Plant Immunity: Plant-Pathogen Arms Race
Lecture 1
Plants come under attack from a wide range of pathogens
- Bacterial
o Pseudomonas syringae (attacks apples)
- Fungal
o Botrytis cinerea (attacks strawberries)
- Viral
o Maize streak virus
o Tobacco mosaic virus
Why Should We Care
- Potato Famine
o Potatoes taken to Europe from South America
o Pathogen Phytophthora infestans (semi fungus pathogen) was brought in
300 years later
o Potato light blight caused by the pathogen caused the Irish Great Famine
of the 1840s
o Decline in population as a result of:
§ Immigration
§ Starvation
§ Slow response by British government
- Stem Rust attacking wheat crops in Africa à impact on bread industry!
o Massive spread from Uganda à SA à Middle East
- Plant pathogens can spread quickly and widely
- Ability to respond to pathogens: scale of farms and available resources
What turns an interaction into disease: epidemiological triad
- Pathogen
o Can pathogen evade host immune system
o Can pathogen suppress host immune system
- Host
o Can plant detect and respond to the pathogen
- Environment
o Humid, warm weather can favour pathogen prevalence
o Can tip the balance in favour of plant or pathogen
o Most pathogens = seasonal
o Climate change: allows new colonization, extension of seasonality of
pathogens
Pathogens Adopt DiXerent Strategies
- Biotrophs
o Feed oX the host but don’t kill it
o Reduce the resources of the plant and result in the plant’s death despite
this not being pathogen’s intention
, - Necrotrophs
o Deliberately kill host and then consume it (consume the remains)
- Hemibiotrophs
o Start of biotrophic but can switch to necrotrophy
Plants have multiple layers of innate immunity
à innate immunity occurs with a fixed set of receptors present throughout lifespan
à rely on the detection of non-self (alien/foreign)
- PAMP-triggered immunity (PTI)
- EXector triggered immunity (ETI)
- RNA interference (RNAi)
- Systemic-acquired resistance (SAR)
*PTI and ETI mainly used against bacterial and fungal pathogens
*RNAi used against viral pathogens
VS animals have an innate and adaptive immune system (adaptive response comes
from somatic recombination, memory and specificity to a pathogen)
- Plant immune response is cell autonomous
o Every cell can elicit an immune response
o No mobile response cells
o Each cell is responsible for mounting a necessary response
PTI and ETI
- Case Study: Pseudomonas syringae (Gram – Bacteria) and Arabidopsis Thaliana
o Pseudomonas syringae has flagellum for movement + Hrp pilus
§ Biotroph/hemibiotroph
o Arabidopsis = easily genetically manipulated
o DC3000 gene of pathogen = important for infection
§ Can knockout genes
• Allows us to move from correlation to causation (need
functional analysis)
o Can edit Arabidopsis via CRISPR
- Pst Infection Cycle:
o Bacteria arrive on the leaf surface (epiphytic growth) via raindrop
§ Hostile environment (waxy cuticle on leaf surface)
o Bacteria can enter via stomata (normally used for gas exchange and
photosynthesis) or via wounds on plant surface
§ Grow between plant cells (in the extracellular space: apoplast)
NOT IN THE PLANT CELL = intercellular growth
• Forces plant cells to export nutrients and water to the
apoplast
o Disease development
§ Nutrients are being used up
, § Water being exported to apoplast à tissues get waterlogged
§ Wilting
§ Loss of chlorophyll
o Dissemination via rain splash or infected cells
§ Can lead to new infection
o In the lab: 2 ways to infect Arabidopsis with Pst
§ Pressure infiltration (syringe)
• Liquid culture of pseudomonas
• Syringe culture without needle and press onto leaves
• Allows for infiltration of a known amount of bacteria: easy to
monitor over time if a certain concentration of bacteria can
replicate
• BUT bypasses the stomata: unnatural infection
§ Spraying
• Spray bottle of culture
• Spray tops of leaves
• Cannot control amount of bacteria infiltrating
• Mimics natural infection
o Bacteria must bypass stomata
o To find cfu/ cm in a leaf: grind up leaf and perform serial dilution
3
§ Symptoms of the plant are not a good measure for the number
of bacteria within the plant
• Measure pathogen growth!!
• Disease symptoms lag bacterial growth
o Plant may appear healthy but is infected by
numerous bacteria
PAMP-Triggered Immunity (PTI)
- PAMP = pathogen associated molecular pattern (MAMPs = microbial associated
molecular pattern)
- = Highly conserved and widely distributed molecules found in microbes but
not in plants that are essential to the pathogen/microbe for survival
§ Widely distributed à can use smaller number of PAMPs (wide
range of surveillance by plants)
§ Highly conserved à can use smaller number of PAMPs
§ Leads to eFiciency!!
§ Recognition of essential molecules à unlikely to evolve rapidly
or change over time (loss would be deadly to the microbe as
they attempt to change under selective pressure)
o Eg. Flagellin (subunit of bacterial flagellum; protein)
o Eg. Lipopolysaccharide (component of Gram – bacterial cell walls)
o Eg. EF-Tu (translation elongation factor; protein)
§ Intracellular protein
§ Released as the bacteria die within the plant
o Eg. Chitin = essential to fungal pathogens
, - Induction of PTI is dependent on recognition of these non-host molecules by the
plant
Flg22: the first PAMP
- If plant cell culture was exposed to pathogenic bacterian (eg. Pseudomonas) à
a response was elicited
o Change in media pH à pH increases (alkalization)
§ Either, the bacteria alkalizes the media
§ OR the plant cells are responding to the pathogen’s presence
o To test, add dead pseudomonas and monitor pH change
§ Saw the pH increase
§ Therefore, plant cells recognize a component of the pseudomonas
rather than a process undergone by the pseudomonas
• Could be lipids, carbohydrates, nucleic acids or protein
• To identify which: use enzymes specific to destruction of
specific biomoelcules
o Protease, lipase, RNase/DNase etc
• Destruction of carbohydrates, nucleic acids and lipids saw
no change in response
• Protein molecule was being responded to by the plant
o Ran an SDS PAGE gel alongside total protein extract
o Cut gel
o Eluted proteins from gel slices
§ Small mix of proteins
o Add proteins from slices to the plant cells in
succession to monitor change in pH of cell culture to
see which slice elicits response
o Found one major protein to result in a pH change
§ Flagellin
• Added pure flagellin to culture and saw a massive change in
pH
§ Analyse amino acid sequence of flagellin
• Found 22aa sequence conserved across most organisms in
database but NOT ALL
o Highly conserved sequence is likely the one
recognized (epitope)
o If not conserved in an organism à will not be
recognized by plant if infected with that microbe
• Can test by synthesizing the sequence of aa and adding to
plants to see if the pH changes in response to the aa
sequence
Lecture 1
Plants come under attack from a wide range of pathogens
- Bacterial
o Pseudomonas syringae (attacks apples)
- Fungal
o Botrytis cinerea (attacks strawberries)
- Viral
o Maize streak virus
o Tobacco mosaic virus
Why Should We Care
- Potato Famine
o Potatoes taken to Europe from South America
o Pathogen Phytophthora infestans (semi fungus pathogen) was brought in
300 years later
o Potato light blight caused by the pathogen caused the Irish Great Famine
of the 1840s
o Decline in population as a result of:
§ Immigration
§ Starvation
§ Slow response by British government
- Stem Rust attacking wheat crops in Africa à impact on bread industry!
o Massive spread from Uganda à SA à Middle East
- Plant pathogens can spread quickly and widely
- Ability to respond to pathogens: scale of farms and available resources
What turns an interaction into disease: epidemiological triad
- Pathogen
o Can pathogen evade host immune system
o Can pathogen suppress host immune system
- Host
o Can plant detect and respond to the pathogen
- Environment
o Humid, warm weather can favour pathogen prevalence
o Can tip the balance in favour of plant or pathogen
o Most pathogens = seasonal
o Climate change: allows new colonization, extension of seasonality of
pathogens
Pathogens Adopt DiXerent Strategies
- Biotrophs
o Feed oX the host but don’t kill it
o Reduce the resources of the plant and result in the plant’s death despite
this not being pathogen’s intention
, - Necrotrophs
o Deliberately kill host and then consume it (consume the remains)
- Hemibiotrophs
o Start of biotrophic but can switch to necrotrophy
Plants have multiple layers of innate immunity
à innate immunity occurs with a fixed set of receptors present throughout lifespan
à rely on the detection of non-self (alien/foreign)
- PAMP-triggered immunity (PTI)
- EXector triggered immunity (ETI)
- RNA interference (RNAi)
- Systemic-acquired resistance (SAR)
*PTI and ETI mainly used against bacterial and fungal pathogens
*RNAi used against viral pathogens
VS animals have an innate and adaptive immune system (adaptive response comes
from somatic recombination, memory and specificity to a pathogen)
- Plant immune response is cell autonomous
o Every cell can elicit an immune response
o No mobile response cells
o Each cell is responsible for mounting a necessary response
PTI and ETI
- Case Study: Pseudomonas syringae (Gram – Bacteria) and Arabidopsis Thaliana
o Pseudomonas syringae has flagellum for movement + Hrp pilus
§ Biotroph/hemibiotroph
o Arabidopsis = easily genetically manipulated
o DC3000 gene of pathogen = important for infection
§ Can knockout genes
• Allows us to move from correlation to causation (need
functional analysis)
o Can edit Arabidopsis via CRISPR
- Pst Infection Cycle:
o Bacteria arrive on the leaf surface (epiphytic growth) via raindrop
§ Hostile environment (waxy cuticle on leaf surface)
o Bacteria can enter via stomata (normally used for gas exchange and
photosynthesis) or via wounds on plant surface
§ Grow between plant cells (in the extracellular space: apoplast)
NOT IN THE PLANT CELL = intercellular growth
• Forces plant cells to export nutrients and water to the
apoplast
o Disease development
§ Nutrients are being used up
, § Water being exported to apoplast à tissues get waterlogged
§ Wilting
§ Loss of chlorophyll
o Dissemination via rain splash or infected cells
§ Can lead to new infection
o In the lab: 2 ways to infect Arabidopsis with Pst
§ Pressure infiltration (syringe)
• Liquid culture of pseudomonas
• Syringe culture without needle and press onto leaves
• Allows for infiltration of a known amount of bacteria: easy to
monitor over time if a certain concentration of bacteria can
replicate
• BUT bypasses the stomata: unnatural infection
§ Spraying
• Spray bottle of culture
• Spray tops of leaves
• Cannot control amount of bacteria infiltrating
• Mimics natural infection
o Bacteria must bypass stomata
o To find cfu/ cm in a leaf: grind up leaf and perform serial dilution
3
§ Symptoms of the plant are not a good measure for the number
of bacteria within the plant
• Measure pathogen growth!!
• Disease symptoms lag bacterial growth
o Plant may appear healthy but is infected by
numerous bacteria
PAMP-Triggered Immunity (PTI)
- PAMP = pathogen associated molecular pattern (MAMPs = microbial associated
molecular pattern)
- = Highly conserved and widely distributed molecules found in microbes but
not in plants that are essential to the pathogen/microbe for survival
§ Widely distributed à can use smaller number of PAMPs (wide
range of surveillance by plants)
§ Highly conserved à can use smaller number of PAMPs
§ Leads to eFiciency!!
§ Recognition of essential molecules à unlikely to evolve rapidly
or change over time (loss would be deadly to the microbe as
they attempt to change under selective pressure)
o Eg. Flagellin (subunit of bacterial flagellum; protein)
o Eg. Lipopolysaccharide (component of Gram – bacterial cell walls)
o Eg. EF-Tu (translation elongation factor; protein)
§ Intracellular protein
§ Released as the bacteria die within the plant
o Eg. Chitin = essential to fungal pathogens
, - Induction of PTI is dependent on recognition of these non-host molecules by the
plant
Flg22: the first PAMP
- If plant cell culture was exposed to pathogenic bacterian (eg. Pseudomonas) à
a response was elicited
o Change in media pH à pH increases (alkalization)
§ Either, the bacteria alkalizes the media
§ OR the plant cells are responding to the pathogen’s presence
o To test, add dead pseudomonas and monitor pH change
§ Saw the pH increase
§ Therefore, plant cells recognize a component of the pseudomonas
rather than a process undergone by the pseudomonas
• Could be lipids, carbohydrates, nucleic acids or protein
• To identify which: use enzymes specific to destruction of
specific biomoelcules
o Protease, lipase, RNase/DNase etc
• Destruction of carbohydrates, nucleic acids and lipids saw
no change in response
• Protein molecule was being responded to by the plant
o Ran an SDS PAGE gel alongside total protein extract
o Cut gel
o Eluted proteins from gel slices
§ Small mix of proteins
o Add proteins from slices to the plant cells in
succession to monitor change in pH of cell culture to
see which slice elicits response
o Found one major protein to result in a pH change
§ Flagellin
• Added pure flagellin to culture and saw a massive change in
pH
§ Analyse amino acid sequence of flagellin
• Found 22aa sequence conserved across most organisms in
database but NOT ALL
o Highly conserved sequence is likely the one
recognized (epitope)
o If not conserved in an organism à will not be
recognized by plant if infected with that microbe
• Can test by synthesizing the sequence of aa and adding to
plants to see if the pH changes in response to the aa
sequence
