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Basal plant immunity
Lecture overview
• How bacteria and filamentous plant pathogens penetrate and colonize the plant tissue
• How cell polarization/focal immunity contributes to elimination of pathogens
• Describe the components of plant innate immunity
• General features of pathogen associated molecular patterns
• Explain the plant mechanisms for sensing microbial invasion at plant surface
Part 1 – Cell and Molecular Biology of plant-microbe interactions
Global hunger
• Food crisis, hunger is on the rise
• 1bil people at the limit of starvation
• Not an issue in developed countries
• Rise in food prices
• Rise in population (world population of 9 billion by 2020), need in increase food production to
meet this
• Lots of technologies in agriculture but not meeting the global need for food production –
another approach is to prevent crop losses
Plants are attacked by a diverse range of organisms as they produce food
• Bacteria
• Filamentous plant pathogens (fungi and oomycetes) – eukaryotic parasites
• Viruses
• Nematodes
• Insects
• Parasitic plants
Filamentous plant pathogens
• Filamentous plant pathogens (fungi and oomycetes) cause destructive crop diseases
• Five of the top seven most dangerous plant pathogens are filamentous plant pathogens
• Rice blast, huge impact on rice production globally
• Black sigatoka disease on bananas
• Asian soyabean rust – soybean production
• Wheat stem rust
• Potato blight – oomycete pathogen, triggered great Irish famine in the 1850s
• Panama disease – destruction of bananas, huge threat for banana production
o 70% of banana production is one variety, resistance carried by this variety overcome by
panama disease (fungal pathogen)
o Happened in 1965 – there was a better banana variety but fully susceptible to this fungi
o Evolved over time and fungi can infect new variety
Global trade routes boost distributions of plant and animal pathogens
• Global pathogens spread by transport (shipping routes, roads, air travel)
• Food security problems, harm to environment, forests being destroyed by pathogens
introduced from different parts of the world

Infection strategies of filamentous plant pathogens
• Filamentous plant pathogens can enter plants in several ways
• Plants have cuticles on leaves to prevent entry of bacteria, virus and other pathogens, but
filamentous plant pathogens can breach this by physical means
• Fungi can enter through stroma (natural openings) and live in the extracellular space, leaching
nutrients from hosts
• Can directly penetrate in between
two cells, breach cuticle, can
grow in extracellular space and
produce extra penetrations to go
further through plants
(haustoria)
• Intracellular entry, direct
penetration of epidermal cell
layer, appressorium (specialised
cell that produced high turbor
pressure to breach cuticle) and
secretes enzymes to degrade the
cell wall, directly penetrate cell
Waxy epidermal cuticle and cell wall
• Prevents water loss and microbes from entering the epidermal later
• Filamentous plant pathogens can break this cuticle and cell wall
• Spore germinates and forms appressorium and can breach the cell wall
• Can genetically modify with fluorescent proteins to watch
• Intracellular region of plants is highly dynamic
o Lots of intracellular vesicular movements – good weapon to defend
o Can reprogram trafficking routes to overcome external changes (eg
...
) – bacteria produce the
extracellular polysaccharide xanthan to protect from environmental factors
• Form type III secretion machinery (essential pathogenicity factor) which spans the bacterial
membrane and is associated with an extracellular pilus
• The pilus is connected to a channel-like translocon that is inserted into the plant plasma
membrane
• Penetrates cell membrane and injects virulence factors (approximately 30 different effector
proteins) into the plant cell, refold and have different compartments to target
• Bacterial effector proteins are transported to different cellular compartments and manipulate
host cellular pathways
• Can alter plant gene expression in nucleus or vesicular trafficking in golgi
• Effector proteins suppress plant defence responses and promote multiplication at the infection
site
• Microbe can successfully take control of the plant cell, immune system shut down, take sugars
from the leaves
• Multiplication leads to necrotic spots on the plants and fruit
Xanthomonas infection in resistant plant
• Bacteria can also invade resistant plants and inject effector proteins (virulence factors)
• In resistant plants individual effector proteins are mostly recognised by cognate resistance
receptors (intracellular) which sense virulence factors (directly or indirectly) and activate
defence reactions
• Effector protein triggered plant defence leads to death of the plant cells at the site of infection
and bacterial starvation and arrest of growth
• Resistant plant stays healthy – few cells sacrificed at point of infection but remaining plant
stays healthy
Part I summary
• Plant pathogens use various different routes to enter host tissue:
o Through wounds and natural openings (stomata)
o By breaching cuticle and penetrating in between two cells
o Directly penetrating epidermal cells
• Cell polarization plays a major role in non-host resistance by preventing pathogen penetration,
whereas it also contributes to defense against adapted pathogens to an extent

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Focal immunity includes cytoplasmic streaming of endomembrane system and nucleus
towards pathogen ingress sites, followed by targeted cell wall fortifications and defense
related secretion
Focal immunity is suppressed during susceptibility
Pathogens penetrating the plant cells are enveloped by host derived membranes with
unknown origin

Part II – Surface immune recognition in plants
Most plants are actively resistant to most pathogens
• Disease is the exception, not the rule
• Disease occurs certain environment conditions favouring the pathogen and specialised
pathogens that are able to infect the plant
• Plants have an innate ability to recognise potential invade pathogens and to mount effective
defences
o Plants can produce their own food so simply produce a barrier to keep everything out
Local defence
• Occurs at the site of infection – local immune response to prevent spread of parasites
• First line of protection – innate immunity, cell autonomous immunity
• Very early detection of invaders in infected cells through specific or generic interactions
• Restricts pathogen grown and spread (programmed cell death, production of defensive
molecules)
• Local response can include hypersensitive response – induced cell death at the point of
infection, programmed cell death
o Can also happen in animals – cell suicide to warn the immune system
Systemic defence
• Protects non-infected tissues from secondary infection
• Signals transmitted throughout plant cell – probably multiple types of signals
• Discovered that plants have an electric singlaling system using glutamate-like receptors
o Not as fast as our signalling response but similar system
• Systemic activation of plant activation following damage
• SAR – local defence activator alerts the plant
o Another challenge with a pathogen in a different place – plant is resistant
o Upregulation of defence systems throughout the plant
• SAR = systemic acquired resistance
• Prioritise this over growth
Surface receptors mediate basal immunity – pattern recognition is key
• Microbes come from outside the cell so makes sense to have receptors on the cell surface
• Extracellular domains to sense pathogens
• Transmembrane domain to transmit signalling
• Cytoplasmic domain to mediate downstream signalling and activate immune response
• PRRs recognise PAMPs (pathogen-associated molecular pathogens)
• PAMPs are conserved molecules essential for lifestyle of pathogen (common molecule) but not
necessarily required for infection process; associated with a group of pathogens; recognised by
cells of the immune system
o LPS – protects bacterial cell

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•
•
•

o Bacterial flagellin is a typical PAMP
o Remove flagellin and inject bacteria, will still give an infection but cannot move around
and survive – bacteria cannot remove flagella
o Chitin is a typical fungal PAMP
Common feature – one receptor can detect many pathogens if they all carry a common feature
PRR-triggered immunity (PTI) activated
DAMPs (damage-associated molecular patterns) sense own molecules that tell the plant cell
there is damage
PAMP activation combined with DAMP activation – more confidence that there is a threat
o Symbiotic bacteria may carry flagellin but will not damage the cell

General features of PAMP
• Not fast evolving, widely or narrowly conserved
• Invariant or highly constrained sequence
• PAMP is essential for microbial fitness and survival
• Do not need to be contribute to virulence by targeting host (defence) physiology
• Do not necessarily need to be secreted
o Elongation factor in bacteria is not secreted, inside bacteria but highly abundant, one of
the most expressed proteins in bacteria, when they invade plant intracellular space,
many bacteria die and bacterial EF is released
• PAMPs need to be abundant
The N terminus of bacterial EF Tu elicits innate immunity in Arabidopsis plants
• P
...
syringae isolates
• Flg22 is recognised by FLS2
• Extracellular domain binds to the ligand
• Co-receptors are required for signalling – in the absence of partners, the receptor is unable to
signal
Surface immune receptors in plants
• Immune receptors have extracellular domains, transmembrane domains and cytoplasmic
domains
• Receptor-like kinase (RLK)
o Extracellular leucine-rich repeat (LRR) attached to a cytoplasmic kinase
• Receptor-like protein (RLP)
o No intracellular kinase domains
o Interacts with receptor-like kinase co-receptors to trigger downstream signalling
• Symbiosis/immunity – Extracellular LYSm domains for binding sugars (PGN in bacteria and
chitin in eukaryotes)
o Involved in immunity and symbiosis (for commensal bacteria/fungi)
• Many contain LRRs, important for protein-protein interactions
Activation of surface receptors leads to cellular reprogramming
• PAMPs are released/secreted by pathogens and PRRs are activated
• Early response in PTI is production of ROS
o Highly reactive chemicals which disrupt membranes, generally toxic to microbes
o Cause DNA damage
o Effective against microbial pathogens

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•

o Also important for signalling- can warn neighbouring cells of pathogen threat
o ROS also mediate cross-linking of cell wall polymers around papilla and gives strong cell
wall fortification
Defence-related gene expression
Activation of defence hormones
Cell wall fortification through formation of papilla with callose deposition (ROS also involved)

Activation of FLS2 by bacterial flagellin and downstream signalling leads to ROS production
• FLS2 requires a co-receptor – BAK1
• BAK1 is a co-receptor for many different PRRs
o FLS2 and EF receptor are examples
o Without BAK1 there is only a low level of signalling as this the main signalling partner
• FLS2 forms a complex with cytoplasmic kinases such as BIK1
o BIK1 has a kinase domain but no transmembrane region – it is a membrane-associated
protein but not embedded in the membrane
• Bacterial flagellin activates FLS2 and BAK1 associates, resulting in a strong interaction
• Once FLS2 is activated and is in complex with BAK1, they phosphorylate each other
• FLS2 directly phosphorylates BIK1 (following cross-activation by BAK1)
• BIK1 activation leads to phosphorylation of RBOHD (NADPH oxidase), leading to production of
reactive oxygen species (ROS)
o Only RBOHD involved in immunity
• ROS has antimicrobial function and is also important for the stomata closure to stop entry of
pathogens
• FLS2 activation also stimulates BAK1 to activate super uptake through activation of sugar
transporter
o BAK1 directly phosphorylates sugar transporter STP13
o STP13 has multiple transmembrane domains and internalises sugars
o Sugar is no longer accessible to pathogen
• FLS2 activation results in upregulation of STP13 gene expression so internalisation of sugar is
more effective – this removes sugar from the extracellular environment so the pathogen cannot
access it
Activation of surface immune receptors leads to upregulation of defence related genes
• A group of defence-related plant genes are up-regulated upon pathogen challenge
o Take plant and challenge with PAMP or microbe
o Extract extracellular fluid at different time points (0-72 hrs) and monitor protein
production
• Many proteins upregulated over time – pathogenesis-related (PR) proteins
o Only secreted after pathogen challenge
• Involve many different enzymes
o β-1,3-glucanases
o Chitinases – degrade chitin; usually leads to fungi destruction but some have strategies
to fight this
o Proteases – prevent microbial colonization by degrading proteins in microbes
o Thaumatin-like proteins
o Peroxidases – produce hydrogen peroxide leading to formation of ROS
o Ribosome-inactivating proteins
o Thionins
o Nonspecific lipid transfer proteins
o Oxalate oxidase

Some apoplastic effectors ‘trip the wire’ and activate immunity in particular plant genotypes
• PAMP activation in plants does not necessarily lead to cell death activation
• Surface receptors in plants that bind to specific molecules secreted by the pathogen to subvert
plant processes – virulence factors
o Required for virulence function of the pathogen (unlike PAMPs)
o High selective pressure, evolving fast
o Not conserved like PAMPs
• Plants have strategies to capture these – generally use receptor-like proteins
o No kinase domain for signalling
• Receptor-like proteins signal through structural change; cytoplasmic tail can interact with
signalling molecules
• BAK1 can also pair with this type of receptor to mediate signalling
• Activation through specialised virulence factors can be direct or indirect
o Factors target guardee, which is recognised by the plant receptor
o Receptor recognises guardee to trigger basal defences and induce PCD (hypersensitive
response)
o Guardee detection acts as a confirmation signal – no question that there is a real threat
present as detects specific virulence factors
• This is effector-triggered immunity
o Involves cell surface receptors but the majority of signalling is intracellular
Part II summary
• Plants rely on surface immune receptors to sense and respond to PAMPs released by invading
pathogens
• PAMPs are conserved to a degree which are able to report presence of a group of pathogens
rather than being specific to certain pathogen strain
• PTI leads to various cellular outputs including ROS production, defense gene induction and cell
wall fortifications etc
...
infestans)
o AVR3aEM – virulent allele
o AVR3aKI – avirulent allele
• Clone virulence genes into Agrobacteirum to deliver the gene to plants and express in plant
cells
o AVR3aKI allele (avirulent) in the presence of the R3a receptor resulted in a good
immune response
o Virulent allele does not show any response
o No R3a receptor = no response
Most plant resistance genes encode NLR proteins
• NLRs are modular proteins that contain three classical domains
o N-terminal coiled-coil (CC) domain or TOLL/interluekin-1 receptor (TIR) domain
▪ Signal transduction and downstream signalling
▪ Protein-protein interactions
▪ Cell death executer
▪ TIR domain may be enough to initiate a response
o Central nucleotide binding pocket (NB)
▪ Regulator of activation
▪ Nucleotide binding
▪ Mediates large conformation changes
▪ May drive oligomerisation
o C-terminal leucine-rich repeat (LRR) domain
▪ Implicated in effector recognition
▪ Role in auto-inhibition

•
•

NLR activation involves intra- and inter-molecular
conformational changes
Intramolecular interactions are critical to regulate NLR activity
o Without an effector molecule, the NB domain is
sandwiched between the CC and LRR domains; ATP is
masked in the central region
o Binding of the effector results in a structural change,
triggering the optening of the structure; ATP can be
replaced with ADP
o This results in the hypersensitive response

Effector detection by NLRs can be direct or indirect
• Direct detection by NLRs

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o Direct binding of the effector protein to the NLR
o Rare occurrence
Indirect detection
o Effector binds to a host protein ‘target’
o Modification of host target by effector protein – cleavage, structural change
o NLR/R gene recognises modified self – R proteins monitor the integrity of host cellular
targets (guard hypothesis)
Both direct and indirect detection leads to activation of immunity

Example of indirect recognition of an effector
• AvrPphB is an effector protein (avirulence factor) secreted by bacteria via a type III secretion
system
• AvrPphB is a protease that targets a host cytoplasmic kinase PBS1
• AvrPphB cleaves PBS1, leading to an immune response triggered by RPS5 (R protein)
o RPS5 can be activated by mimicking cleavage of PBS1 (without AvrPphB), leading to
activation of the immune response
o This is proof of indirect recognition as there is a response in the absence of the effector
protein
• In the absence of PBS1 (effector target), there is no hypersensitive response and no immunity
Integrated domains – the merger of effector biology and NLR biology
• Although the majority of NLR receptors share a conserved domain architecture, anywhere
from 3 to 10% of plant NLRs turned out to include extraneous domains in addition to the
classic NLR architecture
• These so-called “integrated domains” are thought to have evolved from effector targets to
mediate pathogen detection, either by binding effectors or by serving as substrates for the
effector’s enzymatic activity
o Rice RGA5, Pik1 and RRS1 from Arabidopsis
• The discovery that NLR proteins contain unconventional domains that have evolved by
duplication of an effector target, followed by fusion into the NLR, signals the merger of two
fields – NLR biology and effector biology
...
oryzae effectors AVR-Pia or AVR1-CO39 to RGA5 results in the release
of RGA4, which leads to immune signaling and disease resistance
• Cooperation (positive regulation) – the sensor and helper NLRs work together and when the
effector binds and is recognised by the sensor NLR, the helper NLR is activated to get a immune
response
o In the rice Pik pair, neither Pik-1 nor Pik-2 exhibit autoimmunity, and thus presumably
operate through a different mechanism
o Although the precise mechanics of the activation of the Pik pair remains to be
elucidated, both NLRs are required to trigger an immune response following AVR-Pik
binding to Pik-1
o This suggests that the Pik pair may function through cooperation between the NLR
mates
• The mechanisms by which NLR pairs function is likely to impose different constraints on the
evolution of the corresponding NLR genes
o NLRs that operate through negative regulation, such as Pia, are likely to function as a
single unit and remain genetically linked to ensure fine-tuned co-regulation and prevent
the accumulation of deleterious effects
...


Constraints and plasticity in plant NLR evolution
• NLR evolution must be constrained by its mode of action
• Some NLR pairs are known to operate by negative regulation with the helper NLR exhibiting
autoimmunity (NLR*) and the sensor NLR acting as a helper inhibitor; in such cases, expansion
of the pair will be constrained throughout evolution due to the genetic load caused by
autoimmunity
o It is favourable to have genes next to each other with the same promtor
• In contrast, NLRs that function through a different mechanism (e
...
, positive regulation of the
NLR helper by the sensor) will be less constrained to evolve into networks beyond genetically
linked pairs of NLRs
o Genes do not need to be side by side
Plant immune system can drive the formation of new species
• Breeding of certain plants leads to dwarf plants
• NLR combinations can lead to autoimmunity and hybrid necrosis
• Plant pathogens may also be driving plant evolution – receptors evolve to protect against
pathogens but this leads to restrictions on breeding
From pairs to networks
• One-to-one – one sensor NLR acts on one helper NLR
• Many-to-one – many sensor NLRs act on one helper NLRs
• Many-to-many – sensor and helper NLRs can form many different pairs
• NLR networks mediate immunity to diverse plant pathogens
o An effector strategy is to have effectors that target helper NLRs

Why form a network?
• Evolvability
o Increases adaptive landscape of sensor NLR
o Enables more rapid evolution of NLR immune system
• Robustness
o More stable and stronger immune responses
o Tolerates environmental disturbances
o Evades immune suppression by pathogen

Part I summary
• Plants utilize a ubiquitous disease resistance toolkit known as NLRs to defend against
unrelated pathogens
• NLRs are key components of plant immunity and their activities tightly controlled by both
intramolecular and intermolecular interactions
...
fulvum only lives in the extracellular space
o It secretes apoplastic effector AVR4, which binds to chitin, preventing chitinase binding
to chitin – protects fungi against chitinase
o C
...
fulvum
o Protease PiP1 does contribute to defence
• Fungi secretes effector protein AVR2 – protease inhibitor that binds that active site of the
protease and inhibits both of them
• Some surface receptors recognise effectors indirectly by guarding secreted plant proteases
o CF-2 is a plant surface receptor evolved to recognise AVR2
o Leads to hypersensitive response
o Only guards RCR3 protease (the one that does not contribute to defence)
o Decoy in this case as RCR3 does not contribute to immunity
o Absence of RCR3 does not impair immune response and only serves as a decoy
o CF-2 recognises RCR3 bound to AVR2 and activates basal defences and induces HR
• Diverse pathogens can target the same host targets – AVR2 and EPIC1
o In response to an oomycete infection, RCR3 contributes to defence
o EPIC1 effector protein inhibits RCR3
o CF-2 does not recognise EPIC1
• Two protease inhibitors for the same protease – one activates immunity, one does not
• CF-2 can only sense the interaction between ARV2 and RCR3, not EPIC1 and RCR3 – oomycete
has developed a better inhibition
o RCR3 interaction with AVR2 may alter the structure of RCR3 so that it can be recognised
by CF-2
o EPIC1 has evolved to inhibit RCR3 without detection by CF-2
Part III – Molecular arms race between plants and pathogens
Effector-targeted pathways – functional redundancy among pathogen effectors
• Pathogens generally target pathways
• Effectors from a given pathogen tend to converge on particular host pathways
• Many effectors seem functionally redundant – they effect different steps or converge on the
same target
• Effectors from phylogenetically unrelated pathogens may converge on the same host targets
• Example – FLS2
o Effectors do not only target FLS2 directly but target different steps in the pathway
o FLS2 recognises bacterial flagellin; upon recognition, FLS2 is phosphorylated
o There are receptors that directly target FLS2, could block activation/phosphorylation or
lead to degradation
o Effectors also target downstream pathways, such as MAPK pathway

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o MAPK signals to nucleus leading to activation of immune genes – effectors can supress
the activation of gene transcription
o Production of transcripts for PRR – effectors also target these transcripts
AvrPtoB secreted by bacteria targets FLS2
o Behaves as a ubiquitin ligase – evolved to mimic this, ubiquitinates FLS2
o Ubiquitinated FLS2 is internalised by endocytosis and is degraded in the lysosome – no
receptor on the cell surface so suppression of immunity
HopB1 targets activated BAK1-FLS2 complexes (does not target inactivated BAK1)
o Leads to cleavage of cytoplasmic cleavage domain resulting in immune suppression –
BAK1-FLS2 complex is inactivated by the bacteria

PR1 – sterol binding protein
• Pathogenesis-related protein expressed in almost all plants
• Many pathogens are sterol auxotrophs so requires sterols
• Expression of PR1 limits pathogen growth
• PR1 binds to sterols so that the microbe cannot get them and they starve to death
o Works with sterol auxotroph
• P

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