Hi,
Please discuss the following questions in group.
1. Fatimah group
Q: Discuss the plant-pathogen interactions.
2. Melanie group
Q: Prokaryotes as plant pathogens.
3. Kenneth group
Q: Fungi, water mold, viruses as plant pathogens.
4. Quek group
Q: Discuss the existing approaches to combat plant disease.
Friday, November 4, 2011
Biotechnological Approaches to Disease Resistance
Biotechnological Approaches to Disease Resistance
Aim: to enhance plant defence systems
Approaches:
(1) Protection against pathogens
PR protein show some activity at inhibiting disease, but have limited activity, generally do not switch on disease resistance pathway
The PR proteins: eg chitinase, glucanase and ribosome-inactivating proteins (RIPs)
Provide protection and tolerance over fungal infection
chitinase, glucanase degrade cell wall of many fungi
RIP – enzyme that remove an adenine residue from a specific site in the large rRNA of eukaryote & prokaryote ribosome à inhibit protein synthesis
Isolated chitinase, glucanase genes fr plant (rice, barley), bacteria (Serratia marcescens), fungi (Trichoderma harzianum)
Common promoters used: 35s promotor, wound-inducible promotor (potato prp1-1 promotor – mediate rapid & localized expression in response to pathogen, restrict gene expression to where the protein is required & may reduce any potential hazards associated with the protein)
Advantage: gene expression only turn on during infection à limiting gene expression à no serious drain on plant biosynthetic capability & yield reduction
Common promoters used: 35s, wound-inducible
Most widely used in fruit trees
Challenge?
Greenhouse field
(2) Antimicrobial proteins
Antifungal and antibacterial, eg lysozyme, defensins
A. Lysozyme
Degrade chitin and peptidoglycan
B. Defensins
Small antimicrobial peptides
Display lytic activity difficult to develop resistance
Eg alfalfa defensin, cecropin, melittin, gallerimycin, temporins etc
Problem? Limited spectrum
Case study:
Combating the bacterial disease fireblight
Cause by Erwinia amylovora
Solution? Transgenic crop containing gene code for antimicrobial protein
2 Approaches:
(1) Lytic approach – produce transgenic fruit trees contain gene for T4 lysozyme + insect antimocrobial pt attacin E + cecropin
(2) Bovine lactoferrin gene (to compete with microbial siderophores) + depolymerase gene (degrade specific exopolysaccharides)
Siderophores - small, high-affinity iron chelating compounds secreted by grasses and microorganisms such as bacteria and fungi,
- amongst the strongest soluble Fe3+ binding agents known.
(3) Transgenic crops for food safety
How? By reduce amount of mycotoxins
Use to combat the effect of fungal infection
Example:
Bt genes into Bt crops minimize insect damage less wound sites due to insect infection lower possibility for fungal infection reduce amount of mycotoxins ensure safety of livestock and human
(4) Introduction of HR and SAR in transgenic plant
Aim: use elicitor receptor system to switch on general resistance pathway
Approach 1: the Guard hypothesis (tomato)
R protein increase its production will increase the expression of resistance systems
Outcomes:
Overexpression of R protein genes were induced differently
Not pathogen-specific
Approach 2: (tobacco) switch on the HR-related defence system
crytogein (a Phytophthora cryptogea gene that codes for highly active elicitor) fuse with pathogen inducible promoter (from tobacco) à introduce into transgenic tobacco
Non-induced condition (eg no pathogen)àno elicitor
Once infected by virulent fungus (Phytophthora parasitica var. nicotianae) à crytogein gene is expressed à elicitor-induced defense gene expressed
Broad-spectrum resistance
Other approaches
Trigger expression of a range of genes by using different stimuli, eg introduction of Arabidopsis gene NPR1 into tomato, wheat, rice etc.
The product of this gene lies at a node in the regulatory pathways that link SAR, induced systemic resistance, R-gene mediated resistance, ethylene, salicylic acid, jasmonic acid etc
it activates gene expression by interacting with members of the TGA family of basic leucine zipper (bZIP) transcription factors
The introduction of the gene enhanced resistance to a range of plant pathogens in tomato, but in rice the gene had a detrimental effect
The mode of action for the transgene seems to be a priming function: the plant responds more rapidly to attack rather than having its defence system switched on constitutively, which would have an effect on yield
Switching on cascade of defense genes (sunflower)
Involve of O sp eg H2O2 as a messenger in HR reaction has been tested by transforming plant with oxalate oxidase from wheat
oxalate oxidase
oxalic acid+ O2 -----------------------------------> CO2+ H2O2
transgenic plant could degrade oxalic acid & generate H2O2, and an HR-like response along with salicylic acid synthesis & defence-gene expression occur
The genes were identified as being PR & defensin protein
Transgenic sunflower exhibited enhanced resistance against the oxalic acid-generating fungus Sclerotinia sclerotiorum
Aim: to enhance plant defence systems
Approaches:
(1) Protection against pathogens
PR protein show some activity at inhibiting disease, but have limited activity, generally do not switch on disease resistance pathway
The PR proteins: eg chitinase, glucanase and ribosome-inactivating proteins (RIPs)
Provide protection and tolerance over fungal infection
chitinase, glucanase degrade cell wall of many fungi
RIP – enzyme that remove an adenine residue from a specific site in the large rRNA of eukaryote & prokaryote ribosome à inhibit protein synthesis
Isolated chitinase, glucanase genes fr plant (rice, barley), bacteria (Serratia marcescens), fungi (Trichoderma harzianum)
Common promoters used: 35s promotor, wound-inducible promotor (potato prp1-1 promotor – mediate rapid & localized expression in response to pathogen, restrict gene expression to where the protein is required & may reduce any potential hazards associated with the protein)
Advantage: gene expression only turn on during infection à limiting gene expression à no serious drain on plant biosynthetic capability & yield reduction
Common promoters used: 35s, wound-inducible
Most widely used in fruit trees
Challenge?
Greenhouse field
(2) Antimicrobial proteins
Antifungal and antibacterial, eg lysozyme, defensins
A. Lysozyme
Degrade chitin and peptidoglycan
B. Defensins
Small antimicrobial peptides
Display lytic activity difficult to develop resistance
Eg alfalfa defensin, cecropin, melittin, gallerimycin, temporins etc
Problem? Limited spectrum
Case study:
Combating the bacterial disease fireblight
Cause by Erwinia amylovora
Solution? Transgenic crop containing gene code for antimicrobial protein
2 Approaches:
(1) Lytic approach – produce transgenic fruit trees contain gene for T4 lysozyme + insect antimocrobial pt attacin E + cecropin
(2) Bovine lactoferrin gene (to compete with microbial siderophores) + depolymerase gene (degrade specific exopolysaccharides)
Siderophores - small, high-affinity iron chelating compounds secreted by grasses and microorganisms such as bacteria and fungi,
- amongst the strongest soluble Fe3+ binding agents known.
(3) Transgenic crops for food safety
How? By reduce amount of mycotoxins
Use to combat the effect of fungal infection
Example:
Bt genes into Bt crops minimize insect damage less wound sites due to insect infection lower possibility for fungal infection reduce amount of mycotoxins ensure safety of livestock and human
(4) Introduction of HR and SAR in transgenic plant
Aim: use elicitor receptor system to switch on general resistance pathway
Approach 1: the Guard hypothesis (tomato)
R protein increase its production will increase the expression of resistance systems
Outcomes:
Overexpression of R protein genes were induced differently
Not pathogen-specific
Approach 2: (tobacco) switch on the HR-related defence system
crytogein (a Phytophthora cryptogea gene that codes for highly active elicitor) fuse with pathogen inducible promoter (from tobacco) à introduce into transgenic tobacco
Non-induced condition (eg no pathogen)àno elicitor
Once infected by virulent fungus (Phytophthora parasitica var. nicotianae) à crytogein gene is expressed à elicitor-induced defense gene expressed
Broad-spectrum resistance
Other approaches
Trigger expression of a range of genes by using different stimuli, eg introduction of Arabidopsis gene NPR1 into tomato, wheat, rice etc.
The product of this gene lies at a node in the regulatory pathways that link SAR, induced systemic resistance, R-gene mediated resistance, ethylene, salicylic acid, jasmonic acid etc
it activates gene expression by interacting with members of the TGA family of basic leucine zipper (bZIP) transcription factors
The introduction of the gene enhanced resistance to a range of plant pathogens in tomato, but in rice the gene had a detrimental effect
The mode of action for the transgene seems to be a priming function: the plant responds more rapidly to attack rather than having its defence system switched on constitutively, which would have an effect on yield
Switching on cascade of defense genes (sunflower)
Involve of O sp eg H2O2 as a messenger in HR reaction has been tested by transforming plant with oxalate oxidase from wheat
oxalate oxidase
oxalic acid+ O2 -----------------------------------> CO2+ H2O2
transgenic plant could degrade oxalic acid & generate H2O2, and an HR-like response along with salicylic acid synthesis & defence-gene expression occur
The genes were identified as being PR & defensin protein
Transgenic sunflower exhibited enhanced resistance against the oxalic acid-generating fungus Sclerotinia sclerotiorum
Natural Disease-Resistance Pathways
Natural disease-resistance pathways: overlap between pests and diseases
Table 7.1
No immune system that produce specific cells to attack invading microbes; rather
Adopted more general defence systems and show many similarities with the innate defence systems of mammals.
E.g. cellular damage caused by plant’s response to pathogen and plant pests trigger general defence systems
Model plants such as tobacco and Arabidopsis in which the most recent molecular work has been done.
Four different levels of defence will be considered
(1) Anatomical defences;
(2) Pre-existing protein and chemical protection;
(3) Inducible systems;
(4) Systemic responses
Levels 1 and 2 were the basis general defences.
Inducible also contribute to these but also show more-specific defence responses.
(1) Anatomical defences
Pathogen invade plants through wound due to plants have developed morphological and structural systems that preclude pathogen access to living cells.
This can be thick layers of protective material, e.g. cuticle, bark, or waxes.
If this defence breached, cascades of defence systems come into play.
(2) Pre-existing protein and chemical protection
made up of antimicrobial proteins produced by the plant in defence against infectious agents
structure : conserved 3-d folding pattern, a superfamily of peptides that pre-dates the divergence of plants and animals
frequently associated with seeds at germination period
(3) Inducible System
Third level defence - a switch to counterattack that relies on protein systhesis de novo.
Normally switch off, but when some mechanisms of plant detect infection, this level of defence will be switch on. Why?
Costly for plant to permanently switched on its defence system.
Elicitor response
The inducers, or elicitors (pathogen) that gain entry or arrives into a living cell, it may induce resistance to infection.
The elicitors are of a number of different types, and the responses are complex.
Local response involves interaction with molecules released by pathogen (exogenous elicitor). OR
Host itself response to pathogen attack (endogenous elicitor) by the receptor molecules on the host .
This will stimulate general defense systems in both host and non-host plants OR race-specific interactions inducing defense responses to specific cultivars.
Both interaction may lead to cascade of reaction that include hypersensitive response (HR).
Or, induction of systemic resistance & even passage of signals to other plants
Fig 7.1, table 7.2
General response
Damage of plant cell wall in first stage of infection.
Fungi or bacteria (invaders) release proteolytic enzymes (e.g. oligogalacturonides) cause release wall fragments pectic-oligomers – general signals of endogenous elicitors, bind to specific receptor & induces cascade of reactions of specific defense genes.
Specific defense genes involve in coding :
(1) Enzymes – synthesize structural components for cell wall thickening (to repair damage)
(2) Enzymes for 20 metabolism,
(3) Lectins (multimeric sugar-binding pt that agglutinate cells)
(4) And many pathogenesis-related (PR) proteins.
• Fig 7.3
• PR protein include chitinases & β-1,3-glucanases, protease inhibitors, non-specific lipid-transfer proteins, ribosomal inhibitor proteins & various antimicrobial protein (eg, defensins eg SN1 - active against bacterial & fungal pathogens in potato
• A number of PR gene products have been identified as latex allergens
• Function of the defence proteins depends on the pathogens.
• E.g. chitinases and glucanases may cause degradation of fungal hyphae cell wall. Causing production of chitin and β-1,3-glucan oligomers which act as signal molecules (exogenous elicitor), which bind to membrane receptor & re-enforce the induction of defence systems.
• The cell may also produce phytoalexins (phenolic compound or terpenes) that kill any pathogen & cell in the vicinity of the infection, therefore limiting the spread
Race-specific response
Pathogens carried the avirulence gene (Avr) codes protein recognized by a specific receptor proteins in plant cell, encoded by resistance gene (R).
Most important!!! Either of the proteins is absent on this gene-gene system will cause disease in the plant.
Fig 7.2
This interaction induces
(a) the HR, which is manifested as a local necrosis that develops through a NADPH-dependent oxidative burst and/or the release of phenolics & nitric oxide
(b) many pathogenesis-related (PR) proteins
The gene-gene system is one of the most important ways plants have of switching on resistance systems eg for the interaction between plants & aphids, nematodes, fungi, viruses & bacteria
Many of the bacterial avirulence protein fall into a class of proteins transported into the plant cell by the type III protein-secretion system (TTSS)
They might function as virulence factors, subverting cellular functions through interactions with plant-encoded pathogenicity targets
Ex, a type III efector from Pseudomonas syringae (pv.) phaseolicoli, VirPphA, blocks other masked Avr proteins from inducing the HR response
Different type effectors able to suppress PAMP-induced defences. (PAMP – pathogen-associated molecular patterns)
AvrPto has been shown to suppress the expression of a set of genes that encode cell wall & defence proteins
Elicitor receptors (R proteins)
Fit into five basic structural groups.
Biggest group having a nucleotide-binding site and a leucine-rich repeat region (NB-LRR proteins).
Other four groups are structurally diverse but the R protein also contain an LRR region or R protein interacts with anther protein that contains he LRR domains.
Fig 7.3
Plant use receptors of similar structure comparison for the sensing of different types of elicitor that have evolved from a limited number of ancient receptors.
Many elicitor proteins are key factors in the biology of the pathogen, favour plant defence mechanisms.
Some plant R proteins come from mutation of LRR domain.
(4) Systemic responses
The induction of local defense pathways may lead to the induction of intercellular signals that produce a systemic response –systemic acquired resistance (SAR)
Both avirulent & virulent pathogens may result in the induction of SAR
SAR has two phases :
(1) the initiation phase
(2) the maintenance phase
Initiation and Maintenance phase
Initiation phase
cells at the foci of the infection release signal molecules (typically salicylic acid) phloem transported to target cells where SAR genes (eg, certain PR protein) are expressed
Maintenance phase
quasi-steady-state resistance against virulent pathogens
Fig 7.4
Induced systemic resistance
independent of salicyclic acid
induced by avirulent pathogens through pathway which induce ethylene or jasmonic acid (JA) as messengers
result in induction of different classes of defence proteins
Evidence : signals carried by volatile chemicals, such as methyl jasomenate, can travel between plants to warn of an attack
Fig 7.5 (pg 171)
Development from transcriptomics study
Significant levels of crosstalk between biotic (pathogen wounding, mechanical wounding by insects) and abiotic (drought, high salinity, cold) stress signaling networks.
Same gene are expressed in response to different stresses & same hormone regulate these processes
Reactive oxygen species (ROS) regulate these phenomena
ROS + nitric oxide establishment of HR
ROS – a common signal that trigger downstream processes in many stress responses
Host Resistance
Different plants defence systems have significant levels of overlap – plant innate immune system
Host resistance is dependent upon the interaction of an R gene with a TTSS protein HR reaction
TTSS – type III protein-secretion system
Non-host Resistance
Non-host resistance is more complex and composed of various defence genes including R proteins
2 types
(1) Does not induce an HR reaction and involves general defence mechanism eg the physical barriers of the cell wall, antimicrobial compounds, 2⁰ matabolites followed by the response to PAMPs à pathogen cannot get further in its attack
(PAMP – pathogen-associated molecular patterns)
(2) Produce a non-host HR reaction
Pathogen enters the cell
(a) Response to cell surface elicitors, (fungi or oomycetes), or
(b) TTSS-R-gene interaction (bacteria)
Table 7.1
No immune system that produce specific cells to attack invading microbes; rather
Adopted more general defence systems and show many similarities with the innate defence systems of mammals.
E.g. cellular damage caused by plant’s response to pathogen and plant pests trigger general defence systems
Model plants such as tobacco and Arabidopsis in which the most recent molecular work has been done.
Four different levels of defence will be considered
(1) Anatomical defences;
(2) Pre-existing protein and chemical protection;
(3) Inducible systems;
(4) Systemic responses
Levels 1 and 2 were the basis general defences.
Inducible also contribute to these but also show more-specific defence responses.
(1) Anatomical defences
Pathogen invade plants through wound due to plants have developed morphological and structural systems that preclude pathogen access to living cells.
This can be thick layers of protective material, e.g. cuticle, bark, or waxes.
If this defence breached, cascades of defence systems come into play.
(2) Pre-existing protein and chemical protection
made up of antimicrobial proteins produced by the plant in defence against infectious agents
structure : conserved 3-d folding pattern, a superfamily of peptides that pre-dates the divergence of plants and animals
frequently associated with seeds at germination period
(3) Inducible System
Third level defence - a switch to counterattack that relies on protein systhesis de novo.
Normally switch off, but when some mechanisms of plant detect infection, this level of defence will be switch on. Why?
Costly for plant to permanently switched on its defence system.
Elicitor response
The inducers, or elicitors (pathogen) that gain entry or arrives into a living cell, it may induce resistance to infection.
The elicitors are of a number of different types, and the responses are complex.
Local response involves interaction with molecules released by pathogen (exogenous elicitor). OR
Host itself response to pathogen attack (endogenous elicitor) by the receptor molecules on the host .
This will stimulate general defense systems in both host and non-host plants OR race-specific interactions inducing defense responses to specific cultivars.
Both interaction may lead to cascade of reaction that include hypersensitive response (HR).
Or, induction of systemic resistance & even passage of signals to other plants
Fig 7.1, table 7.2
General response
Damage of plant cell wall in first stage of infection.
Fungi or bacteria (invaders) release proteolytic enzymes (e.g. oligogalacturonides) cause release wall fragments pectic-oligomers – general signals of endogenous elicitors, bind to specific receptor & induces cascade of reactions of specific defense genes.
Specific defense genes involve in coding :
(1) Enzymes – synthesize structural components for cell wall thickening (to repair damage)
(2) Enzymes for 20 metabolism,
(3) Lectins (multimeric sugar-binding pt that agglutinate cells)
(4) And many pathogenesis-related (PR) proteins.
• Fig 7.3
• PR protein include chitinases & β-1,3-glucanases, protease inhibitors, non-specific lipid-transfer proteins, ribosomal inhibitor proteins & various antimicrobial protein (eg, defensins eg SN1 - active against bacterial & fungal pathogens in potato
• A number of PR gene products have been identified as latex allergens
• Function of the defence proteins depends on the pathogens.
• E.g. chitinases and glucanases may cause degradation of fungal hyphae cell wall. Causing production of chitin and β-1,3-glucan oligomers which act as signal molecules (exogenous elicitor), which bind to membrane receptor & re-enforce the induction of defence systems.
• The cell may also produce phytoalexins (phenolic compound or terpenes) that kill any pathogen & cell in the vicinity of the infection, therefore limiting the spread
Race-specific response
Pathogens carried the avirulence gene (Avr) codes protein recognized by a specific receptor proteins in plant cell, encoded by resistance gene (R).
Most important!!! Either of the proteins is absent on this gene-gene system will cause disease in the plant.
Fig 7.2
This interaction induces
(a) the HR, which is manifested as a local necrosis that develops through a NADPH-dependent oxidative burst and/or the release of phenolics & nitric oxide
(b) many pathogenesis-related (PR) proteins
The gene-gene system is one of the most important ways plants have of switching on resistance systems eg for the interaction between plants & aphids, nematodes, fungi, viruses & bacteria
Many of the bacterial avirulence protein fall into a class of proteins transported into the plant cell by the type III protein-secretion system (TTSS)
They might function as virulence factors, subverting cellular functions through interactions with plant-encoded pathogenicity targets
Ex, a type III efector from Pseudomonas syringae (pv.) phaseolicoli, VirPphA, blocks other masked Avr proteins from inducing the HR response
Different type effectors able to suppress PAMP-induced defences. (PAMP – pathogen-associated molecular patterns)
AvrPto has been shown to suppress the expression of a set of genes that encode cell wall & defence proteins
Elicitor receptors (R proteins)
Fit into five basic structural groups.
Biggest group having a nucleotide-binding site and a leucine-rich repeat region (NB-LRR proteins).
Other four groups are structurally diverse but the R protein also contain an LRR region or R protein interacts with anther protein that contains he LRR domains.
Fig 7.3
Plant use receptors of similar structure comparison for the sensing of different types of elicitor that have evolved from a limited number of ancient receptors.
Many elicitor proteins are key factors in the biology of the pathogen, favour plant defence mechanisms.
Some plant R proteins come from mutation of LRR domain.
(4) Systemic responses
The induction of local defense pathways may lead to the induction of intercellular signals that produce a systemic response –systemic acquired resistance (SAR)
Both avirulent & virulent pathogens may result in the induction of SAR
SAR has two phases :
(1) the initiation phase
(2) the maintenance phase
Initiation and Maintenance phase
Initiation phase
cells at the foci of the infection release signal molecules (typically salicylic acid) phloem transported to target cells where SAR genes (eg, certain PR protein) are expressed
Maintenance phase
quasi-steady-state resistance against virulent pathogens
Fig 7.4
Induced systemic resistance
independent of salicyclic acid
induced by avirulent pathogens through pathway which induce ethylene or jasmonic acid (JA) as messengers
result in induction of different classes of defence proteins
Evidence : signals carried by volatile chemicals, such as methyl jasomenate, can travel between plants to warn of an attack
Fig 7.5 (pg 171)
Development from transcriptomics study
Significant levels of crosstalk between biotic (pathogen wounding, mechanical wounding by insects) and abiotic (drought, high salinity, cold) stress signaling networks.
Same gene are expressed in response to different stresses & same hormone regulate these processes
Reactive oxygen species (ROS) regulate these phenomena
ROS + nitric oxide establishment of HR
ROS – a common signal that trigger downstream processes in many stress responses
Host Resistance
Different plants defence systems have significant levels of overlap – plant innate immune system
Host resistance is dependent upon the interaction of an R gene with a TTSS protein HR reaction
TTSS – type III protein-secretion system
Non-host Resistance
Non-host resistance is more complex and composed of various defence genes including R proteins
2 types
(1) Does not induce an HR reaction and involves general defence mechanism eg the physical barriers of the cell wall, antimicrobial compounds, 2⁰ matabolites followed by the response to PAMPs à pathogen cannot get further in its attack
(PAMP – pathogen-associated molecular patterns)
(2) Produce a non-host HR reaction
Pathogen enters the cell
(a) Response to cell surface elicitors, (fungi or oomycetes), or
(b) TTSS-R-gene interaction (bacteria)
PLANT DISEASSE TOLERANCE
Learning outcomes
Explain plant-pathogen interactions
Explain existing approaches to combat disease
Describe natural disease-resistance pathway:
Anatomical defences
Pre-existing protein and chemical protection
Inducible systems
Systemic responses
Discuss biotechnological approaches to disease resistance
- Protection against pathogens - PR proteins
- Antimicrobial proteins
- Transgenic crops for food safety
- Induction of HR and SAR in transgenic plants
Predict future development
Explain plant-pathogen interactions
Explain existing approaches to combat disease
Describe natural disease-resistance pathway:
Anatomical defences
Pre-existing protein and chemical protection
Inducible systems
Systemic responses
Discuss biotechnological approaches to disease resistance
- Protection against pathogens - PR proteins
- Antimicrobial proteins
- Transgenic crops for food safety
- Induction of HR and SAR in transgenic plants
Predict future development
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