin deposition Involved enzymes: Peroxidase + Laccase Lignin polymers differ according to tissue and plant species CAL = autonomous cell lignification Each lignifying cell controls the whole process of its own lignification and therefore undergoes „Cell-Autonomous Lignification“ NCAL = non-autonomous c. l. In contrast to sclerenchyma fiber cells, which stay alive throughout the lignification process, vessel formation involves rapid programmed cell death. In consequence, vessels require the help of neighboring cells to achieve full lignification after their programmed cell death. This is called the ‘good neighbors’ scenario or the non-cell autonomous lignification

V: vessel; XP: xylary parenchyma; IP: Interfascicular parenchyma; XF: xylary fiber; IF: interfascicular fiber

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Examples of lignified cells or tissues. (A) and (B) cross sections of Arabidopsis thaliana stem (magnification 50×). LP: lignifying parecnhyma; IF: interfascicular fibers; LMV: lignifying metaxylem vessel; MMV: mature metaxylem vessel; Ph: phloem; PX: protoxylem. (C) and (D) cross sections of Populus tremuloïdes secondary xylem (magnification 20× and 50× respectively). PScl: phloem sclereids; CA: cambium area; LX: lignifying xylem; FLX: mature lignified xylem; LV: lignifying vessel; FLV: mature lignified vessel; Fi: fiber; Ra: ray. (E) and (F) cross section of Brachypodium distachyon roots (25× and 100×). Ex: exodermis; En: endodermis; PMX: peripheral metaxylem; CMX: central metaxylem; PX: protoxylem; En: endodermis cell; PC: passage cell in endodermis. Arrows show lignin fluorescence of casparian strip. (G) cross section of Picea abies secondary xylem (80×). (H) tangential section of Picea abies secondary phloem (80×). (I) radial section of Brachypodium distachyon culm (100×). In A, C, D, E, G, H, I, sections were stained with phloroglucinol-HCl that colors lignin aldehydes in red. In B, section was colored with Maüle staining method that colors G unit-enriched tissues in orange/brown and S unit-enriched tissues in red. In F, root section was vizualized under UV illumination

http://www.sciencedirect.com/science/article/pii/S1369526614001587#sec0015

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Suberin layer

Gloeosporium album BOT/ZFP

Abscission

Alternaria brassicicola

Pucciniastrum areolatum - Padus

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Papila

Pochva

Callose deposition sheath - penetration of micromycetes

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Plasmodesmata: connection channels in between cells

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Tobacco mosaic virus movement protein 30 localizes to plasmodesmata

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Trends in Plant Science 2011 16, 201-210DOI: (10.1016/j.tplants.2011.01.004)

Copyright © 2011 Elsevier Ltd Terms and Conditions

Plasmodesmata: defense against pathogens

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(a) viruses

(b) fungi (c) bacteria

Plasmodesmata: defense against pathogens

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Callose deposition at plasmodesmata

Arabidopsis leaf tissue BOT/ZFP

Callose deposition - model of plasmodesmata role in defense against

Pseudomonas syringae

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Tyloses

- outgrowths on parenchyma cells of xylem vessels in secondary heartwood

- when the plant is stressed by drought or infection, tyloses fall from the sides of the cells and "dam" up the vascular tissue to prevent further damage

- Occurrence: tracheomycoses (Dutch elm disease)

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Interaction

between

host cell

and

pathogen

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Biochemical defense

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Hypersensitive

reaction

- a sequence

of steps leading

to run HR after

elicitor contact

with receptor BOT/ZFP

Mechanisms of defense reaction initiation

2. Receptor – in plasma membrane / cytoplasm 3. Signal transduction (secondary messengers) fosfoinositic system (hydrolysis of lipids lipidů inositol-1,4,5-triP diacyl glycerol + increased level of Ca2+ = activation of protein kinases)

formation of hydrogen peroxide +ROS – fosfoinos. system - peroxidation of membrane lipids – JA+MeJA - transcription ethylene – initiation of gene expression

1. Elicitor – induces defense exogenic elicitors – metabolites secreted by a pathogen (polysacharides, specific enzymes, peptides) endogenic elicitors – released following disruption of the cell wall of - pathogen effectors (oligomers of chitin, oligoglucans, glycoproteins) - plant (oligogalacturonans)

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Elicitors

and

supressors

in

resistance

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Activation of defence mechanisms Local at the site of attack - production of reactive oxygen species (ROS) - reactive nitrogen species (RNS) - hypersensitive reaction (HR) - acumulation of phenolics - cell wall impregnation Local reaction of tissue - synthesis of PR-proteinů - phytohormones (SA, ET, JA…) - cell walls impregnation Systemic response - in distant organs (systemic acquired resistance, SAR) BOT/ZFP

Hemzalová, DiP, ČZU, 2012 BOT/ZFP

Sites of formation: chloroplasts, mitochondria, membrane peroxisomes, glyoxysomes, (photosynthesis, respiration, glycolate oxidase-photorespiration, NADPH oxidase in PM, oxalate oxidase,…) Effects - negative: Peroxidation of lipides, proteins (esp. enzymes), nucleic acids, lipids - positive: Signalling, gene regulation

Singlet oxygen (1O2) Superoxide (O˙- 2) Hydrogen peroxide (H2O2) Hydroxyl radical (OH-)

Reactive oxygen species (ROS)

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HOST • Direct antimicrobial action • Signalling molecules in regulation of gene activities for synthesis of - PR-proteins - phenolics - fytoalexins • Cross-linking of precursores of polymers – lignin in CW • HR

PATHOGEN • Signalling – recognition in incompatible interactions? • Penetration – disruption of CW polymers, membrenes

Reactive oxygen species (ROS)

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- Enzyme inhibitors - Induced by injury, infection - Formed in resistant and susceptible genotypes, qualitative and quantitative variation - Antibiotic effects - Defense against UV - Structural polymers – fortification of CW,

lignituber at the site of penetration - Irreversible membrane damage (IMD) - Autofluorescent phenolics - de novo

synthesized during HR

Phenolic compounds

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penetration site periplasmic space invaginated plasma membrane

Phenolic compounds localize to

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Cytoskeleton of host cells • binding to transmembrane receptors, CW cellulose microfibrils

• plant - pathogen recognition

• rapid reorganization affects the process of penetration (AFs gather at the point of penetration, callose deposition = mech. hardening) and further development of the pathogen in the cells after infection (invagination of membrane)

• in resistant plants with cortical microfilaments and microtubules cluster around developing appressoria and thus contribute to the inhibition of pathogen penetration

• microfilaments participate in migration host nuclei and organelles as well as deposition of matrix compounds (phenolics, callose, lignin, etc.) which hardens cell wall

• localized programmed cell death - depolymerization of the cytoskeleton - reducing the development of the pathogen - and HR elicitors (cryptogein etc.).

• resistant cells - synthesis of various post-translationally modified tubulin isoforms, associations with other proteins BOT/ZFP

MT basket 24hai L. sativa UCDM2

MT patches 48hai L. serriola PIVT 1309

L. serriola LSE/18

HR 48hai L. sativa Mariska

Cytoskeleton

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PR-proteins (pathogenesis-related proteins)

- soluble at low pH - low Fw - low or high pI - resistant to proteases - extracellularly localized Classified by their size and function: - Chitinase, β-1,3-glucanase (pathogen CW hydrolysis, production of other elicitors) - Peroxidase - Ribosome inactivating proteins - fungicidal activity - Thionins - form pores in the membranes of pathogens - Lipid transferring proteins BOT/ZFP

Systemic responses

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Principles of SAR

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Inductors of SAR

Used to induce resistance to: viruses – TMV fungi and oomycetes – Phytophthora tabacina bakteria – Pseudomonas syringae BOT/ZFP

Strategies of pathogens to avoid plant defense mechanisms

• Fast growth – not to be affected by plant reaction

• Supressors formation – compounds released to diminish HR, production of PR-proteins, etc.

• Enzyme production

• Cell poisoning - e.g. fusicoccin in Phomopsis (old name Fusicoccum) amygdalii which causes hyperpolarisation of cell membranes and stimulates a quick acidification of the plant cell wall; this causes the stomata to irreversibly open, which brings about the death of the plant,

canker on peach tree

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Transgenic plants – improvement of defenses against fungal pathogens

Punja (2001) Can. J. Plant Pathol. 23:216-235 BOT/ZFP

Strategies to obtain crops resistant to diseases

Gurr a Rushton (2005)

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Molecular mechanisms of resistance 1 / Natural resistance: • the cell wall or membrane structure • the existence of transporter protein for excretion of the substance • absence of inhibited metabolic pathway • the presence of enzymes that metabolize the substance • structure of target site, wherein the compound acts • expression of specific stress proteins • high capacity of correction mechanisms 2 / Acquired resistance - mechanism developed under evolutionary selective pressure against the background of the toxic substances. Individual resistance mechanisms can be summarized into the following types: • reduction of drug transmission • reducing the uptake of the drug • increase of the excretion of the drug • reduction of metabolic drug activation • increasing the de-activation of the drug • sequestration preventing drug hitting the target site • increasing the intracellular concentration of target sites • structural change of target molecule • duplication of functions in target • increase of the repairing mechanisms for damaged targets

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