Programmed cell death in plant defense

N.Jagadeesh Selvam, Ist PhD Biotechnology.,

Programmed Cell Death

Programmed cell death (PCD) is a physiological cell death process involved in the selective elimination of unwanted cells (Ellis et al.,

1991).

In plants, selective cell death is necessary for growth and survival and can occur in local or large scale (Barlow, 1982)

It comprises

cytoplasmic shrinkage, membrane blebbing,

loss of cell to cell contact,

fragmentation of nDNA at interchromosomal sites ordered disassembly

PCD depends on the active participation of the dying cells, and is regulated by genetically controlled, well-orchestrated cell suicide machinery.

At the molecular level, the apoptotic cell death follows two pathways

the interaction of a death receptor with its ligand the participation of mitochondria, most notably release of cytochrome c from the mitochondrial intermembrane space

Typical biochemical hallmarks of apoptosis in plants include

calcium influx, exposure of phosphatidylserine,

activation of specific proteases,

nDNA fragmentation leading to nucleosomal ladders and cytochrome c release from mitochondria

HR

PCD

Programmed cell death (PCD) is an intrinsic mechanism that occurs in nearly all organisms.

In plants endogenous stimuli induce developmental PCD processes during, for instance,

embryogenesis (Bozhkov, 2005), leaf morphogenesis (Gunawardena, 2008), xylem development (Fukuda, 1996), floral development (Rogers, 2006) and

organ senescence (Rogers, 2005).

Exogenous stimuli elicited by abiotic and biotic factors

Sites of PCD in Vascular Plants

(Pennel R.I and C. Lamb, 1997)

Cell death in the plant system in response to abiotic stresses such as waterlogging and hypoxia is a clear example of PCD.

Hypoxia stimulates ethylene production, which in turn activates a signal transduction pathway involving phosphoinositides and Ca2+

Plants defense mechanisms

constitutive inducible

The most robust resistance to pathogens often occurs when a plant

can specifically recognize the pathogen and rapidly induce a variety of potential defenses that limit its growth and/or development.

Plant Defense Responses

Passive (constitutive) defense Active (inducible) defense

PCD occurs when the pathogen unsuccessfully parasitizes the host as well as when the pathogen successfully causes disease.

Plantpathogen interactions depend on the classical gene-for-gene resistance model, requiring an avirulence gene (Avr) in the pathogen and a corresponding resistance gene (R) in the plant.

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Infection by Pseudomonas syringae pv. tomato

HC: host cells. Ba: Bacteria. Arrows

indicate the direction of type III secretion from bacteria in the apoplast into the host cell interior. Host cell wall remains intact, physically separating bacteria and host cells until the very late stages of the
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interaction, when host cells collapse.

The Hypersensitive Response

Resistance protein

Effector protein

Host Cell

Bacterium

Type III secretion

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The Hypersensitive Response

Hypersensitive cell death occurs at the site of attempted
HR lesions attack by an avirulent pathogen, and the HR leads to the formation of dry lesions that is clearly delimited from surrounding healthy tissue (Dangl et al., 1996) Resistant plant

Avirulent pathogen

Incompatible interaction, no disease

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Plant disease

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Disease symptoms

Virulent pathogen

Susceptible plant Compatible interaction, disease

15 http://www.as.wvu.edu/~cbarth/BIOL%20350%20Lecture%2032%20Induced%20Defenses%20080428%20ppt.p

The role of cell death during pathogenesis

When a plant is infected by a pathogen that can grow extensively, the interaction is called susceptible. Cell death during such a susceptible interaction may benefit pathogens that do not rely on living tissue to grow and develop.

PCD plays central role in pathogenesis Cells challenged by pathogens initiate an active PCD response, which is triggered by host-specific signals and requires synthesis of new proteins and/or activation of specific metabolic pathways (He et al.,1994; Greenberg, 1997)

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At least two types of cell death occur following the infection of a plant with a pathogen

The hypersensitive response (HR). A rapid PCD process that is activated in some plants in order to inhibit the spread of invading pathogen

Disease symptoms. This type of cell death which appears relatively late

during the development of some diseases and is considered to result from

toxins produced by invading pathogen.

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HR vs. Disease
Hypersensitive response (HR):
Rapid, localized plant cell death upon contact with avirulent pathogens. HR is considered to be a key component of multifaceted plant defense responses to restrict attempted Tobacco

infection by avirulent pathogens. Disease:

Chlorosis: A common disease symptom in pathogen infection in

which the leaf tissue appears yellow due to the loss of chlorophyll. Necrosis: A common, slow-developing disease symptom caused by necrotrophic pathogens. Tissue necrosis appears at very late stage of disease development.
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Tomato

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(Pennel R.I and C. Lamb, 1997)

HR helps the plant defense by limiting the spread of microorganisms, and represents a form of PCD (Lam et al, 2001).

Several lines of evidence suggest that the death of host cells during HR
results from the activation of an intrinsic cell death programme that is encoded by the plant genome.

HR is also believed to generate a signal that activates host defense mechanisms, and in many cases, induces long-lasting systemic acquired resistance (SAR) (Dong., 2001; Kovalchuk et al., 2003).

Induction of SAR is accompanied by an increase in the rate of synthesis of several pathogenesis-related proteins and the accumulation of a complex network of signaling molecules.
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Recognition of the pathogen avirulence (Avr) gene products by the plant initiates a signal transduction cascade that activates the HR. The final stage of the HR is PCD that play central role in the disease resistance. Critical steps in the HR are:

Interaction of the Avr-gene (X1, X2, X3) with the Resistance gene (R-gene) (RX1, RX2, RX3),

Convergence of the signals from the individual R genes into a conserved HR pathway;

Activation of NADPH oxidase induces the PCD.

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In plant disease response, localized PCD is a characteristic phenotype,and stands for both a symptom of susceptibility and resistance. Sphingolipid and

its phosphorylated derivatives, synthesized in plants through ceramide

biosynthetic pathway, are the signal molecules negotiated by the pathogens to trigger the endogenous PCD during susceptible disease response.

A resistant disease response in most cases is characterized by hypersensitive response (HR)-linked PCD at the infection site of an avirulent pathogen in recognition of a number of intrinsic signals such as caspases, reactive oxygen/nitrogen species, salicylic acid, mitogen-activated protein kinase, membrane ion channels, etc., cascading either alone or in combinations through a coordinated signal transduction pathway.

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Programmed Cell Death

PCD in compatible plant-pathogen interactions.

Ceramide signalling

PCD in incompatible plant-pathogen interactions

Role of caspases Role of ROS/ nitrogen species Role of SA and MAPK Role of ion fluxes

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Ceramide signalling

Ceramide signalling pathway as a critical second-messenger system has been studied in detail to understand apoptosis during both degenerative and

proliferative disease expressions in animal systems.

The balance between the bioactive sphingolipid ceramide and its phosphorylated derivatives has been proposed to modulate the PCD.

As second messengers, sphingolipids and sphingoid bases regulate cell behaviour at many levels, including cell-to-cell communication, growth factor receptors, growth, differentiation and transformation.

The most abundant and active SAM congeners, AAL toxin TA and fumonisins B1 (FB1), are functionally potent inhibitors of ceramide synthase in plants resulting in activation of PCD
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FB1-induced PCD in Arabidopsis protoplasts has been shown to be light-dependent and linked to jasmonate-, ethylene- and salicylatedependent signal transduction pathways.

phosphorylated derivatives of ceramide partially block plant PCD, suggesting the involvement of ceramide phosphorylation in

modulating cell death.

The genetic basis for susceptible disease response to SAMs has been mapped to the co-dominant Asc locus of the tomato plants

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No Apoptosis

Partial Apoptosis

Apoptosis

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(Khurana et al., 2005)

PCD in incompatible plant-pathogen interactions: Role of caspases

A family of cysteine proteases known as caspases are the key enzymes in the regulation of animal apoptosis

they have been shown to be also induced in plant systems undergoing PCD

Chichkova et al., have recently shown a plant caspase-like protease activity during the HR reactions in tobacco. (Chichkova et al., 2004)

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Role of reactive oxygen/nitrogen species

Extra cellularly secreted plant peroxidases catalyse the generation of ROS coupled to oxidation of IAA and defence-related compounds like salicylic acid (SA), aromatic mono-amines and chitooligosaccharides.

Inter and intra-cellular generation of ROS (O2 and H2O2) resulting in oxidative burst is widely accepted to trigger HR reactions

Signalling responses of ROS include the activation of mitogen-activated protein kinases and the up- and down-regulation of gene expression leading to PCD.

In general, ROS and NO interfere in phenylalanine ammonia-lyase (PAL) activity and ascorbate (ASC) and glutathione (GSH) metabolisms, and their

simultaneous increase activates a process typical of hypersensitive PCD

Hypersensitive cell death is activated following the interaction of NO with H2O2 (but not with O2 ) generated from O2 by superoxide dismutase.
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Role of salicylic acid

SA is a critical signaling molecule in the disease resistance pathways, including PCD and local and systemic resistance (Delaney et al., 1994). SA

accumulates more than 100-fold in the challenged area. Treatment of

exogenous SA induces many defense genes, phytoalexins and promotes ROS generation and PCD (Shirasu et al., 1997).

Besides promoting the PR gene expression and activation of early steps in the HR cascade, SA also signals mitochondrial processes in defense reactions.

The effects of SA is related with activation of the SA-inducible MAP kinase or interaction with SA-response elements in promoters of defense genes, and its inhibitory effect on mitochondria, emphasize the involvement of SA in diverse signaling pathways within the HR signal transduction.
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Similar to SA many defense responses are modulated by other plant hormones such as jasmonic acid, ethylene and abscisic acid (ABA) (Dong et

al., 1998; Klessig et al., 2000).

Production of phytoalexins that are low molecular weight secondary metabolites is one of the best defense responses in plants. The specificity of this compound changes depending on the compounds and on the pathogens (Dixon et al., 1994).

Consequently, the observed localization of phytoalexin biosynthesis to the area challenged by pathogens corresponds with the induction of PCD in the same cells (Dorey et al., 1997). Phytoalexins are stable compounds and stay in an active form even after the plant cell die.
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Role of ion fluxes

Membrane channel-related proteins are intimately involved in mediating HRlinked cell death. Ion channels play an important role in regulating PCD in

HR.

The role of cytosolic free Ca ([Ca2+]i) has been investigated in cowpea HR reactions involving PCD.

This study showed that the elevation of [Ca i2+] was involved in signal transduction leading to HR during rust fungal infection. Current line of evidence also supports that plasma membrane anion channels are essential components of early signal transduction processes resulting in PCD triggered by HR.

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Contd.,

The function of these anion channels is assumed to initiate or amplify plasma membrane depolarization, which in turn may activate Ca2+ voltage-dependent channels or K+ channels.

Mitochondria voltage-dependent-gated anion channels, a family of channels involved in the release of cytochrome c during apoptosis in mammals, are also presumed to operate in early stages of HR in plants.

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(Jones A.M, 2001)

Different signal transduction pathways contributing to activation of apoptosis during HR in incompatible plantpathogen interactions.

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(Paul Khurana et al., 2005)

The signaling downstream of the NADPH oxidase is regular to almost all types of plant PCD, including developmental PCD and physiological

responses to abiotic stress. Additional signaling molecules such as calcium

and salicylic acid (SA) regulate NADPH oxidase activation that transforms the extent PCD and associated defense reactions.

Following the recognition of pathogens by plants, which is mediated by plant R gene and pathogen Avr gene interactions, signals need to be transmitted and distributed to compartments involved in defense reactions. Application of protein kinase and/or phosphatase inhibitors indicated that the protein phosphorilation and dephosphorilation are involved in a numerous defense responses. Several protein kinases that participate in the perception of specific induction of defense responses have been identified and cloned.
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Selective inhibition or induction of apoptosis can be successfully employed to control plant diseases caused by necrotrophic or biotrophic pathogens respectively. Transgenic expression of animal antiapoptotic genes in plants provides broad-spectrum disease resistance against compatible obligate pathogens. Similarly, plantand animal-derived proapoptotic genes can be engineered to be expressed in plants for activating HR-linked resistant disease response against biotrophic pathogens.

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The beginning of this movie shows a cell before secondary cell wall synthesis. You can see

This movie shows another TE committing suicide. The focal plane is optimized to allow observation of the vacuole membrane, and you can see the membrane collapsing inward as the cell dies.

cytoplasmic streaming, indicating the cell is alive and happy. Then the movie jumps forward in time, and you can see that the cell has synthesized a secondary cell wall (stripes). The cell will suddenly stop cytoplasmic

streaming, and at the same time you can see that

there is a sudden implosion. The implosion is the large central vacuole collapsing. The cell is now, for all practical purposes, dead 38

http://www.bio.unc.edu/faculty/jones/lab/pcd

We found that tracheary elements commit suicide by triggering a massive ion flux across the plasma membrane, and that this is the driving force behind the collapse of the vacuole. This movie shows cells treated with mastoporan (a bioactive peptide from wasp venom), which triggers an influx of calcium into the cell that mimics the normal tracheary element suicide. The drug acts quickly, and the cells are still settling onto the bottom of the culture chamber during recording. Note that the indicated cell, a nascent tracheary element (see secondary cell wall thickenings) collapses the vacuole just like the tracheary elements undergoing their normal suicide in the previous two movies. 39

This movie shows the dramatic toxic effects of sodium azide, an inhibitor of respiration. You will see the cells move when the drug is applied, then cytoplasmic streaming stops -

but the vacuole does not collapse. Nor do

these cells fragment DNA.

http://www.bio.unc.edu/faculty/jones/lab/pcd

Greenberg J.T, 1997 , Programmed cell death in plant-pathogen interactions. Annu. Rev. Plant Physiol. Plant Mol. Biol.

Paul Khurana S. M., K. Suman., Pandey, S. Debabrata and A. Chanemougasoundharam, 2005, Apoptosis in plant disease

response: A close encounter of the pathogen kind, Current Sci, 88(5), 740-752.

Jones, A.M 2001, Programmed Cell Death in Development and Defense, Plant Physiology, 25; 94-97.

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