Systemic Acquired resistance

Systemic acquired resistance in Plant Pathology

1. WELCOME 1

2. Systemic Acquired Resistance 2

3. Plants defend themselves from pathogen infection through a wide range of mechanisms that can be either systemic or local, constitutive or inducible. Contact with pathogenic and non pathogenic microorganisms trigger a wide range of defence mechanisms in plants. 3

4. 4 SYSTEMIC PLANT RESPONSES : Type of systemic response induced, however, is determined by the identity of the attacking organism. A)Systemic acquired resistance. B)Systemic proteinase inhibitor/wound response. C)Induced systemic resistance (by PGPR).

5. 5 Viruses, fungi, bacteria activate systemically a specific subset of defence responses in a phenomenon known as systemic acquired resistance (SAR). In which local necrosis formation at the initial site of pathogen invasion triggers a local increase in salicylic acid (SA) accumulation i.e the formation of a phloem-mobile signal. Subsequently , in distal plant tissue, SA concentrations increase and volatile methyl- Salicylate (MeSA) is released. Together, these signals induce the synthesis of various pathogenesis related (PR) proteins in the noninvaded parts of the plant.

6. 6 (B) In the systemic PI/wound response activated by chewing insects, the initial tissue damage causes a transient increase in the synthesis of ethylene and jasmonic acid (JA). Volatile methyl jasmonate ( MeJA ) and another phloem- mobile signal called systemin then activate the systemic responses, which include the accumulation of PIs and other systemic wound response proteins (SWRPs). Root attacking nematodes appear to induce a mixture of both the SAR and systemic Pathogen induced/wound responses.

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8. 8 (C) Induced systemic resistance (ISR) caused by soil inhabiting nonpathogenic rhizobacteria colonizing plant roots. ISR requires both JA and ethylene- mediated signaling to induce protective defense responses in the distant leaf tissue. This form of defense does not involve the accumulation of neither pathogenesis-related proteins nor SA.

9. Two main mechanisms have been recognized . Systemic Acquired Resistance Induced Systemic Resistance 9

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11. SYSTEMIC ACQUIRED RESISTANCE Systemic acquired resistance (SAR) refers to a distinct signal transduction process, that plays an important role in the ability of plants to defend themselves against pathogens. After the formation of a necrotic lesion, either as a part of hypersensitive response (HR) or as a symptom of disease , the SAR process is activated. SAR activation results in the development of a broad-spectrum, systemic resistance. 11

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13. Historical development The natural phenomenon of resistance development in response to pathogen infection was first recognized in 1901 by Ray & Beauverie , both who worked with Botrytis cinerea . In 1933, Chester- he first found the concept of SAR and reviewed research on “the problem of acquired physiological immunity in plants” and determined that this phenomenon may play an important role in the preservation of plants in nature 13

14. In 1964, Hecht & Bateman showed that inoculation of tobacco leaves with Thielaviopsis basicola lead to necrosis and subsequent systemic resistance to other pathogens, TMV, and TNV. Accoring to Smedegaard -Peterson et al both virulent and avirulent races of Erysiphe graminis f. sp. hordei could induce resistance in barley towards virulent strains of this fungus. 14

15. Mechanism of sar maintenance SAR can be distinguished from other disease resistance responses by both the spectrum of pathogen protection and the associated changes in gene expression. In 1970,Van Loon and Gianinazzi et al showed that viral infection of tobacco induced the accumulation of a distinct set of proteins , called pathogenesis-related proteins (PR proteins). Van Loon & Antoniw demonstrated that a subset of PR proteins (acidic-extracellular forms) accumulated during the onset of resistance. 15

16. In 1991, steady-state mRNA levels from at least nine families of genes were shown to be coordinatedly induced in uninfected leaves of inoculated plants; these gene families are now known as SAR genes. SAR gene products have direct antimicrobial activity or are closely related to classes of antimicrobial proteins. 16

17. SAR genes include ß-1,3-glucanases, chitinases , cysteine -rich proteins, which lack known biochemical function, but have in vitro activity against Phytophthora infestans . However, all the ß-1,3-glucanases should not be considered to be associated with SAR, rather a gene –specific subset consisting of mostly acidic isoforms are precisely correlated with SAR. 17

18. In tobacco, SAR activation results in a significant reduction of disease symptoms caused by the fungi Phytophthora parasitica , Cercospora nicotianae, and Peronospora tabacina , the viruses tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV), and the bacteria Pseudomonas syringae pv tabaci and Erwinia carotovora . 18

19. Components of SAR Basically SAR has three main components 1. Accumulation of pathogenesis related (PR) proteins (termed SAR proteins) 2. Lignification and cross-linking of the cell walls of tissues distant from the HR 3. Phenomenon of conditioning 19

20. Pathogeneis Related proteins: PR-proteins have been defined as plant proteins that accumulate after pathogen attack or related situations. The role of PRs in defence can be addressed using transgenic plants expressing the corresponding genes. In tobacco, over expression of PR-1 significantly increase resistance against infection by Peronospora tabacina and Phytophthora parasatica var. nicotianae . 20

21. Lignification and other Structural Barriers : The process of lignification is observed in nonhost resistance and SAR. Lignification may contribute resistance in many different ways. Incorporation of lignin into plant cell wall strengthen it mechanically and make it more resistant to degradation by enzymes secreted by an invading pathogen. 21

22. Lignin precursors themselves might exert a toxic effect on pathogens or by binding to fungal cell wall, make them more rigid and impermeable, thus, hindering further growth or uptake of water and nutrients. Conditioning/Sensitizing: When plants are pretreated with a necrotizing pathogen or a synthetic inducer of SAR, the systemically protected leaves react more rapidly and more efficiently to infection with a virulent pathogen. This phenomenon is known as conditioning or sensitizing. 22

23. The biochemical changes occurring in a sensitized plant usually become apparent at the moment of challenge infection. At the cellular level, there appears to be a shift towards a greater sensitivity not only to pathogens but also biotic and abiotic elicitors. Although the phenomenon has long been known, it is a known effect, whereby, a pathogen attempting penetration of SAR tissue meets a more efficient and faster response than it does on non-SAR tissue. The response may also occur at lower limits of elicitors. 23

24. Reactive oxygen species Alvarez et al . had found ROS in Arabidiopsis . H 2 O 2 accumulates in small groups of cells in uninoculated leaves of Arabidiopsis after infection with an avirulent strain of P. syringae . These microbursts occur within two hours after an initial oxidative burst in the inoculated tissue and are followed by the formation of microscopic HR lesions. Using catalase to scavange H 2 O 2, or DPI ( diphenylene iodonium ) to inhibit the NADPH oxidase , it was demonstrated that both the primary and secondary oxidative bursts are required for the onset of SAR. 24

25. Elicitors Molecules produced by a pathogen that induce a defence response by the host. Innate immunity in plants relies on the rapid evolution of membrane-bound or cytosolic disease resistance proteins (R proteins) , which perceive pathogen protein signatures known as 'elicitors’. 25

26. Recognition of the elicitors by the host plant activates a cascade of biochemical reactions in the attacked and surrounding plant cells and lead to new or altered cell functions and to new or greatly activated defence-related compounds. 26

27. In compatible host–pathogen interactions, the elicitor escapes recognition by an R protein, leading to the development of disease. In incompatible interactions, the elicitor is recognized by an R protein, triggering a cascade of defence reactions that culminate in a form of programmed cell death (PCD) or apoptopsis , which is a 'hypersensitive response’ (HR). 27

28. Typically, HR includes signal transduction , PCD, increase activation of defence related genes (e.g., synthesis if phytoalexins , salicylic acid, and antimicrobial proteins), and a distant induction of general defence mechanisms that serve to protect the plant i.e.,Systemic Acquired Resistance. 28

29. 29 Effector Virus Fungi Bacteria Pathogen cell surface Plant cell surface Cell recognition Elicitor receptor site Elicitors Signal transduction pathways Defence genes Gene products PR proteins SAR Phyto alexins Chitinases HR Primary immune Response of Plants.

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31. 31  Hypersensitive response and systemic acquired resistance . Tobacco mosaic virus  infection of tobacco cultivars carrying a disease resistance gene (e.g .   N gene ) lead to the  HR   and subsequent establishment of  SAR . The  HR  is characterized by host cell death and necrosis at the site of infection (left). Several days after the primary infection ,  SAR  is induced throughout the plant. As a result, secondary infections of the plant with the same virus or other unrelated pathogens lead to much smaller lesions or weaker symptoms (right). The leaves are shown four days after viral infection.

32. Role of Salicylic Acid in SAR Salicylic acid (SA) plays a key role in both SAR signaling and disease resistance . Initially, the level of SA was found to increase by several hundred-fold in tobacco or cucumber after pathogen infection, and this increase was shown to correlate with SAR. 32

33. The involvement of SA in signaling SAR has been clearly demonstrated in experiments with transgenic tobacco that express the salicylate hydroxylase gene ( nahG ) from Pseudomonas putida . This enzyme catalyzes the conversion of SA to catechol , which has no SAR inducing activity. The nahG expressing plants do not accumulate SA in response to pathogen infection and they show no SAR gene or resistance expression in the distal portions of the plant. 33

34. 34 Even though SA is not likely to be the translocated signal that triggers SAR in distal plant organs, it is essential for SAR signal transduction. Inoculation of wild-type rootstocks with TMV leads to the induction of SAR in wild type scions but not in n ahG scions, demonstrating that the induction of SAR in systemic tissues is SA dependent. These findings indicate that SA is an essential signal in SAR and that it is required downstream of the long distance signal.

35. Biosynthesis of Salicylic acid The biosynthetic pathway of SA in plants begin with the conversion of phenylalanine to trans- cinnamic acid (t-CA) catalyzed by phenylalanine ammonia lyase (PAL). The conversion of t-CA into SA has been proposed to proceed via chain shortening to produce benzoic acid (BA), followed by hydroxylation at the C-2 position to derive SA . 35

36. The next step is likely to be catalyzed by a cytochrome P450 mono oxygenase , called benzoic acid 2-hydroxylase (BA2H), the activity of which is induced by either pathogen infection or exogenous BA application. The mechanism of BA production from t-CA is unknown, but it may occur in a manner of β-oxidation of fatty acids (or) a nonoxidative mechanism. 36

37. 37 phenylalanine Cinnamic acid

38. Mode of action of SA The mechanism by which SA induces SAR is unknown; however, it has been proposed that H 2 O 2 acts as a second messenger of SA in SAR signaling. Biochemical screens for SA-binding proteins, by Klessig et al. resulted in the identification of multiple enzymes, such as catalase , ascorbate peroxidase , the E2 subunit of α- ketoglutarate dehydrogenase , and glutathione S- transferases , showed that their enzymatic activities are inhibited upon binding to SA. 38

39. SA decreases the expression of ascorbate peroxidase (APX) & catalase (CAT) genes leading to substantial increase in H 2 O 2 . The later causes induction of PR-1 gene expression and induce SAR. 39

40. 40 Fig. 1. SA decreases the expression of ascorbate peroxidase (APX) & catalase (CAT) genes leading to substantial increase in H 2 O 2  which is crucial for the activation of local acquired resistance (LAR) & systemic acquired resistance (SAR).

41. Nitric Oxide (NO) as signal molecule: NO is involved in multiple regulatory processes in plants. In plants, NO and SA appear to function in a positive feedback loop. NO donors induce SA accumulation, and NO signaling in defence requires SA. Supporting this hypothesis nahG expression suppressed NO-inducible local and systemic resistance in TMV-infected tobacco, where as SA –induced SAR was promised by an NO scavenger or inhibitors of NO synthesis. NO is involved locally in the induction of cell death (HR) in conjunction with SA, H 2 O 2, and ethylene. 41

42. Lipid-Transfer Protein The dir1 (defective in induced resistance 1) mutant was discovered in a labor -intensive genetic screen designed specifically to identify SAR Signals. In contrast to other SAR-deficient mutants, dir1 has a normal local defence response but is compromised in SAR. Even though petiole exudates collected from dir1 lack the SAR-inducing activity, the mutant is competent in responding to induction by the petiole exudates collected from induced wild-type plants. 42

43. This suggests that DIR1, which encodes a putative lipid-transfer protein, is probably involved in the synthesis or transport of a lipid molecule, which is a mobile signal for SAR. Recently a tobacco SA-binding protein SABP2 a lipase was discovered and was found that, silencing of SABP2 gene diminishes both SRI and ISR. 43

44. SAR activation with chemicals For the chemical to be considered as an activator of SAR, it should exhibit these characteristics. First, the compound or its significant metabolites should not exhibit direct microbial activity. Second, it should induce resistance against the same spectrum of pathogens as in biologically activated SAR. Third, it should induce the expression of the same marker gene as evident in pathogen-activated SAR. 44

45. Low molecular weight molecules are also capable for SAR induction in plants. The use of chemicals to activate SAR provides novel alternatives for disease control in agronomic systems as well as tools for the elucidation of the SAR signal transduction cascade. 45

46. Natural organic compounds : SA is the only plant derived substance that has been demonstrated to be an inducer of SAR. The chemicals 2, 6-dichloroisonicotinic acid and its methyl ester were the first synthetic compounds shown to activate SAR, providing broad spectrum disease resistance. Synthetic chemical benzo-1,2,3-thiadiazole-7-carbothioic acid S methyl ester (BTH) was demonstrated to be a potent SAR activator that showed protection in the field against a broad spectrum of diseases in a variety of crops. 46

47. Inorganic compounds : Phosphate salts induce SAR in cucumber bean and maize. Calcium sequestration at the site of application by phosphates is thought to generate endogenous SAR signal. 47

48. Synthetic compounds : Probenazole is commercial product used against rice blast. It has a little effect on rice prior to inoculation, but potentiates defence responses upon attack by Magnaporthe oryzae . Benzo -(1, 2, 3)-thiazole-7-carbothionic acid S-methyl ester (BTH) is an inducer of SAR in a number of plants including wheat, rice and tobacco. Its tolerance and efficacy in these crops have warranted its commercialization. 48

49. 2,6-dichloroisonicotinic acid induces changes in rice and barley where treated plants exhibit phenocopies of genetically determined resistance against Erysiphe graminis and Magnaporthe oryzae . 49

50. 50 Salicylic acid 2,6-dichloroisonicotinic acid (INA) Benzo -(1,2,3)-thiodiazole-7-carbothioic acid S-methyl ester (BTH).

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52. SAR compromised mutants Delaney et al., 1994 identified and characterized six allelic recessive mutants named nim . These are not responsive to exogenous application of SA. Cao et al., have also isolated and described recessive Arabidopsis mutant called npr1 (non expresser of PR genes), which exhibits compromised activation of SAR npr 1 and are allelic to nim 1 (non inducible immunity). In these both SAR signaling is blocked before SAR gene expression but subsequent to SA accumulation. 52

53. nim 1 plants infected with the avirulent pathogen Pseudomonas syringae DC 3000 harboring the cloned avr Rpt 2 . nim 1 plants were shown to accumulate both free and glucose conjugated SA levels. nim 1 plants are able to accumulate SA in response to pathogen infection but appear to be defective in SA perception or in subsequent SA sensing events. The nim 1 and npr 1 mutations define genes that act before SAR gene expression substantiated by the lack of PR-1 induction. 53

54. Fitness costs of sar Phenotypes of many mutants show constitutive PR gene expression, accumulation of SA and resistance to pathogen, but have reduced plant size, loss of apical dominance, curly leaves and decreased fertility. Treatment with BTH in plants grown hydroponically, shown a reduction in biomass and number of ears and grains in wheat. Cost of resistance could be due to the allocation of the plant’s resources to constitutive PR protein production. Thus constitutive expression of SAR in uninfected plants is found to be detrimental. 54

55. INDUCED SYSTEMIC RESISTANCE Roots of some plants are colonized by specific strains of non-pathogenic fluorescent Pseudomonas spp. and they develop phenotypically similar form of protection that is called rhizobacteria -mediated induced systemic resistance (ISR). ISR is controlled by signaling pathway in which jasmonic acid and ethylene play key roles. Rhizobacteria are present in the rhizoplane and certain strains of it are referred to as plant growth promoting rhizobacteria (PGPR) because, they can stimulate growth of the plants. 55

56. Rhizobacteria mediated ISR in Arabidiopsis colonization of Arabidiopsis roots by ISR inducing Pseudomonas fluorescens WCS417r bacteria protects the plant against different types of pathogens including the bacterial leaf pathogens Pseudomaonas syringe pv . tomato and Xanthomonas campestris pv . armarociol , the fungal root pathogen Alternaria brassicicola and oomycete leaf pathogen Peronospora parasitica . Protection against these pathogens is typically manifested as reduction in disease symptoms and inhibition of pathogen growth. 56

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58. Synthesis of jasmonic acid   Jasmonic acid (JA), is a growth inhibitor, senescence-promoting substance. Jasmonic acid synthesized from linolenic acid via the octadecanoic pathway. JA is synthesized from alpha- linolenic acid, which is a C18 poly-unsaturated fatty acid.  It is synthesized from plant membranes by enzymes similar to lipase.   Key JA biosynthetic enzymes include lipoxygenase , allene oxide synthase , and allene oxide cyclase . 58

59. JA induces vegetative storage proteins, osmotin , thionin (antifungal) and defensin . Induces protease inhibitors to control pests.  JA and ethylene induce PR-3, PR-4, PDF1.2, chitinases (CHI-B). 59

60. 60 OCCURS IN CHLOROPLAST OCCURS IN PEROXISOME SYNTHES I S OF JASMONIC ACID

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62. ISR signal transduction pathway The role of SA in ISR was studied in SA non accumulating Arabidiopsis NahG plants. In contrast to pathogen induced SAR , Pseudomonas fluorescens WCS 417r-mediated signal ISR against Psuedomonas syringae pv . tomato was normally expressed in these plants. (Van Wee et al ., 1997;Pieterse et al ., 1996). SA induction deficient mutants Sid 1-1 and Sid 2-1 expressed normal levels of ISR. 62

63. Determination of SA levels in ISR expressing Arabidopsis plants revealed that in contrast to SAR, ISR is not associated with increased accumulation of SA ( Pieterse et a l., 2000). This has led to the conclusion that Pseudomonas fluorescens WCS417r mediated ISR is an SA independent resistance response, and that ISR are regulated by distant signaling pathways. 63

64. Wounded leaves produce an 18-amino acid peptide called systemin from carboxyl terminal of prosystemin (200 AA precursor) in plant and it elicits production of JA in companion cell- seive element complex. JA moves through out the plant in the phloem. Covalently modified JA (JA-x) play important role in systemic signaling. Signal is recognized at the target cell e.g. mesophyll (leaf). Jasmonic acid turns on defence related genes (genes for proteinase inhibitor) in target cells. 64

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66. Crosstalk between SA and JA Both synergistic and antagonistic interactions between SA and JA have been reported, suggesting that the interaction is either concentration dependent or tissue specific. Application of low concentrations of SA and JA resulted in synergistic expression of both the SA target PR1 and the JA marker PDF1.2 and Thi1.2. 66

67. In contrast, higher concentrations of SA and JA have antagonistic effects on expression of the same genes. It is generally believed that this antagonism between SA and JA allows plants to prioritize defence against either biotrophic or necrotrophic pathogens. Host plants rely on SA-mediated defence against biotrophic pathogens, whereas JA-mediated signaling participates in defence against necrotrophic pathogens. 67

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69. Comparison of ISR and SAR: Both ISR and SAR are dissimilar with respect to their induction, signaling pathways and their co-ordination, level of defense elicited in host plant and protein expression. ISR is induced by jasmonic acid and ethylene molecules coordinated signaling pathway; proteins other than pathogenesis related are too expressed. Level of defense generated is lesser and also dependent on host plant genotype and presence of non-pathogenic bacteria. 69

70. SAR is induced by salicylic acid. SAR and PR proteins are induced together. The defense level produced by SAR is high, long lasting, broad spectrum and independent of plant genotype but requires necrotic pathogens or presence of inducer analogs. It has been indicated that ISR and SAR show phenotypic similarity and together can produce protection of higher level than they produce alone. 70

71. Article: The effects of riboflavin on defence responses and secondary metabolism in tobacco ( Nicotiana tabacum cv. NC89) cell suspensions and the effects of protecting tobacco seedlings against Phytophthora parasitica var. nicotianae and Ralstonia solanacearum were investigated. Defence responses elicited by riboflavin in tobacco cells induced an oxidative burst, alkalization of the extracellular medium, expression of 4 defence -related genes and 2 phenolic compounds. When applied to tobacco plants riboflavin treatment gives protection. 71

72. Finally conclude that riboflavin can both induce a series of defence responses and secondary metabolism in cell suspensions and protect tobacco against P. parasitica and R. solanacearum Exogenously applied sphinganine produced similar effects on these markers. This suggests that fumonisin production contributes to the colonization of this necrotroph by activating the SA pathway and inducing cell death 72

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74. References: Agrios,G.N ., (2005). Plant Pathology; Fifth Edition; 341-348. Helmut Kessmann et al ., (1994). Induction of Systemic Acquired Disease Resistance in Plants by Chemicals, Annual Review of Phytopathology . 32:439-459. Michal Shoresh , Gary E.Harman , and Fatemeh Mastouri .,(2010). Induced Systemic Resistance and Plant Responses to Fungal Biocontrol Agents, Annual Review of Phytopathology, 48:21-43. 74

75. Rajan Katoch .,(2010). Biochemistry and Molecular Biology of Plant Disease Resistance; First Edition; 73-89. Raskin I.,(1992).Role of salicylic acid in plants, Annual Review of Plant Physiology and Plant Molecular Biology ,43:439-463. W.E. Durrant and X. Dong.,(2004). Systemic Acquired Resistance, Annual Review of Phytopathology, 42:185-209. 75

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