Wangkhem Tampakleima Chanu Ph.D Scholar, Department of plant pathology, COA, CAU,Imphal

1. CREDIT SEMINAR
ON
PROGRAMMED CELL DEATH IN PLANT
DISEASE PERSPECTIVE
PRESENTED BY
WANGKHEM TAMPAKLEIMA CHANU
Ph.D 1st yr 2nd sem
Adm. No.2A-16(Ph.D)
DEPARTMENT OF PLANT PATHOLOGY
COLLEGE OFAGRICULTURE,CAU

2. 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. PHASES OF PCD IN VASCULAR PLANT
Source: http://science.howstuffworks.com/life/cellular-microscopic/apoptosis.htm

6. What is the purpose of this PCD ?
1. Essential for successful development & growth of complex
multicellular organisms.
2. Regulates the rate of cell division.
3. Shaping of cells, tissues & organs.
4. Control of cell populations & defense against invading
microbes.
5. PCD is needed to destroy the cells that represent a threat
to the integrity of the organism.
Examples: Cells infected with virus
Cells with DNA damage

7. Morphological difference between Necrosis (Death by
injury) and PCD (Death by suicide)
Source: Studzinski, 1999

8. Table 1: Pathological features of necrosis and apoptosis
Necrosis Apoptosis
1. Pattern of death Group of neighboring cells Single cells
2. Cell size Swelling Shrinkage
3. Plasma
membrane
Smoothing Blebbed
4. Mitochondria Swelling disordered
structure
Contents released into the
cytoplasm and contracted
5. Organelle shape Disruption Apoptotic bodies
6. Nuclei Membrane disruption Clumps and fragmented
7. DNA
degradation
DNA fragmentation is
random or smeared
Ladder like- DNA
fragmentation
8. Cell degradation Macrophage invasion
Inflammation
Phagocytosis
No inflammation
Source: Unsal, N.P. et al., 2005

9. Classes of plant PCD

10. Two classes of plant PCD
PCD Class Examples
Autolytic Developmental PCD.
Example: PCD that occurs during the formation of the
male and female zygotes, in seeds (except endosperm in
cereals), in embryonic structures, and during development
of roots and shoots.
Non-
autolytic
It is a class of PCD where tonoplast rupture may or may
not occur and is not followed by complete clearance of the
cytoplasm.
Example: Hypersensitive response (HR)-related PCD.
Endosperm in cereal seeds is an example of non- autolytic
PCD (no tonoplast rupture).
Source: Wouter G. van Doorn, 2011

11. PROGRAMMED CELL DEATH
MECHANISM

12. PCD REGULATORS
Most important player
Proteases
Aspartate-
directed
Cysteine
dependent
Cysteine
dependent
Aspartate-
directed
Proteases
Caspases

13. Caspases
Initiator caspases Executioner caspases
Initiate the process of
apoptosis
Execute the process of
apoptosis
Caspases 2,8,9 & 10 Caspases 3,6 & 7

14. All caspases are in inactive form. These are called
procaspases
The initiator caspases are activated by extrinsic or intrinsic
pathway
The executioner caspases are activated by initiator
caspases

16. Programmed cell death in response to biotic stress
Two types of cell death occur following the infection of a plant
with a pathogen:
1. Hypersensitive response: HR is a process of PCD associated with
plant resistance to pathogen infection and occurs at incompatible and
sometimes in compatible plant pathogen interactions.
2. 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.
Source: Unsal, N.P. et al., 2005

17. Hypersensitive response (HR) - mechanism, used by plants,
prevent the spread of infection, microbial pathogens
Strong resistance reaction of plants against pathogens
Interaction, ‘avr’ gene (pathogen) and ‘R’ gene (plant)
E. C. Stakman (1915) credited, hypersensitive reaction (HR)
HR - plant disease, bacteria was 1st recognized, Klement
(1963)

18.  HR, result, ‘incompatible reaction’, ‘R’ gene (non-host) plant
corresponds to ‘avr’ gene, (pathogen)
 ‘Compatible reaction’ ‘R’ gene (host plant ) does not match
with ‘avr’ gene (pathogen) resulting, spread of pathogen,
disease occurs.
When the virulent pathogen
artificially injected to non-host or
resistant plant and when
Avirulent strain into susceptible
plant, it induce HR
Occurs – vertical resistance

19. Source: Collmer et al., 2000

20. Hypersensitive Response

21. Hypersensitive
responses kill small
parts of the leaf
Source: res2.agr.gc.ca/ecorc/ corn-
mais/images/fig-22.jpg

22. HR - programmed cell death (PCD) associated with death of small no.
of cells at and around site of infection (Fig.1)
HR – inhibit, growth of invading pathogen, killing infected and
uninfected cells, producing a physical barrier composed of dead cells.
Fig 1: Hypersensitive response
on a tobacco leaf during
pathogen infection
HR manifested with development
of necrotic lesions (stained with
Evans blue), localized desiccation
and browning of the affected cells.
A) initial hours of infection B) late
hours of infection
Source: Wright et al., 2000

23. During HR, dying plant cells strengthen their cell walls and
accumulate certain toxic compounds, phenols and phytoalexins
Fig 2: Accumulation of phenolic
compounds during HR
Autofluorescence of phenolic
compounds and cell wall
thickenings in plant tissues
during HR, UV light
 A) initial hours of pathogen
infection (absence of phenols
shown with asterix)
 B) late hours of infection
(phenol compounds shown with
arrows)
Source: Soylu, 2006

24. HR also occurs when pathogen-derived molecules or unique
proteinaceous bacterial elicitors like ‘harpin’ interact with non-host
plants
Fig 3: HarpinPss infiltration induces
HR on tobacco leaves
Reaction of tobacco leaves with
(A) Harpin Pss
(B) control leaf (buffer alone).
Source: Greenberg et al., 1994

25. Characteristics
1. Rapid death of cells: Seals of the wounded
tissue, prevent the pathogen, moving into the
plant
2. Restrict growth and spread of the pathogens
3. Analogous to the innate immune system,
animals and precedes a slower systemic (whole
plant) response, leads to SAR

26. 1. Interaction of the Avr-gene (X1, X2, X3) with the
Resistance gene (R-gene) (RX1, RX2, RX3),
2. Convergence of the signals from the individual R genes
into a conserved HR pathway;
3. Activation of NADPH oxidase induces the PCD.
Critical steps in the HR are:

27. 1. The activation of cell death in the absence of pathogens
by mutations in certain genes thought to be involved in
the cell death pathway.
2. The activation of cell death upon recognition of elicitors
produced by the pathogen, and
3. The activation of the HR by expressing of transgenes in
plants
Evidences suggesting that HR results from PCD process are:
Source: Heath, 2000

28. MECHANISM of HR
Phase 1: The activation of R genes triggers an ion flux,
involving an efflux of hydroxide and potassium outside the cells,
and an influx of calcium and hydrogen ions into the cell.
Phase 2: These generate an oxidative burst by
producing reactive oxygen species(ROS), superoxide
anions hydrogen peroxide hydroxyl radicals and nitrous oxide.
Compatible interaction between R gene product and Elicitor
activates a cascade of biochemical reaction, activate defense
related compound

29. These results in death of affected cells & formation of local lesions.
ROS also trigger deposition of lignin and callose as well as the cross-
linking of pre-formed hydroxyproline-rich glycoproteins.
These event leads to biosynthesis of SA, JA, & ET, leads to long
lasting SAR
Secondary metabolite
Inhibitory protein
Phytoalexin

30. Pathogen Defense – Hypersensitive Response

31. Abiotic stress Ultrastructural changes
Hypoxia-lysigenous Chromatin condensation and DNA fragmentation
Aerenchyma
formation
Organelle surrounded by membranes
Plasma membrane invagination and tonoplast degradation
Cell wall degradation
Light radiation Oligonucleosomal fragmentation of DNA
Migration of nuclear contents to cell periphery
Mechanical stress TUNEL positive material around nuclear periphery
Oligonucleosomal fragmentation of DNA in chloroplast and
nuclei
Cold stress Chloroplast swelling, thylakoids distort and swell, grana unstuck
and chloroplast lyse, nuclei swell, chromatin fragments, ER and
golgi cisternae swell, cytoplasmic condensation occurs
Ozone Ozone enters the apoplast where it elicits generation of ROS,
mainly hydrogen peroxide
Programmed cell death in response to abiotic stress
Source: Evans, 2004.

32. CASE STUDY….

33. Case study 1- Beneficial effect of Programmed
cell death in plant
Mittler et al., 1997 worked on ‘’Pathogen-
induced programmed cell death in tobacco’’

34. Activation of cell death, following recognition of invading
pathogens, results in the formation of a zone of dead cells localized
around the site of infection also called a hypersensitive response
(HR) lesion.

35. Material and methods
Plant material and pathogen infection
1. Wild-type tobacco plants were grown under continuous
illumination provided by cool-white fluorescent lamps .
2. Fully expanded young leaves of 5- to 6-week-old plants were
infected with TMV strain U1 and kept at 30°C for 4 days
under continuous light.
3. PCD was induced by shifting of TMV-infected and control
uninfected plants from 30°C to 25°C and cell death was
assayed by measuring ion leakage from leaf discs.

36. Fig. 4. Cross sections through stems (A,B,C) or leaves (D,E,F) fixed 0 (A,D), 24 (B,E)
and 48 (C,F) hours after induction of PCD (a shift from 30°C to 25°C of TMV-infected
plants). Cells at different stages of cell death can be seen at the border of lesions,
indicated by arrows in B,C. E, epidermis; P, parenchyma.
RESULTS

37. Case study 2- Detrimental effect of Programmed
cell death in plant
Lincoln et al., (2002) worked on “Expression
of the antiapoptotic baculovirus p35 gene in
tomato blocks programmed cell death and
provides broad-spectrum resistance to
disease”

38.  The sphinganine analog mycotoxin, AAL-toxin, is
the primary determinant of the Alternaria stem
canker disease of tomato, thus linking apoptosis to
this disease caused by Alternaria alternata f. sp.
lycopersici.
 The product of the baculovirus p35 gene is
a specific inhibitor of a class of cysteine
proteases termed caspases, transgenic
tomato plants bearing the p35 gene were
protected against AAL-toxin-induced death
and pathogen infection.

39. Pathogenicity Assays
Alternaria alternata f. sp lycopersici
 This forma specialis of A. alternata causes the Alternaria stem canker
disease of tomato and secretes the host-selective AAL toxins as primary
chemical determinants of the disease
 Spore concentrations of 1 X 104, 1 X 105 and 1 X 106 spores per ml were
used to assess the dosage dependent contribution of p35 to resistance.
A. alternata.
 The strain of A. alternata that causes the black mold disease of tomato
does not produce any detectable AAL-toxin, can only infect ripening
tomato fruit, and has no known genetic resistance.
 Mean lesion diameter of lesions on p35 expressing fruit was compared with
mean lesion diameter on susceptible control fruit.

40. RESULTS
In case of A. alternata f.sp. lycopersici
Given the host-selective nature of AAL-toxin, it was reasoned that
decreased toxin sensitivity would translate into decreased disease
symptoms caused by the toxin-producing fungus, Aal.
Fig 5: Expression of p35 and pathogenicity of Aal fungus.

41. In case of A. alternata :
 The size of the lesions is at least 50% smaller in p35-expressing
fruit than in control fruit (Fig.6), and in some cases they were
free of black mold symptoms.
 This means that p35 expression provides postharvest protection
against this fungal necrotroph.

42. Fig 6: Expression of p35 and pathogenicity of black mold

43.  From this report it is evident that in vivo
expression of the baculovirus p35 gene
effectively blocks apoptotic cell death as
induced by either a host-selective toxin or any
of several necrotrophic pathogens, leading to
protection against the disease caused by these
pathogens.

44.  The process of PCD is essential for ensuring the proper
development of plants as well as ensuring a robust defense
response against invading pathogens.
 But major contributions will come from research focused
specifically on how cell death occurs with in unique biological
contexts (Hypersensitive response, etc.) that are of interest to
plant biologists & important for improving agriculture
CONCLUSION