Defense mechanism palm7016 - copy

Post-Infectional Biochemical
Defense Mechanism in Plant Pathogen
Interaction
UNIVERSITY OF AGRICULTURAL SCIENCES, BANGALORE
COLLEGE OF AGRICULTURE, V C FARM, MANDYA
SEMINAR - 2
Chaithra,M
PALM7016
Dept. of Plant Pathology

Flow of Seminar
Introduction
Plant – Pathogen interaction
Types of plant-pathogen interaction
Types of Biochemical defense mechanism
Case studies
Conclusion

1Post-infectional Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
Plant represent a rich source of nutrients.
Plant have developed a structural, chemical and protein- based defense
mechanism.
Introduction:

2Post –infectional Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
The host-pathogen interaction is defined as how pathogens
sustain themselves within host organisms on a molecular and
cellular level.
Host- Pathogen interaction
Elicitors: A molecule produced by the pathogen that induces a
defense response by the host.
Receptors: A protein that recognizes and binds an elicitor; any organ
or molecular site that sensitive to a specific signal molecule.

Outcomes of Plant-Pathogen Interaction
Compatible
reaction
Incompatible
reaction
(Agrios, 2008)

Defense
Structural
defense
mechanism
Pre- existing
structural
defense
Post-inflectional
or induced
structural
defense
Biochemical
defense
mechanism
Pre-existing
Biochemical
defense
Post- infectional
or Induced
biochemical
defense
Types of defense mechanism
(Agrios, 2015)
7

Post-infectional changes in host cells involve production and modification of
large number of molecules.
• Oxidative burst
• Phenolics
• Phytoalexins
• Pathogenesis-related proteins
• Systemic acquired resistance
• Induced systemic resistance
Post infectional or induced biochemical defense mechanism

Oxidative burst: an early plant response to pathogen infection

7Post – infectional Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
• The oxidative burst is generally defined as rapid production of high levels
of ROS in response to external stimuli.
Doke (1983) : on potato plant infected with Phytopthora infestans
(Wojtaszeb, 1997)

• Free radical: each molecule or its fragment, which can exists
independently and contains one or two unpaired electrons.
• ROS: species, which contain one or more oxygen atoms and are much
reactive than molecular oxygen.
ROS
(Wojtaszeb, 1997)

• These are formed as normal aerobic processes that occur in the body; some
as necessary intermediates of enzymatic reaction.
• Most are produced in the ETC when oxygen is reduced to water in the
mitochondria.
O2 reduction H20
• During this conversion various reactive oxygen specie are formed.
ROS formation in cells
(Streller et al., 1994)

Cellular and extracellular sources of H202 :
H202 is produced not only through the disproportionation of superoxide, but
also due to the reduction of O2
- by a reductant such as ascorbate, thiols,
ferredoxins and others
• Chloroplast:
The photosynthetic electron transport (PET) chain in the chloroplast is
responsible for H202 production. ROS in chloroplasts, and the rate of
photoreduction depends on environmental condition.
Origin of H202
(Foyer et al., 1994)

• Peroxisomes:
The main function of peroxisomes in the plant cell is photorespiration, which is
light-dependent uptake of O2 and the associated release of C02 connected with the
generation of H202
• Mitochondria:
In plant mitochondria superoxide anion radical production occurs mainly at two sites
of the electron transport chain:
NADPH dehydrogenases
Cytochrome complex

• The amount of H202 produced in plant mitochondria is less than of
chloroplast or peroxisomes when exposed to light, but in the dark or in
non green tissues, mitochondria can be a major source of ROS.
• Other sources of H202 in the plant cell:
H202 is also produced in the cytoplasm, plasma membrane and in the
extracellular matrix(ECM)

Source of ROS in the oxidative burst
(Baker et al., 1995)
NADPH oxidase system
Oxalate oxidase
pH dependent cell wall binding peroxidase
lipoxygenase

14Post-infectional t Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
Schematic representation of major hypotheses describing the possible
origin of ROS building the oxidative burst (Baker et al., 1995)
Cell wall binding
peroxidase
NADPH oxidase

ROS scavenging enzymes: to avoid the cellular disintegration by ROS
• Superoxide dismutase (SOD)
• Catalase (CAT)
• Guaiacol peroxidase (GPX)
• Ascorbate peroxidase (APX)
• Phenolic compound (free radical)
• Lipid peroxides : decompose to produce malondialdehyde, volatile hydrocarbons such
as ethane, pentane and are precursors for the synthesis of jasmonic acid.
Investigate the intensity and timing of the ROS formation, lipid peroxidation
and expression of antioxidant enzymes as initial response of tomato
(Solanum lycopersicum L.) against the invading necrotrophic pathogen
Fusarium oxysporum f. sp. lycopersici.
(Mandal et al., 2008)

Fig.1. H2O2 generation in roots of tomato plants on a time course after inoculation of the plant with
Fusarium oxysporum f. sp. lycopersici and in the control.
(Mandal et al., 2008)
Fig: 2 Catalase activity in roots of tomato plants on a time course after inoculation of the plants with
Fusarium Oxysporum f. sp. lycopersici and in the control
Fig: 3 Total Phenolic content in roots of tomato plants on a time course after inoculation of the plants with
Fusarium oxysporum f. sp. lycopersici and in the control

plant Pathogen/type of elicitor ROS observed comments
Potato tuber discs Phytophthora infestans zoospores or
hyphal wall components
O2
- Reaction only to incompatible
race or hyphal wall component
Tomato Cladosporium fulvum race-speciﬁc
secreted elicitors
H202 and O2
- Lipid peroxidation; increased
peroxidase activity in
incompatible interaction
Soybean Pseudomonas syringae pv. glycinea H202 Psg (avrA) induces biphasic
oxidative burst;cell death
observed
Tobacco leaf discs Tobacco mosaic virus
Soybean and cotton Oligo galacturonide H202 Homologous and heterologous
desensitisation observed
Plant systems generating ROS when challenged with various pathogens/elicitors or
in response to mechanical stress
(Wojtaszeb, 1997)

• In plants, successful limiting of the spread of pathogen is often manifested
by:
Oxidative burst and other plant responses
(Tenhaken et al., 1995)

• The term hypersensitivity was first used by Stakman(1915) in wheat infected by rust
fungus, Puccinia graminis
• The HR is a localized induced cell death in the host plant at the site of infection by a
pathogen, thus limiting the growth of pathogen.
• HR occur only in incompatible host-pathogen combination.
Hypersensitive response (HR)
(Heath, 2000)

Sequential events in HR
(Heath, 2000)

• Muller and Borger (1940): Potato plant infected with Phytophtora
infestans
• Low molecular weight antibiotics produced by plants in response to
infection.
• They are broad spectrum inhibitors and are chemically diverse with plant
species
• The accumulation occurs in the living cells surrounding the dead cells.
• Phytoalexins tend to fall into several classes including terpenoids,
glycosteroids and alkaloids
Phytoalexins : Phyton= plant; alien= to ward off
(Ahuja et al., 2012)

Fungitoxic and bacteriostatic at low concentration.
Produced in host plants in response to stimulus (elicitors) and metabolic products.
Absent in healthy plants or present in very minute quantity
Remain close to the site of infection.
Produced in quantities proportionate to the size of inoculum.
Produced within 12-14 hours reaching peak around 24hours after inoculation.
Host specific rather than pathogen specific.
Characteristics of Phytoalexins:

Examples of Phytoalexins:

• Seed and seedling root inoculation of Fusarium ear rot (FER) of
• CML444 : Moderately resistant to FER
• B73: Moderately resistant to FER
• CML144 : Susceptible to FER
Evaluate the induction of Kauralexins and zealexins in
Fusraium verticillioides inoculated sub-tropical maize lines
(Veenstra et al., 2019)

Leaf of (a) control and ( b) F. verticillioides-inoculated tissue.
roots of (c) control and (d) F. verticillioides-inoculated tissue
Susceptibility of B73 shoot and root tissue to F. verticillioides at 10dpi
following seed inoculation
Fig: 1The amount of F. verticillioides growing in B73 maize (ng.μg-1) was quantified using FvEF1α and
ZmMEP primers respectively.

Fig: 2 qPCR analysis for quantification of F. verticillioides growth in (a) CML444 and ( b) CML144 root
tissue at 10 and 14days post seed inoculation(dpi) using FvEF1α and ZmGST3 primers respectively.

Fig: 3 GC-MS analysis of total kauralexin accumulation in (a) CML444 and (b) CML144 roots 10 and 14
days post F. verticillioides seed inoculation (dpi).

Fig: 4 GC-MS analysis of total zealexin accumulation in (a) CML444 and (b) CML144 roots 10 and 14
days post F. verticillioides seed inoculation (dpi).

Fig: 5 a) GC-MS analysis of total kauralexin accumulation and b) RT-qPCR analysis of ZmAn2 gene
expression in the root tissue of mockinoculated (control) W22,W22 and an 2mutant lines 14days post seed
inoculation with F. verticillioides

Fig: 6 GC-MS analysis of total zealexin accumulation and b) RT-qPCRanalysis of ZmTPS6 gene
expression in the root tissue of mock inoculated W22 (control), and F. verticillioides inoculated W22 and
an2 mutant lines 14 days post seed inoculation with F. verticillioides.

Phenolics
 Secondary metabolites that encompass structurally diverse natural products those have one or more
benzene rings with hydroxyl groups.
 Plants need these phenolic compounds for pigmentation, growth, reproduction and resistance to
pathogens.
Madhu et al., 2012)
Eg: Onion smudge: Catechol and Protocatechoic acid

Phenolics are formed by three different pathway:
 Shikimate/ chorizmate or succinylbenzoate pathway, which produces the phenyl propanoid derivatives.
 Acetate/ malonate or polyketide pathway, which produces the side chain elongated phenyl propanoid,
including the large group of flavonoids and some quinones.
 Acetate/ mevalonate pathway, which produces the aromatic terpenoids.
Phenols are often produced and accumulated in the sub-epidermal layers of plant tissues exposed to stress
and pathogen attack.
www. Plant-cyc.org

33Post- infectional Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
Phenolics Functions
Coniferyl alcohol, sinapinic acid, cinnamic acid Vir gene inducers, determinants of scent and attractants of
pollinators
Acetosyringone, alfa-hydroxyacetosyringone, p-
hydroxybenzoate
Chemoattractants in Agrobacterium and Rhizobium, and vir gene
inducers in Agrobacterium
Hydroquinones Allelochemical for plant competition
Coumarins, xanthones, anthocyanidins Determinants of colour and attractants of pollinators in plants
Chlorogenic acid Precursor for lignin and suberin synthesis in plants
Isoﬂavonoids Chemoattractants and nod gene inducers in Rhizobium
Cajanin, medicarpin, glyceoline, rotenone,
coumestrol, phaseolin, phaseolinin, limonoids,
tannins, ﬂavonoids
Phytoalexins, phytoanticipins and nematicides in plant defence
Lignin, tannins and suberins Structural components of plant cells
Examples of different types of phenolics and their diverse functions
(Bhattacharya et al., 2010)

Post-infectional Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
Determination of Phenolic content in healthy and infected tissue of
Apple infected with Venturia inaequalis
(Petkovsek et al.,2008)
34

Post Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
Date Treatme
nt
Chlorogenic
acid
P-
coumaric
acid
Caffeic
acid
Catechin Epicatechin Rutin Phloridzin Quercetin-3-
rhamnoside
May 27 Healthy
infected
26.4±3.4
66.8±19.6
14.4±0.4a
27.1±1.2b
28.6±0.9a
57.4±2.4b
62.2±2.5a
99.8±5.1b
99.2±2.5a
175.6±6.2b
5.7±0.2a
11.1±0.5b
93.8±3.7a
203.3±11.4b
7.1±0.3a
13.6±0.4b
June 2 Healthy
infected
30.8±1.5a
162.4±12.1b
18.3±2
25.1±0.8
29.6±1.4a
55.9±2.1b
68.2±4.1a
126.5±5.3b
98.8±5.9a
215.4±8.8b
6.2±0.4a
10.7±1.2b
114.5±4.8a
299.2±9.4b
10.8±0.6a
19.7±0.6b
June 23 Healthy
infected
67.1±3.7a
108±16.3b
25.2±0.9
25.4±1.6
30.3±1.5a
45.2±5.4b
82.5±6.6
100.7±12.8
156.9±5.7
201.5±14.4
6.1±0.2a
8.9±0.8b
88.4±6.2a
222.2±22.4b
8.4±0.4a
13.9±1.3b
July 25 Healthy
infected
57.4±16.2a
133.8±7.2b
17.1±3.1
23.3±3.1
29.4±2.7a
48.1±3.8b
54.8±11.9a
97.1±12.7b
162.2±22.7
225.8±17.2
7.4±1.1
12.9±1.2
109.2±11.8a
213.4±27.4b
6.6±0.8a
16.5±1.96
August
22
Healthy
infected
41.5±2.8a
173.3±21.03
b
16.2±0.4a
28.5±2.4b
26.1±0.8a
50.2±3.4b
38.7±4.5a
76.4±7.8b
166.5±21.7a
269.8±25.7b
10.4±1.1a
17.4±1.2b
121.3±10.2a
247.3±20.0b
10.2±0.6a
16.4±1.3b
Septem
ber 12
Healthy
infected
28.7±4.4a
129.5±10.6b
14.1±0.9a
27.8±1.9b
21.7±1.4a
41.6±1.2b
32.4±4.6ba
70.2±8.8b
136.9±11.3a
261.4±10.9b
7.1±0.6a
13.6±0.6b
87.5±12.8a
176.4±13.5b
6.3±0.7a
12.8±0.8b
Table: Content of phenolic compounds( mg/g dry weight) in healthy and infected leaves of the cv. Golden
Delicious at various times
(Petkovsek et al.,2008)

Post –infectional Biochemical Defense Mechanism during plant pathogen infection, PALM7016; 2018-2019,ACM, UASB
Systemic resistance
36

Systemic Acquired Resistance (SAR)
 SAR : The phenomenon in which uninfected systemic plant parts become more resistant in response to
localized infection elsewhere in the plant.
 long-lasting protection against a broad spectrum of microorganism
 Enhance resistance against subsequent attack by a wide array of pathogen.
 The vascular provide the excellent channel for transport of systemic signals.
37

Systemic signal in plant Defense
 Methyl salicylate
 Jasmonate
 Azelaic acid
 Terpenoids
Inorganic compounds
 Phosphate salts
Synthetic compounds:
 Benzo-1,2,3-thiadiazole-7-carbothioic acid S
methyl ester (BTH)
 Probenazole
 2,6-dichloroisonicotinic acid and its methyl ester (Mayers et al., 2005)
38

Methyl salicylate (MeSA)
 Mobile signal, moves systemically, it found in phloem exudates of
infected leaves and is required in systemic tissue for SAR.
 Accumulation of salicylic acid induces the secretion of pathogenesis-
related (PR) proteins with antimicrobial activities.
 SAR requires SABP2’s MeSA esterase activity in the systemic tissue to
convert biologically inactive MeSA to active SA(SABP2’s = Receptor in
systemic tissue)
 Nicotiana tabaccum contains N resistance gene that governs gene- for-
gene resistance to TMV, MeSA functions as enhance the resistance to
subsequent infection by TMV
(Mayers et al., 2005)
39

Jasmonate
 Methyl jasmonate (MJ) function as a volatile signal and also translocated through the
plant through vascular tissue.
 JA activates gene encoding protease inhibitor which protect plants against pathogen
attack
 Treatment of potato with jasmonate increase resistance to Phytophthora infestans.
Signal generation and transmission in SAR
(Mayers et al., 2005)
40

Determination of salicylic acid, hydrogen peroxide activity, superoxide
desmutase, peroxidase, catalase, Polyphenol oxidase(PPO), Phenylalanine
ammonia lyase (PAL), Lipoxygenase(LOX), Callose content in wheat plant
against Fusarium graminearum (Fusarium head blight)
(Sorahinobar et al., 2016)
41

Fig: 1 Effect of Fusarium graminearum inoculation and salicylic acid application on content of salycilic
acid in spikelets of Falat and Sumai3
(Sorahinobar et al., 2016)
42

Pathogenesis-related (PR) proteins
 Van Loun and Van Kammen (1970)- worked on discovery of PR proteins -while
working on HR of tobacco plants infected- TMV.
 PR protein are the proteins encoded by plants in response to infection by pathogens and
associated with the development of systemic acquired resistance.
(Ryals et al., 1996)
43

 Proteinase resistance protein.
 Low molecular weight : 14000-30000
 Low pH : 2-3
 Rich in aromatic amino acids
 Pathogenesis-related proteins are present in low levels.
 Acid PR protein located in the intercellular spaces, whereas, basic PR proteins are
located in vacuole.
Characteristics of PR protein
(Loon et al., 1999)
44

Classification of pathogenesis related proteins
(Ebrahim et al., 2011)
45

(Chandrasekaran and Chun, 2016)
Expression of PR-protein genes and induction of defense-related enzymes by
Bacillus subtilis CBR05 in tomato
(Solanum lycopersicum) plants challenged with
Erwinia carotovora subsp. carotovora
46

Fig:1 The β-1,3-glucanase (a) and PAL (b) activity in tomato treated with B. subtilis
CBR05 and challenged with Erwinia carotovora subsp. carotovora.
(Chandrasekaran and Chun, 2016)
47

Polyvalent cation of pectin-protein in the middle lamella : pectic enzymes
(polygalactouranase)
Phenols, tannins and proteins
Eg: Resistance of immature grape fruit against Botrytis cinerea : presence of catechoal-
tannin (enzyme inhibitors)
Eg: Pyricularia grisea (paddy blast)
:picolinic acid : picolinic acid ester and N-methyl picolinic acid
pyricularin : non toxic substance by phenolics (chlorogenic acid and ferulic acid)
Fusarium f. sp. vasinfectum (cotton wilt) and Fusarium f. sp. lycopersici (tomato wilt)
: fusaric acid : N-methyl fusaric acid amide
 Formation of substrates resistant to enzymes of the pathogen:
 Inactivation of pathogen enzymes:
 Detoxification of pathogen toxins:
48

RNAi- mediated defense against plant viruses:
 Post- transcriptional gene silencing
 Transcriptional gene silencing
49

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