Programmed cell death (pcd)

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

9. 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. .

10. 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.

11. 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 .

12. 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 )

13. 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 .

14. 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.

15. BAG PROTEIN FAMILY

16. 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 .

17. 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).

18. Pathogen Defense – Hypersensitive Response

19. 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.

20. 23 A simplified model for the potential coupling of Plasma membrane depolarization and Ca2+ Influx.

21. 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 .

22. 25 Interconversion of active oxygen species (AOS) derived from O 2

23. 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 Nitric Oxide (NO)

24. 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.

25. 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.

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

27. Pathological features of necrosis and apoptosis

28. 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 ,

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

30. 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)

31. 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)

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33. 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.

34. 37 HR involving the TTSS

35. 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

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

37. Mammalian PCD Regulation

38. 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.

39. 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).

40. 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.

41. 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

42. 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.