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Autophagy and its role in plants - By Tilak I S, Dept. of Biotechnology, UASD.
- 1. Autophagy and its role in plants TILAK I. S. PGS17AGR7335 Ph. D. Dept. of Biotechnology University of Agricultural Sciences, Dharwad DOCTORAL SEMINAR -II
- 2. Contents Introduction History Types of autophagy Mechanism of autophagy Functions of autophagy Future prospects Conclusion 1/17/2020 2Department of Biotechnology, UASD.
- 3. AUTOPHAGY • Autophagy (Greek word)- “Self-eating” • Catabolic process of intra cellular degradation and recycling of cytoplasmic contents under stress conditions or during development. - Role: Creates energy for cells, Cell homeostasis, Nutrient recycling, Turnover of dysfunctional organelles & Defense against pathogens and toxic products. (Li et al., 2012) Christian de Duve 1/17/2020 3Department of Biotechnology, UASD.
- 4. Vacuole Unwanted cellular material (Cytoplasm) Degradation Degradaded / broken cellular components (Klionsky and Ohsumi, 2014)1/17/2020 4Department of Biotechnology, UASD.
- 5. (Mizushima et al., 2019)1/17/2020 5 Department of Biotechnology, UASD.
- 6. Timeline of autophagy research (Soto-Burgos et al., 2018)1/17/2020 6 Department of Biotechnology, UASD.
- 8. How autophagy is activated ? 1) Nutrient starvation 2) Oxidative, salt, ER stress 3) Pathogen invasion 4) Protein aggregation/ organelle aggregation ATG proteins can be divided into 3 classes i) ATG1-ATG13 kinase complex ii) ATG6/vps30 complex iii) ATG5-ATG12 complex and ATG8-PE complex (two ubiquitin like conjugation systems) (Frake et al., 2016)1/17/2020 8 Department of Biotechnology, UASD.
- 9. PAS Amino acids/Lipids Sugars Cytoplasm (Suzuki et al., 2017) 1/17/2020 9Department of Biotechnology, UASD.
- 11. Types of autophagy Macroautophagy (Autophagy) Microautophagy Plants Chaperone mediated autophagy Animals (Li et al., 2012)1/17/2020 11Department of Biotechnology, UASD.
- 12. (Okomoto et al., 2014) Types of autophagy Selective & Non selective Vacuole Vacuole 1/17/2020 12 Department of Biotechnology, UASD.
- 13. Selective autophagy “It is an autophagic process that degrades specific cytoplasmic components such as protein complexes, aggregates, organelles and pathogens” Types of selective autophagy: 1. Aggrephagy: Misfolded protien aggregates are degraded. 2. Chlorophagy: Eliminate nonfunctional chloroplasts. 3. Mitophagy: Eliminate nonfunctional mitochondria. (Vierstra et al., 2018)1/17/2020 13 Department of Biotechnology, UASD.
- 14. 4.Pexophagy: Degradation of nonfunctional perioxisomes via autophagy. 5.Ribophagy: Degradation of non functional ribosomes. 6.Proteaphagy: Degradation of ubiquitylated protiens via the Ub- proteasome system. 7.Xenophagy: Cell directs autophagy against pathogens. 1/17/2020 14Department of Biotechnology, UASD.
- 15. Two types of selective autophagy receptors (UBD) ubiquitin binding domain , SQSTM1: Cargo receptor (Fujita et al., 2012) ATG8 ATG40 1/17/2020 15Department of Biotechnology, UASD.
- 16. (Li et al., 2012) Ub-dependent & independent selective autophagy 1/17/2020 16 Department of Biotechnology, UASD.
- 17. Regulator: TOR Stages of autophagy Mechanism of autophagy
- 18. Target of Rapamycin (TOR) (Burgos et al .,2018) IRE1b: Inisitol-requiring enzyme-1. Snf1: related protein kinase 1 1/17/2020 18 Department of Biotechnology, UASD.
- 19. (Ustun et al., 2017) Induction Autophagosome formation Phagophore initiation Breakdown (Degradation) Docking and fusion with vacuolar membrane Stages of autophagy 1/17/2020 Department of Biotechnology, UASD.
- 20. Induction Dephosphorylation of ATG13 under stress condition leads to high affinity to ATG1 & ATG17 This complex enhance kinase activity Inhibition of TOR under starvation leads to ATG1 complex activation (Ustun et al., 2017)1/17/2020 Department of Biotechnology, UASD.
- 21. 1/17/2020 21Department of Biotechnology, UASD.
- 22. 1/17/2020 22Department of Biotechnology, UASD.
- 23. SNARE (snap receptor like protein complex) and Rab protein (family of Gprotien GTPase) primary role in vesicle fusion with vacuole Autophagic bodies are then degraded via ATG15, lipase and vacuolar hydrolases Docking and fusion (Ustun et al., 2017)1/17/2020 23 Department of Biotechnology, UASD.
- 24. Identification and characterization of ATG genes Sequences of ATG genes from Arabidopsis and rice were used as queries to search against corresponding genomic sequences for most of 14 crop species ATG proteins in crops are typically encoded by a single gene To date, core ATG genes have been identified in 14 crop species (Tang and Bassham., 2018)1/17/2020 24 Department of Biotechnology, UASD.
- 25. (Tang and Bassham., 2018) Newly identified autophagy genes in crop species 1/17/2020 25Department of Biotechnology, UASD.
- 26. Crop species with identified ATG genes and potential processes that require autophagy (Tang and Bassham., 2018)1/17/2020 26 Department of Biotechnology, UASD.
- 27. Functions of autophagy • Leaf senescence • Seed development • Reproductive development Development • Nitrogen starvation • Drought stress • Endoplasmic reticulum stress Abiotic stress • Plant-Pathogen interaction Microbe interaction (Tang and Bassham., 2018)1/17/2020 27Department of Biotechnology, UASD.
- 28. Role of autophagy during seed development Crop ATG genes (Autophagy) Stage Result Arabidopsis Upregulated in siliques Seed maturation Good growth, No Seed abortion. Maize ATG8–PE adducts in endosperm Endosperm development Enhance the seed size (Tang and Bassham., 2018)1/17/2020 28 Department of Biotechnology, UASD. PE: Phosphatidyllinositol
- 29. Induction of ATG genes during seed maturation in arabidopsis 1) Columbia wild-type 2) Atg5 mutant (Autophagy defective) Induction of ATG genes during seed maturation. (WT). Presence of many GFP-ATG8 autophagosomes confirmed the presence of autophagic activity in WT seed embryos. (Not seen in atg5 mutant) Plant grown under low & high nitrate to check seed abortion. (No/less seed abortion: Wt) 12S globulin accumulated earlier in atg5 seeds than in WT. (Berardino et al., 2018)1/17/2020 29Department of Biotechnology, UASD.
- 30. • Relative expression of almost all the ATG genes increased during seed maturation Autophagy was clearly induced from the early stage of silique development and that it increased sharply after 20 DAF Involved in the ATG4 conjugation system Induction of the autophagy process. (AT1/ATG13 complex) (Berardino et al., 2018) 1/17/2020
- 31. Immunodetection of autophagosomes in the WT (a,c,e), that were absent in atg5 (b,d,f) (Berardino et al., 2018) WT Atg5 mutantWT 1/17/2020 31 Department of Biotechnology, UASD.
- 32. Plants grown under Low Nitrate (2mM) High Nitrate (10mM) Col No seed abortion Seed abortion 6% Atg5 Seed abortion 21% Seed abortion 33% Atg5 mutant exhibit early browning 16DAF Col seeds remained green longer (Berardino et al., 2018)1/17/2020 32 Department of Biotechnology, UASD.
- 33. Autophagy related gene expression increases during seed maturation, especially in the embryo (Wt) Wt 1) Autophagsome activity. 2) Increased expression of all ATG genes. 3) No/less Seed abortion 4) Seeds remained greener 5) 12S globulin accumulation delayed 6) Good growth 7) Low N content Atg5 mutant 1) Autophagy defective. 2) -- 3) Higher seed abortion. 4) Early browning. 5) Early accumulation of 12S globulin. 6) Abnormal growth 7) High N content (Berardino et al., 2018)1/17/2020 33 Department of Biotechnology, UASD.
- 34. Abiotic stress induces autophagy Stress Crop Nitrogen starvation First studied in Arabidopsis.(apple , barley, foxtail millet , grapevine , maize , pepper, rice and wheat ) Drought stress First elucidated in Arabidopsis, indicated by the upregulation of ATG8a. Endoplasmic reticulum stress First elucidated in Arabidopsis, (Tang and Bassham., 2018)1/17/2020 34 Department of Biotechnology, UASD.
- 35. Overexpression of autophagy related gene SiATG8a from foxtail millet (Setaria italica L.) confers tolerance to both nitrogen starvation and drought stress in Arabidopsis. SiATG8a is mainly expressed in stems and its expression was dramatically induced by drought stress and N starvation treatments. (Li et al., 2015) (A) Expression patterns of SiATG8a in plants treated for 1, 6, 24, 36, and 48 h with 6% PEG 6000. (B) Expression patterns of SiATG8a in plants cultured on N-deficient liquid medium for 1, 2, 3, and 6 days. 1/17/2020 35 Department of Biotechnology, UASD.
- 36. 36 Overexpression of SiATG8a in transgenic Arabidopsis enhanced tolerance to nitrogen starvation
- 37. 37 Overexpression of SiATG8a enhanced tolerance to drought stress Malondialdehyde
- 38. Wild type : Plants with AtATG18a confers resistant during salt and drought stresses. RNAi : AtATG18a plants are autophagy defective. (Yimo Liu et al., 2012)1/17/2020
- 39. Role of autophagy in plant immunity 1/17/2020 39Department of Biotechnology, UASD.
- 40. Plant defense mechanism Pathogen/Microbe-associated molecular patterns Detection Ethylene production, Callose deposition, Cell wall thickening, Antimicrobial proteins, Pathogen inject effectors (to evade) Another layer of defense to recognize effector modifcations R-mediated defenses Hypersensitive response (HR) to limit spread of the pathogen. (Kabbage et al., 2013) 1/17/2020 40Department of Biotechnology, UASD.
- 41. Autophagy is engaged in various aspects Functions as Anti and promicrobial Antiviral Antibacterial and Antifungal mechanism Interestingly, phytopathogens are able to manipulate plant autophagy. 1. To target defense compounds 2. To enhance nutrient acquisition 1/17/2020 41Department of Biotechnology, UASD.
- 42. Anti-microbial Pro-microbial (Tang and Bassham., 2018) The dual role of autophagy during plant–pathogen interactions in crops REM: Remorin1/17/2020 42
- 43. Antiviral defense by targeted degradation of VSR protein Antibacterial: Avirulent (Pst) (avrRps4) induces autophagy, which contributes to HR Autophagy is an integral part of plant immunity (Hofius et al.,2014)1/17/2020 43Department of Biotechnology, UASD.
- 44. Cell Death Control: The Interplay of Apoptosis and Autophagy in the Pathogenicity of Sclerotinia sclerotiorum Sclerotinia sclerotiorum (Necrotrophic fungal pathogen) OA production : Pathogenic Defective in OA production: less virulent. Inhabits autophagy pathway. Induces apoptotic cell death (Kabbage et al., 2013) 1)Inoculation of wild type S. sclerotiorum to transgenic Arabidopsis (CED-9) anti- apoptotic gene resulted in a resistant phenotype. 2) Inoculation of Arabidopsis with an OA deficient non-pathogenic mutant strain of S. sclerotiorum (A2) : No effect on penotype 1/17/2020 44Department of Biotechnology, UASD.
- 45. (Kabbage et al., 2013)1/17/2020 45 Department of Biotechnology, UASD.
- 46. Expression of ced-9 in Arabidopsis inhibits wild type infection but does not affect the A2 phenotype -Growth of WT S. sclerotiorum was markedly inhibited in CED-9 leaves compared to Col- 0 control plants. -A2 phenotype remained unchanged when inoculated onto wild type or transgenic plants expressing CED-9. (Kabbage et al., 2013) 46
- 47. S. sclerotiorum A2 mutant triggers the production of autophagosomes TEM analysis: Presence of autophagy structural features following inoculation with the A2 mutant BUT not in the wild type challenge (Kabbage et al., 2013) 1/17/2020 47 Department of Biotechnology, UASD.
- 48. • Research on applied part of plant autophagy is still in its infancy. • Regulators (TOR) characterized in Arabidopsis have not yet been well-studied in other crops. • Need to develop drugs to manipulate plant- specific autophagy. Future prospects 1/17/2020 48Department of Biotechnology, UASD.
- 49. Conclusions 1/17/2020 49Department of Biotechnology, UASD.
- 50. Thank You 1/17/2020 Department of Biotechnology, UASD.
Editor's Notes
- Plants have developed sophisticated mechanisms to survive when in unfavorable environments Autophagy, literally defined as “self-eating,” , is a universal catabolic process of intra cellular degradation of macromolecules and organelles, functions as a degradation process by recycling cytoplasmic contents (cargo) (proteins, protein aggregates and organelles) under stress conditions or during development. Upon activation of autophagy, a membrane structure known as a phagophore forms and expands, finally closing to form a double-membrane vesicle called an autophagosome The completed autophagosome, which contains the autophagic cargo, is delivered to the vacuole (plants and yeast) or lysosome (animals), Which contains digestive hydrolytic enzymes cathepsin, hydroleases, proteases , lipases etc Which separate junk into small component parts . The outer membrane fuses with the vacuolar/lysosomal membrane, and the inner membrane and contents are released into the vacuole/lysosome as an autophagic body and are degraded by hydrolases. The breakdown products are transported back into the cytoplasm for reuse by the cell . Role of Autophagy it is the main housekeeper that removes cellular debris and replenishes essential nutrients needed for new growth Nutrient recycling, development, cell homeostasis, and defense against pathogens and toxic products. -dysregulated autophagy (human) -pathophysiologies, such as cancer, myopathies, neurodegeneration, heart and liver diseases, and gastrointestinal disorders dysregulated autophagy (Plants) – Early senescence
- during stress conditions or specific developmental processes. Autophasome which collect the thrash and transport into vacuoles (plant and yeast) or lysosome(animals) Which contains digestive hydrolytic enzymes cathepsin, hydroleases, proteases , lipases etc Which separate junk into small component parts ,, this can be used again for further development Explain about Apoptosis, necrosis & autophagy
- Discoveries of mechanisms for autophagy in Yeast – Saccharomyces cerevisiae Characterisation of autophagy in yeast: Demonstrated that autophagy in yeast is similar to that in mammalian cells The group used electron microscopy to identify and characterize double- membraned „autophagosomes‟ as the precursors of „autophagic bodies‟ in yeast
- The core autophagic machinery consists of autophagy-related (ATG) proteins, which function during the induction of autophagy and formation of autophagosomes. A conserved set of proteins encoded by autophagy-related (ATG) genes facilitate the initiation, elongation, maturation, and fusion of the autophagosome with the vacuole ATG proteins can be divided into three major groups ATG1/ATG13 kinase complex:responds to upstream signals and induces downstream autophagosome formation ) Phosphatidylinositol-3-kinase (PI3K) complex /ATG6/vps30 complex : essential for phosphorylating phosphatidylinositol to produce phosphatidylinositol-3-phosphate (PI3P), which is required to recruit proteins involved in autophagy Eg: Arabidopsis, SH3 domain-containing protein 2 (SH3P2) is a recently identified protein that binds to PI3P and is potentially involved in autophagosome membrane remodelling and autophagosome fusion with the vacuole . -ATG8 conjugation to the membrane lipid phosphatidylethanolamine (PE) requires two ubiquitin conjugation-like pathways 3) ATG5-ATG12 complex and ATG8-PE complex The core machinery for autophagosome formation includes ATG1, which forms a complex with ATG13 for the induction of autophagy (Kamada et al., 2000); two ubiquitin-like conjugates, ATG12-ATG5 and ATG8-PE, which are recruited to the phagophore assembly site and play an important role in autophagosome formation (Yin et al., 2016); andATG9, which may function in the recruitment of other ATG components and membrane to the forming autophagosome (Reggiori et al., 2005).
- Vacuole: a large, membrane-bound organelle found in plant and fungal cells; it houses hydrolytic enzymes that degrade autophagic bodies Microautophagy: a degradative pathway mediated by direct vacuolar engulfment of cytoplasmic components by invagination and subsequent scission of the tonoplast Macroautophagy: degradation of cytoplasmic components following engulfment by a phagophore and delivery of the resulting autophagosome to the vacuole
- Pre-autophagomal-structure/site (PAS)- the putative site for autophagosome formation Several types of autophagy have been described in many species, including Microautophagy (84), Macroautophagy (135), Caperone-mediated autophagy (92), and In plants, microautophagy and macroautophagy have been shown to occur In macroautophagy :The principal characteristic of autophagy is the formation of double-membrane structures called autophagosomes (Figure 1). Upon induction of autophagy, phagophore sequesters cytosolic components and then seals to form a double membrane–bound autophagosome. Phagophore initiation and expansion are directed by the pre-autophagosomal structure (PAS), which is generated by a hierarchical assembly of autophagy-related proteins. Autophagosomes fuse with the tonoplast to release the internal vesicle as an autophagic body into the vacuolar lumen, where its cargo is degraded by resident hydrolases. These digestion products are either stored in the vacuole or transported back to the cytosol for re-use. autophagosomes .( hereafter referred to as autophagy ) In microautophagy, Microautophagy proceeds by invagination of the tonoplast to engulf portions of the cytosol directly into autophagic bodies within the vacuole (formation of a small intravacuolar vesicle called an autophagic body by invagination of the tonoplast, thus engulfing cytoplasmic components ) (By Invagination of the lysosome membrane, cytosolic components are directly taken up by the lysosome itself through.) (The lysosome/vacuole itself engulfs small components of the cytoplasm by inward invagination of the lysosomal membrane) (Lysosomes directly engulf cytosolic components via lysosomal membrane invagination. ) In chaperone-mediated autophagy (CMA), the Hsc70/co-chaperone complex delivers specific substrate proteins to lysosomes. (Lysosomes directly engulf cytosolic components via lysosomal membrane invagination. (Chaperone proteins (e.g., heat shock cognate 70 protein; HSC-70) interact with cytosolic proteins destined for degradation. This complex is recognized by a lysosomal-associated membrane protein-2A (LAMP-2A), resulting in the translocation of the unfolded cytosolic protein into the lysosome.) resulting in their unfolding and degradation. The through the lysosomal membrane protein Lamp-2A and digested in the lysosomal lumen. found only in mammals.involved in reducing aging process degrade only proteins but not whole organelles Selective autophagy pathways in plants. The selective degradation of proteasomes (proteaphagy) in response to nutrient starvation. The proteasome subunit RPN10 selective autophagy receptor interaction with ATG8. Selective autophagy further mediates degradation of whole organelles such as chloroplasts (chlorophagy), mitochondria (mitophagy) or peroxisomes (pexophagy), but the specific cargo receptors remain to be identified. Single proteins are also subjected to selective autophagic degradation. These include the brassinosteroid-responsive transcription factor BES1 via the cargo receptor DSK2, the aquaporin PIP2;7 via TSPO, and plastid-specific proteins via ATI1. Finally, NBR1/Joka2 mediates selective degradation of poly-ubiquitinated aggregates (aggrephagy) and viral particles (xenophagy).
- Cytosolic components are targeted for degradation in bulk or by selective autophagy. Selective autophagy in mammals depends on two main components: selective-autophagy receptors and lipidated LC3 (microtubule-associated protein light chain 3) proteins. Their interaction ensures specifc degradation of various cellular components including mitochondria, endoplasmic reticulum, aggregated proteins and ribosomes, among other components The quality and quantity of mitochondria in several organisms including yeast and mammals are maintained via selective mitophagy Aggrephagy: An essential aspect of protein homeostasis is the sequestration of nonfunctional and potentially cytotoxic proteins away from the cellular milieu, which is often achieved by their accumulation in distinct, spatially organized aggregates (157). These aggregates are sufficiently large to prevent their clearance by proteasomes and are instead degraded by a specialized autophagic route termed aggrephagy, which often exploits Ub as the signal. (Degradation of intracellular protein aggregates that arise spontaneously or following abiotic perturbations that impair protein folding. Aggrephagy is triggered by aggregate ubiquitylation followed by binding of autophagy receptors such as NBR1 or Joka) Chlorophagy: chloroplasts harbor the photosynthetic machinery and perform other essential metabolic functions . Chloroplast turnover through chlorophagy not only provides a quality control mechanism to eliminate nonfunctional chloroplasts, but also provides a major reservoir of nitrogen and fixed carbon that can be remobilized during senescence and starvation. Degradation of chloroplasts occurs through piecemeal-type degradation of stromal fragments in Rubisco-containing bodies (RCBs) during senescence or nutrient starvation, the formation of which may be mediated by ESCRT components such as CHMP1; through engulfment of whole chloroplasts in response to oxidative damage, possibly mediated by PUB4-dependent ubiquitylation; or through formation of ATI1/2 bodies containing thylakoid proteins. Mitophagy: Mitochondria are the primary organelles responsible for energy generation in eukaryotes via the tricarboxylic acid cycle and oxidative phosphorylation. In addition, mitochondria play crucial roles in intracellular signaling, stress responses, and regulation of cell death (74). As such, maintenance of a healthy mitochondrial population is imperative, especially as mitochondria are a major source of ROS that can lead to oxidative damage if unsupervised (Degradation of mitochondria is induced during senescence. Although no proteins targeting plant mitochondria for autophagic clearance have been identified, ATG11 has been implicated. ) Pexophagy: Degradation of peroxisomes occurs in young seedlings upon the onset of photomorphogenesis and in response to oxidative damage. No pexophagy receptors have yet been described in plants, although the LON2 chaperone likely plays a role in peroxisome stress sensing, whereas PEX6 and PEX10 interact with ATG8. Ribophagy: Autophagic degradation of ribosomes has been implied by increased ribosomal protein levels in autophagy mutants and by the accumulation of ribosomal RNA (rRNA) in the vacuoles of an rns2-2 mutant. RNS2 is a vacuolar RNase T2 enzyme required for the decay and recycling of rRNA in plants Proteaphagy Xenophagy: Numerous examples of intracellular pathogens being degraded by autophagy, such as NBR1-mediated elimination of the cauliflower mosaic virus P4 protein, in plants have been reported. Additionally, pathogens themselves can secrete effectors that interfere with the host autophagy machinery (e.g., PexRD54 from Phytophthora infestans).
- Ubiquitin-Dependent Autophagy: FAM134B=ATG40 receptor proteins : which molecularly bridge the autophagosome and the cargo Selective autophagy receptors interact with ubiquitinated cargo via their ubiquitin binding domain (UBD). This selective autophagy pathway cooperates with the ubiquitin-proteasome system for the elimination of protein aggregates. Many other cellular components are targeted for degradation by these receptors. The factors that determine the specifcity of the receptor to cargo interactions have not been completely elucidated. Considerable overlap exists in the selectivity of the receptors Ubiquitin-Independent Autophagy: Receptors target directly a variety of molecules and organelles as cargo including proteins, lipids, peroxisomes and lysosomes, among many other cellular components.
- Negative regulator of Autophagy In all organisms studied, under high-nutrient conditions, TOR activates protein synthesis and growth pathways and inhibits autophagy, whereas under low-nutrient conditions, growth is blocked and autophagy is activated. TOR is so named because it was identified in screens for yeast mutants that are resistant to the effects of rapamycin. TOR is a member of the phosphatidylinositol kinase-related kinase family based on its sequence but functionally is a serine/ threonine protein kinase. Upon TOR activation (Presence of abundant nutrients): Enanches Mrna translation, ribiosome biogenesis, cellwall synthesis and inahits autophagy. S6K(S6 kinase ) is a substrate of the TOR kinase complex that inhabits autophagy Tap46 interacts with PP2A(protein phosphatase type 2A ) to inhabit Autophagy
- TOR: A Serine/threonine protein kinase that negatively regulates autophagy Induction of autophagy requires the action of the Atg1kinase, that is negatively controlled by the TOR (target of rapamycin) i.e active TOR inhabits autophagy by phosphorylating ATG13. (Prevents association with ATG1) Inhibition of TOR (Dephosphorylation) under starvation leads to ATG1 complex activation Induction: Starvation or reduced nutrient availability is one of the most studied triggers in autophagy research. The induction of autophagy in response to starvation is often dependent on the inhibition of mTOR kinase (mammalian target of rapamycin), a key regulator of nutrient signaling. Upon TOR inhibition, the concomitant decrease in the phosphorylation status of a protein complex including ULK1 (unc-51-like kinase 1), ATG13 and RB1CC1 (RB1-inducible coiled-coil 1), increases the activity of ULK1 and induces autophagy Note: TOR inactivation by RANi, chemical inhabitors (Rapamycin, AZD8055) or genetic elimination of RAPTOR/LST8 enhanches Autophagy. E ATG1–ATG13 kinase complex initiates the formation of autophagosomesg: Atg13 proteins are no longer phosphorylated by TORC1 once autophagy has been induced
- phosphatidylinositol 3-phosphate (PI3P) generated by PI3K, recruits several binding proteins (ATG13,1,VE4,ATG37 etc) ATG9 ( transmembrane protien ) required for membrane delivery along with ATG2 & A8 . Growing phagophore and very imp role in formation of early autophagosome structure and development ATG9 and associated proteins may function in acquiring lipids from the Golgi, endoplasmic reticulum (ER), mitochondria, plasma membrane, and endosomes to expand the phagophore membrane;
- Phagophore assembly site (PAS). After phosphatidylethanolamine (PE) binding with ATG8 it recruits selected recptor process there is complet autopagsome formation Nucleation and Phagophore Formation: The membrane that forms the nascent phagophore may originate from the Golgi, endoplasmic reticulum, mitochondria or endosomes. Formation of the initial engulfng organelle or phagophore depends on the release of Beclin1 and AMBRA1 (activating molecule in BECN1 regulated autophagy protein 1) from their inhibitor Bcl-2. Release from inhibition allows for the formation of the Ptdlns3K (class lll phosphatidylinositol 3-kinase) complex including ATG14, VPS15 and VPS34 (vacuolar sorting proteins). This complex gives rise to phosphatidylinositol-3-phosphate and promotes the recruitment of ATG proteins into the nascent membrane . Elongation, Autophagosome Formation: Vesicle elongation is facilitated by the activation of two ubiquitin-like conjugation complexes. In the frst complex, ATG7 and ATG10 participate in the covalent binding of ATG12 to ATG5. Interaction of the ATG12/ATG5 covalently-bound complex with ATG16L1 (ATG16-like 1) may promote the second ubiquitin-like conjugation complex. In the second complex, ATG4, ATG7 and ATG3 participate in the processing of soluble LC3 to its lipid conjugated form LC3-II. LC3 is lipidated to phosphatidylethanolamine groups associated with the phagophore’s membrane. Together, these two ubiquitin-like conjugation complexes participate in the formation of the autophagosome. By the end of the elongation stage, the phagophore’s membrane has expanded and closed onto itself completing the sequestration of cytosolic content. All ATG proteins dissociate from the autophagosome and return to the cytosol. Lipidated LC3 is the only ATG protein known to remain associated with the autophagosome’s membrane. the phosphoinositide 3-kinase complex for vesicle nucleation; ATG8–PE (ATG8 covalently linked to phosphatidylethanolamine (PE)) involved in phagophore membrane expansion and cargo selection; and the ATG5–ATG12 and ATG16 complex that functions as an E3 ligase in ATG8–PE conjugation.
- docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex SNARE snap receptor large protein compplex primary role in vesicle fusion with with vacuole Rab protein small family of Gprotien GTPase regulate many steps .. Involed in fusion Docking (Binding) orientation of 2 molecules each other like ligand Fusion and Degradation: During starvation, the SNARE protein STX17 (syntaxin 17) localizes to completed autophagosomes. Interaction of STX17 with SNAP29 and lysosomal VAMP8 allows fusion of the autophagosome with lysosomes to form the mature hydrolytic organelle. Upon fusion, proton pumps and hydrolases are added to the autophagosome for formation of the autolysosome, enabling cargo acidifcation and hydrolysis.
- Senescence Nitrogen: Predominantly remobilized element during senescence. Remobilized N contributes -45% of the total N in new rice leaves Role of autophagy during N remobilization was first studied in Arabidopsis Chloroplast recycling (RCB pathway to allow efficient amino acid recycling ). (from senescing leaves and used in newly-forming and storage organs) Increase in ATG genes expression: Delayed senescence Reproductive development The first evidence connecting autophagy to reproductive development was found in wheat. Endoplasmic reticulum stress In Arabidopsis, ER stress triggers activation of autophagy, dependent on inositol-requiring enzyme 1 (IRE1), an ER stress sensor. Fragments of ER containing unfolded proteins are delivered to the vacuole by autophagosomes, suggesting that autophagy may function to degrade them during ER stress Tunicamycin, a chemical that disrupts protein folding and leads to ER stress, was applied to the roots of maize seedlings over a time course of up to 48 h. Analyses of gene expression and cellular events suggested a transition over time from adaptive activities to cell death under persistent ER stress. Autophagy was activated at both the pro-survival stage and the cell death stage
- 12 DAF Gene expression levels were monitored by RT-qPCR using specifc primers . Relative expression/
- Autophagy-related gene: SiATG8a ; is mainly expressed in stems and its expression was dramatically induced by drought stress and nitrogen starvation treatments -full-length CDS sequence -Promoter : 35s -binary vector pCAMBIA1302 -introduced into Agrobacterium tumefaciens strain GV3101 For the nitrogen starvation treatments, foxtail millet seeds were germinated on MS and cultured for 14 days. On the 15th day, some seedlings were treated for 1d, 2d, 3d, or 6d on N-deficient liquid medium (modified MS medium with 5 mM KCl, 3 mM CaCl2, 1.5 mM MgSO4, and 1.25 mM KH2PO4, pH (5.8)). Seedlings were grown in normal MS medium as a control [20]. For the drought stress treatments, 3-week-old seedlings were treated with 6% PEG 6000 for 1 h, 6 h, 24 h, 36 h, and 48 h, as previously described [21]. The primers used in this study are listed in To study the response to nitrogen starvation, 7-day-old seedlings grown in the long-day conditions (16 h/8 h) were transferred to either high N medium (3 mM NH4NO3) or low N medium (0.1 mM NH4NO3) with 1% sucrose, 0.8% agar, pH 5.8, supplemented with 4 mM CaCl2, 1 mM MgSO4, 1.5 mM KH2PO4, 1.5 mM K2HPO4, and 2 mM K2SO4 [26]. The drought tolerance test was carried out in a greenhouse. Transgenic and wild-type Arabidopsis seedlings were watered normally for 14d. Seedlings in the drought treatment group then had water withheld for 21 d, and were subsequently rewatered for 7 d. Overexpression of SiATG8a in Arabidopsis conferred tolerance to both nitrogen starvation and to drought stress. For the nitrogen starvation - Seeds were germinated on MS medium and 15 days old seedlings were treated with N-deficient liquid medium Under nitrogen starvation conditions, the SiATG8a transgenic plants had larger root and leaf areas and accumulated more total nitrogen than wild-type plants. The transgenic plants had lower total protein concentrations than did the WT plants. For the drought stress treatments, 3-week-old seedlings were treated with 6% PEG for 1 h, 6 h, 24 h, 36 h, and 48 h Under drought stress, the SiATG8a transgenic plants had higher survival rates, chlorophyll content, and proline content, but had lower MDA content than wild type plants.
- Wild-type and transgenic Arabidopsis seedlings grown under nitrogen starvation conditions. Lateral root numbers (B), the total surface area of roots (C), the total surface area of leaves (D), root length (E), nitrogen content (F), and protein content (G) of wild-type and transgenic plants. Values are means ± standard deviation (SD) (n ¼ 3 independent experiments, t-test; identical and different letters represent, respectively, non-significant and significant differences).
- Overexpression of SiATG8a enhanced tolerance to drought stress in transgenic Arabidopsis. Seven day old seedlings were placed in the greenhouse for 14 d wildtype (WT) and transgenic (OE-1, OE-2) plants were not watered for 21 d (B) and were then rewatered for 7 d (C). The survival rates (D), proline content (E), chlorophyll content (F), and MDA content (G). Values are means ± standard deviation (SD) (n ¼ 3 independent experiments, t-test; identical and different letters represent, respectively, non-significant and significant differences). Reactive oxygen species degrade polyunsaturated lipids, forming malondialdehyde - The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism
- RNA interference (RNAi)–AtATG18a plants are hypersensitive to salt and drought stresses. Wild-type and RNAi-AtATG18a plants were grown in short-day conditions with regular watering every 2 days for 3 weeks, followed by 0.16-M salt or drought treatments for 5 weeks. In control conditions, little difference was observed between wild-type and RNAi-AtATG18a plants. However, under salt and drought stresses, the RNAiAtATG18a plants showed decreased growth and survival. Autophagy is induced under salt and osmotic stresses. A fluorescence microscope was used to visualize autophagy induction in 7-day-old green fluorescent protein (GFP)–AtATG8e transgenic Arabidopsis roots. After treatment with 0.16-M NaCl or 0.35-M mannitol, numerous GFP-AtATG8elabeled autophagosomes appeared, whereas few were present in control conditions. Arrows indicate GFP-labeled autophagosomes.
- Fungi, bacteria, and viruses are all known to cause cell death in plants. Plant cells: Multilayered system for dealing with microbial pathogens. The first layer: Involves detection of pathogens by sensing pathogen-associated molecular patterns (PAMPs) or microbe-associated molecular patterns (MAMPs) through receptors in the plant cell membrane. Upon detection, a signaling cascade initiates PAMP triggered immunity responses including ethylene production, mitogen-activated protein kinase activation, callose deposition, cell wall thickening, production of antimicrobial proteins, and immune marker gene expression. To evade PAMP and MAMP-triggered immunity, pathogens can inject effectors into plants that manipulate host machinery for pathogen benefit . As a counter to effectors, plants have evolved another layer of defense to recognize effector modifcations of host target proteins via host surveillance proteins (termed resistance (R) proteins). R-mediated defenses often include localized PCD known as the hypersensitive response (HR) to limit spread of the pathogen. HR is a defense mechanism upon tissue compromise, whereby the invading microorganism is inhibited by a combination of a layer of dead cells, local production of antimicrobial compounds, and induction of systemic acquired resistance in the host. If successful, the host is non-susceptible to the invading pathogen
- During the last years, it has become evident that autophagy is engaged in various aspects of plant immunity Most notably, to regulate basal resistance as well as immunity and disease-related cell death responses to microbial pathogens. However, due to the concomitant involvement of plant autophagy in various activity, the autophagic mechanisms underlying host immunity and microbial pathogenesis is still in its infancy. Interestingly, phytopathogens are able to manipulate plant autophagy to their own advantage. the activation of autophagy pathways to target defense compounds or to potentially enhance nutrient acquisition.
- The dual role of autophagy during plant–pathogen interactions in crops. -----------Remorins (REMs) Autophagy can play an anti-microbial role. Plant viruses express RNA silencing suppressors (RSSs) to inhibit the host RNA silencing pathway, such as the HCpro protein produced by turnip mosaic virus (TuMV) and 2b protein produced by cucumber mosaic virus (CMV). In Nicotiana tabacum, a calmodulin-like protein, rgs-CaM, can detect and bind to RSSs, preventing them from suppressing the host RNA silencing mechanism and promoting their degradation by autophagy. Meanwhile, rgs-CaM is degraded along with the RSSs. In tomato, autophagy is involved in degrading the coat protein (CP) of tomato yellow leaf curl virus (TYLCV). Autophagy can play a pro-microbial role. In rice, group 1 remorin (REM1) undergoes S-acylation and is located in the plasma membrane and plasmodesmata (Pd), inhibiting the cell-to-cell movement of viruses. rice stripe virus (RSV) expresses a protein called NSvc4 that can bind to REM1, block its S-acylation, and retain REM1 in the ER. Decreased REM1 at the plasmodesmata enables RSV to move to another cell. The accumulation of non-acylated REM1 at the ER finally triggers autophagy for degradation.
- There is a compelling evidence . Says that autophagy positively controls plant resistance to necrotrophic pathogens. Autophagy deficiency resulted in spreading necrotic lesions and enhanced fungal growth. Hence, pathogen-induced necrotic cell death and disease development is restricted by autophagy. BAG6 forms a complex with two additional partners, BAGP1 (for BAG Associated GRAMProtein) and an aspartyl protease APCB1 (for Aspartyl Protease Cleaving BAG). Once cleavage triggers autophagy which interact with HSP70 molecular chaperon n modulate apoptosis, stress response ..less known structure of BAG in plants B. cinerea triggers cleavage of the BAG6 protein leads to induction of autophagy. Hence, pathogen-induced necrotic cell death for disease development is restricted (Li et al., 2016). It WILL TRIGGER THW CHAIN OF EVENTS FOR APOPTOSIS.
- Plant survives if it is HR, if not infection(OA) spreads and whole plant dies.
- OA is a pathogenicity determinant in Sclerotinia that has a number of functions that facilitate fungal pathogenicity. OA inhibits plant defense responses (Eg callose deposition) and modulates the host redox environment by blocking the host oxidative burst and creating reducing environment. OA also suppresses autophagy. At later stages, OA accumulation lowers the pH, activates cell wall degrading enzymes and a MAP kinase required for pathogenic sclerotial development. This process culminates in OA induced ROS leading to elicitation of apoptotic cell death and disease.
- Inoculation of Arabidopsis with an OAdeficient non-pathogenic mutant strain of S. sclerotiorum (A2)
- Considering its importance in development and stress responses, autophagy is a promising target to manipulate for agricultural benefits like higher yield. Increased expression of ATG genes may be valuable in agricultural applications, as this can confer a number of benefits to plants, including enhanced growth, higher yield and increased stress tolerance