pcd

1. Doctoral Seminar II
Course No. Pl. PATH-692
Programmed Cell Death (PCD)
Presented by
Sharad Shroff
Ph.D. Scholar
Department of Plant Pathology

2. Introduction
• Plants possess an innate program for controlled
cellular demise is accomplished by activation of
genetically regulated cell suicide machinery that
requires the active participation of the cell in a suicide
process known as programmed cell death (PCD).
• In mammals, a genetically regulated, signal
transduction–dependent programmed cell death
process, commonly referred to as apoptosis.

3. Definition of Programmed cell death
(PCD)
• Programmed cell death (PCD) has been defined as
a sequence of (potentially interruptible) events
that lead to the controlled and organized
destruction of the cell.
(Lockshin and Zakeri, 2004)
The term apoptosis –A Greek word originally means that
‘’fall of the leaf or leaf falling’’ .
(John et al., 1972)

4. What is the purpose of this PCD ?
Developmental PCD
 Essential for successful development & growth of complex
multicellular organisms.
 Regulates the rate of cell division.
 Shaping of cells, tissues & organs.
Defensive PCD
 Control of cell populations & defense against invading microbes.
 PCD is needed to destroy the cells that represent a threat to the
integrity of the organism.

5. Two Classes of Plant PCD

6. Mechanism of PCD in Plants

7. Regulation of PCD in Plants
• Plant Proteases
Metacaspases: A family of cysteine proteases in plants that are most similar
to animal caspases.
Subtilisin-like serine proteases.
VPE family of protease
• Bcl-2-associated athanogene (BAG) family: an evolutionarily conserved
family of co-chaperones in mammals and plants distinguished by a
characteristic BAG domain that mediates direct interaction with HSP70.
• Hypersensitive mediated PCD: Rapid, localized plant cell death upon contact
with avirulent pathogens.
• Necrotrophic pathogen mediated cell death: Death of cell caused by
hydrolytic enzymes and host selective toxins.

8. Family of Plant protease
The Plant protease family can be subdivided into
• Metacaspases
Type 1 Metacaspases (AtMC 1, AtMC 2)
Type 2 Metacaspases (AtMC 4, AtMC 9)
• Subtilisin-like serine proteases.
• VPE family of protease

10. Metacaspase medaited PCD
• Pesudomonas syringae AtMC1 and -2 are positive
and negative regulators of HR-PCD, respectively.
• One of the most highly expressed type 2
metacaspase genes in Arabidopsis, Arabidopsis
thaliana Metacaspase 4 (AtMC4), is a positive
regulator of cell death induced by numerous
abiotic an biotic stresses.
• .

11. VPE family of protease
• Another family of plant proteases implicated in PCD is the
vacuolar processing enzyme (VPE) family of cysteine
proteases.
• This VPE is a positive regulator of HR induced by TMV
through its role in vacuolar collapse.
• The Arabidopsis δVPE protein is implicated in
developmental PCD of the inner integument layer of the
seed coat that occurs during embryo development.

12. Subtilisin-like serine proteases
• Subtilisin-like serine proteases (subtilases or
saspases) with caspase-like activity have also
garnered interest for their potential role in plant
PCD regulation.
• The first of the subtilases characterized from
plants, SAS-1 and -2, were purified from
extracellular extracts of Avena sativa (oat)
challenged with the PCD inducing fungal toxin
victorin.

13. Localization of Protease in Plant Cell
• Proteolytic enzymes that are involved in
PCD are localized in different compartments
of plant cells:
 The cytoplasm (metacaspases),
 The vacuoles (VPE),
 The intercellular fluid (phytaspases)

15. Role of Vacuoles in PCD
• The vacuole tonoplast can fuse with the plasmalemma to
release vacuolar contents extracellularly, or it can
disintegrate to release its contents to the cytosol.
• The vacuole also plays a crucial role in autophagic cell
death.
• During viral infection, the tonoplast is lysed with the
release of lytic enzymes of vacuoles into the cytosol.
Induction of rapid cell death pathways may be an effective
way of cleansing cytoplasm from viral growth.
• Sudden release of vacuolar contents into the cytoplasm
causes rapid cell death as was also noted for the HR
response to TMV in tobacco.

16. BAG family and role in PCD
• The role of Arabidopsis BAG family proteins in abiotic and biotic stress
responses and cell death modulation.
• The functions of BAG 1–3 are unknown.
• BAG 4 is involved in cell death inhibition in response to abiotic stress and binds
HSP70 molecular chaperones.
• BAG 5 forms a complex with CaM/HSC70 and regulates plant senescence.
• BAG 6 is proteolytically activated via aspartyl protease activity and links fungal
or chitin perception to the induction of autophagy.
• The ER-localized BAG 7 binds the molecular chaperone BIP2 and is an essential
component of the unfolded protein response.

17. BAG PROTEIN FAMILY

18. Hypersensitive response
mediated PCD
• 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 infection by avirulent pathogens.
• Rapid - within 24 h.
.
• HR also contributes to the establishment of the long-
lasting systemic acquired resistance against subsequent
attack by a broad range of normally virulent pathogens.

20. Mechanism Of Hypersensitive
Response Includes
• Ion flux (Increase in Ca+ ion in cytosol due to
activation of several ion channels)
• Oxidative bust (production of reactive oxygen species)
• Disruption of cell membranes opening of ion channels
• Cross linking of phenolics with cell wall component
• Production of anti-microbial phytoalexins and PR
protein
• Apoptosis (programmed cell death).

21. Pathogen Defense – Hypersensitive
Response

22. Disruption of Cell Membranes
Opening of Ion Channels
• The earliest detectable cellular events are ion fluxes
across the plasma membrane and a burst of oxygen
metabolism.
• Elicitor increases the open probability of plasma
membrane located ion channel and may thereby
stimulate elevated cytosolic calcium levels, as well as
activate additional ion channels and pumps.
• Mediated through the regulation of plasma
membrane-bound enzymes. These include changes
in Ca2+-ATPase and H+- ATPase activities.

23. 23
A simplified model for the potential coupling of Plasma
membrane depolarization and Ca2+ Influx.

24. Reactive oxygen species in
cell death processes
• Transient elevation of cytosolic calcium levels
necessary for elicitor stimulation of the oxidative
burst.
• Extracellular generation of ROS is a central
component of the plants defense machinery.
• ROS act as direct toxicants to pathogens, catalyze
early reinforcement of physical barriers and are
involved in signaling later defense reactions, such as
phytoalexin synthesis and defense gene activation,
programmed cell death and protective reactions .

25. 25
Interconversion of active oxygen species (AOS)
derived from O2

26.  Cyclic nucleotides may act as second messengers for NO
signaling.
 In tobacco nitric oxide synthase (NOS)-like activity was
strongly correlated with the expression of PR-1
 NO------------ phytoalexins, PAL, SA
 Both NO & SA----------- PR-1
26

27. Endogenous secondary signals in
plant disease resistance
• Salicylic acid: Plays a critical role in the activation of
defense responses.
• Increases in the levels of SA and its conjugates have
been associated with the activation of resistance
responses in a wide variety of plant species.
• These increases slightly precede or parallel the
expression of PR genes in both the infected tissue as
well as the uninfected tissues exhibiting SAR.

28. Necrosis is different from
Programmed cell death (PCD)
• Plant PCD occur as a function of pathogen invasion; these
include the hypersensitive response (HR), cell death–
inducing toxins, and responses to necrotrophic pathogens.
• In contrast to PCD, necrosis is most commonly defined as
cell death that results from exposure to highly toxic
compounds, Enzymatic degradation, severe cold or heat
stress, or traumatic injury that leads to immediate damage
to membranes or cellular organelles.
• The key distinction is that necrosis does not require the
active participation of the cell in its own demise.

29. Morphological difference between Necrosis
(Death by injury) and PCD (Death by suicide)

30. Pathological features of necrosis
and apoptosis

31. Necrotrophic Fungal Pathogens and
Host Cell Death
• Necrotrophic fungi kill host tissue using a plethora of toxins and
lytic enzymes to achieve pathogenic success.
• A classic example is victorin-induced PCD imposed by the fungal
pathogen Cochliobolus victoriae.
• Victorin is a host-selective toxin that is required for
susceptibility to this pathogen and the development of Victoria
blight disease in oats.
• Victorin sensitivity is genetically conditioned by the Vb gene,
which paradoxically has been proposed to be inseparable from
an R gene (Pc2) that controls HR-PCD against another fungal
pathogen,

32. Victoria blight of oat
Causal Organism- Cochliobolus victoriae
Necrosis caused by
Host specific Victorin toxin

33. Signaling pathway of Necrotrophs
• Generally it is assumed that no gene for gene resistance for
necrotrophic pathogen.
• PRRs(Perception of Recognition Receptors) involves in perception of
necrotrophic pathogens such as Receptor like protein Kinases (RLKs).
• Host selective toxins (HSTs) and Hydrolytic enzymes are considered as
efficient weaponry of necrotrophic fungi and the diseases caused by
Necrotrophs are manifested by the appearance of necrotic lesion.
• The defense response to necrotrophic pathogen conferred by RLKs,
defensin, Phytoalexin and JA/ET signaling.
(John et. al. 2003)

34. Contd….
• JA and Ethylene Signaling mediated defense responses are
expected to play key roles in resistance to necrotrophic
pathogens.
• JA and Ethylene Signaling leads to activation of defense related
genes i.e. PDF 1,2 which reduce hyphal elongation and triggering
of fungi permeability to suppress the necrotrophic fungi growth.
• HR response is extreme level of susceptibility for necrotrophic
pathogen.
• The high level of HR activated in biotrophs plant pathosystem
may also provide entry for Necrotrophs in the local
environment.
(John et. al. 2003)

35. 35

36. Hypersensitive Response By
Bacterial Pathogens
• Bacteria like Pseudomonas syringae inject effector
proteins (bacterial avirulence and virulence proteins)
into plant cells using the Type-III secretion system.
• Plants that are resistant to the bacteria have resistance
proteins that recognize the effector proteins and cause
the infected cell to commit suicide
(apoptosis/PCD/Hypersensitive Response).
• Prevents the bacteria from infecting the rest of the
plant by directly killing them and depleting nutrients.

37. 37
HR involving the TTSS

38. The Hrp pathway
• A type III secretion pathway, broadly conserved among
gram negative pathogens of plants and animals.
• Macromolecular structure, Hrp pilus, acts as conduit for
traffic (called needle complex in animal pathogens).
• Encoded by clustered hrp genes.
• Required for hypersensitive reaction and pathogenicity.
• Expression induced in plant and in defined minimal media.
• Capable of delivering proteins into host cells Secretes and
delivers “effector proteins”.
a) virulence factors
b) avirulence factors

40. How Mechanism of PCD
Regulation different in Plants
then Mammals ?

41. Mammalian PCD Regulation

45. Extrinsic apoptotic pathway in
mammals
• There are two major pathway routes for
mammalian PCD: so-called intrinsic and
extrinsic pathways.
• In brief, the extrinsic pathway is mediated by
extracellular “death” receptors that bind
various activating ligands, leading to
cytoplasmic processing and activation of
upstream caspases, which activate downstream
caspases, leading to cellular execution.

46. Intrinsic apoptotic pathways
• The intrinsic apoptotic pathway in animals is generally
associated with the mitochondrion, It is switched on in the
case of internal cell defects(DNA damage, various stresses,
cytotoxic agents).
• Regulation of this pathway involves a large group of
protein from BCl-2 family which includes both pro and anti
apoptotic proteins.
• Cytochrome c loss in mammals occurs through the
mitochondrial permeability transition pore complex when
a death-promoting protein, such as Bax (proapoptotic Bcl-2
family member), induces the opening of the mitochondrial
voltage-dependent anion channel (VDAC).

47. Contd….
• The subsequent release of cytochrome c stimulates the
formation of an apoptosome complex in mammals,
which is composed of cytochrome c, procaspase-9, and
apoptotic protease-activating factor 1 (APAF-1).
• After activation in apoptosome complex caspase 9
triggers the processing of caspase 2, -3,-6,-7, -8.
• APAF-1 is related to the NB-LRR family of proteins, of
which plant R genes are also notable members.

48. APOPTOTIC FEATURES ANIMALS PLANTS
Cell shrinkage Yes Yes
Chromatin condensation Yes Yes
Phosphotidylserine externalization Yes Yes
DNA laddering Yes Yes
TUNELS-DNA cleavage Yes Yes
Caspases Yes No
Mitochondria permabilization Yes Yes
Mitochondria depolarization Yes Yes
Cytochrome c release Yes Yes
Cytochrome c–dependent activation of cell
death
Yes No
Apoptotic bodies Yes Yes
Vacuole leakage and fusion with
plasmalemma
No Yes
FEATURES ASSOCIATED WITH ANIMAL AND
PLANT APOPTOTIC CELL DEATH

49. References
Kabbage M. et. al. (2017): The Life and Death of a Plant
Cell. Annual Review of Plant Biology. 2017. 68:375–404.
Martin B. et. al. (2013): Centrality of Host Cell Death in
Plant-Microbe Interactions. Annual Review of
Phytopathology. 2013. 51:543–70
John M. et. al. (2003) Plant disease resistance genes:
recent insights and Potential applications. Trends in
Biotechnology Vol. 21 No.4 April 2013. 178-183.
Agrios G N; Plant Pathology. Elsevier Academic Press,
Burlington, MA, 2005.