Molecular interaction
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Transcript of Molecular interaction
Evolutionary Ecology of Infectious Disease: Current Topics
B. McDonald, S. Bonhoeffer, R. R. Regös, P. Schmid‐Hempel
Course organization
New theme each semester, often a topic of current research in our labs
Draw connections between diseases of plants, animals, insects
Focus on evolutionary and ecological processes
Recent scientific literature
Review assigned paper (45 minutes Powerpoint) and prepare 1‐2 page summary
Group discussion of paper (45 minutes)
Contributed questions/discussion points from each student in audience
The Evolutionary Ecology of Pathogen Effectors
Number of papers with the words “innate immunity” in their title, 1950‐2011
The Evolutionary Ecology of Pathogen Effectors
Number of citations to papers with the words “innate immunity” in their title, 1950‐2011
First plant R‐genes cloned (NB‐LRR)
Plant Pathologists show conserved structure of plant R‐genes and identify similar motifs in animal and insect proteins involved in signaling pathways activated during host defense. Propose that all of these pathways are involved in innate immunity. (Science 268:661‐667, 1995).
Toll discovered in DrosophilaShown to be involved in development
Proof that TLRs are involved in innate immunity of humans
TLR1 discovered in humans
The Evolutionary Ecology of Pathogen Effectors
The connection between innate immunity in plants and animals was made relatively recently following the discovery of NB‐LRR receptors in plants
Gene‐for‐gene theory developed by plant pathologists during the 1940s‐1950s led to
our current understanding of innate immunity in plants and animals
The Gene‐for‐Gene Hypothesis to Describe Genetic Basis of Pathogen‐Plant Interactions
Major gene resistance was first recognized in 1905 by Rowland H. Biffen(PBI, Cambridge, UK) soon after re‐discovery of Mendel’s Laws of inheritance.
Greg Lawrence CSIRO Plant Industry
H.H. Flor
Harold H. Flor conducted studies in 1940s‐1950s with flax (Linum usitatissiumum) and the flax rust pathogen (Melampsora lini) to understand the genetic basis of the interaction between resistance and virulence.
Flor originated gene‐for‐gene theory based on observations from his experiments with flax rust, making crosses between both plants and pathogens to determine the inheritance of resistance and avirulence.
Resistant Flax Plants Can Be Differentiated from Susceptible Plants
Greg Lawrence CSIRO Plant Industry
X
Resistance is due to a hypersensitive response (flecks)
Susceptible and resistant plants can be crossed to determine the inheritance of resistance
Results From Flor’s Crosses
25 resistance alleles distributed across 5 loci were identified
Locus # AllelesK 1L 11M 6N 3P 4
For every resistance allele found in the plant, a corresponding avirulence allele was found in the pathogen
There is a gene‐for‐gene interaction between host and parasite
L and M loci cloned and sequenced in 1995. 13 different alleles characterized at L locus gave important insights into functions of R‐genes
The Gene‐for‐Gene Hypothesis
Complete set of interactions (diploid host + diploid pathogen)
Pathogen Host genotypegeno. RR Rr rrAA ‐ ‐ +Aa ‐ ‐ +aa + + +
The “Quadratic check”
Pathogen Host genotypegeno. R‐ rrA‐ ‐ +aa + +
R = resistant allele at locus (dominant, has function)
r = susceptible allele at locus (recessive, loss of function)
A = Avirulence allele at locus (dominant, has function)
a = virulence allele at locus (recessive, loss of function)
+ = compatible response (disease results)‐ = incompatible response (no disease)
The Receptor‐Elicitor Model To Explain GFG Interactions
Pathogen Host genotypegeno. R- rrA - +a + +
PathogenAvr allele
elicitor present
Host resistantPlant defenses activated
Host susceptibleNo recognition, no defense
Resistance allele
receptor present
Plant cell
Host susceptibleNo recognition, no defense
Pathogenvir allele
elicitor lost
susceptible allele
no receptor present
Plant cell
Host susceptibleNo recognition, no defense
Resistance allele
receptor present
Plant cell
R-
Plant receptor: product of R allele (NB-LRR or TLR)
susceptible allele
no receptor present
Plant cell
rr
Pathogen elicitor: product of A allele (effectors)
PathogenAvr allele
elicitor present
A
Pathogenvir allele
elicitor lost
a
Major gene resistance
Receptors Can Be Inside Cell or In Cell Wall (membrane)Recognition of pathogen can occur before or after penetration of cell.
After binding with elicitor, signal transduction cascade occurs that turns on a series of genes involved in defense responses, including hypersensitive response,apoptosis.
Diagram illustrating proteins present in plants, Drosophila, and mammals that initiate signal transduction cascades after pathogen infection, which ultimately lead to innate disease resistance
Conservation of LRR‐types of Recognition Systems
Variation In The LRR Region Of An R‐gene Is Generated By Recombination, Deletions, And Duplications
L9 allele of L R‐gene locus in flax shares sequences with L1, L2, LH, and L10 alleles of this locus, suggesting it is a “patchwork” that originated by recombination of existing, older R‐genes. Each allele recognizes a different strain of Melampsora lini.
Variation in number of LRR repeat units between related resistance genes within species.
a. L6, L2, L1 alleles at L locus in flax (11 alleles total)
b. RPP5 gene from Arabidopsishas 13‐25 individual LRR units
c. Cf2/5 locus in tomato has 25‐38 LRR units, due to recombination.
Recombination at other complex R‐genes has been shown to create new specificities
L6: 2 copies of 6‐unit repeat, 26 LRR unitsL2: 4 copies of 6 unit repeat, 38 LRR unitsL1: 1 copy of 6 unit repeat, 14 LRR units
Long arrows are direct repeatsShort arrows are LRR units
The Guard Hypothesis for R‐protein Function
a. A cellular complex of proteins (blue), which includes both the 'guardee' molecule (red) and an NB‐LRR protein, is a target for an effector of disease (orange).
b. Binding of the effector to its targets results in disassociation and activation of the NB‐LRR protein and thus disease resistance.
c. Alternatively, the NB‐LRR protein may not be part of the target complex until after effector binding.
d. Recruitment to the effector/target complex would then activate the NB‐LRR protein.
J.L. Dangl & J.D.G. Jones 2001. Nature.
Avirulence protein(effector molecule involved in pathogenicity)
Support for the Guard Hypothesis is Accumulating (Arabidopsis and bacteria)
Indirect recognition of pathogen effector proteins by plant immune receptors in Arabidopsis (1) RIN4 binds to and suppresses the activity of the R receptor RPS2 in healthy plants. (2) The bacterial effector protein AvrRpt2 is secreted by P. syringae into host plant cells by a specialized secretion system. (3) Arabidopsis cyclophilins (for example, ROC1) interact with AvrRpt2 and activate this bacterial effector protein.The AvrRpt2 protease becomes localized to the plasma membrane after autoproteolytic cleavage of its secretion signal peptide. (4) Activated AvrRpt2 then cleaves RIN4 and at least three other Arabidopsis proteins. (5) RIN4 cleavage products can no longer suppress RPS2 activity.
Schulze‐Lefert and Biere 2005 Science
Support for the Guard Hypothesis is Accumulating (Tomato and Fungi)
Indirect recognition of pathogen effector proteins by plant immune receptors in tomato. (1) The fungal effector protein Avr2 of C. fulvum is secreted into the apoplastic space surrounding host tomato plant cells. Avr2 associates with the secreted plant protein Rcr3pim and inhibits its protease activity. (2) There is a binding‐dependent conformational change in Rcr3. (3) Altered Rcr3 then binds to the extracellular R receptor Cf‐2, which is activated by indirect recognition of Avr2. (4) Altered Rcr3 binds to Cf‐2 and activates it. (5) Alternatively, Rcr3pim may inhibit Cf‐2–triggered immune responses. (6) In this case, conformational changes in Rcr3pim enforced by Avr2 binding sequester Rcr3pim away from Cf‐2, thereby de‐repressing the R receptor.
Schulze‐Lefert and Biere 2005 Science
General Elicitors of Plant Defense
Pathogen‐Associated Molecular Patterns (PAMPs)
Fungi: chitin, ergosterol
Bacteria: lipopolysaccharides, flagellin
Activate “basal” or “innate” recognition and resistance pathways for plant and animal defense
Do not always activate hypersensitive response or apoptosis
ergosterol
Flagellin Is A Classic PAMPFlagellin is the basic protein subunit of the bacterial flagellum
TLR5 in mammals recognizes flagellin and activates human defensesHumans with mutated TLR5 are susceptible to Legionnaire’s disease
A conserved amino acid sequence of flagellin is recognized by FLS2 (encoded by an NBS‐LRR gene) in Arabidopsis
Recognition of the flg22 peptide restricts bacterial invasion at an early stage and contributes to plant resistance
Flagellin Activates Innate Resistance In Plants(Li et al. 2005 PNAS)
The Flg22 peptide induces transcription of NHO1 (nonhost resistance) in Arabidopsis, a gene that encodes resistance to many nonhost bacteria.
A Pseudomonas syringae pv tabaci strain engineered to lack flagella could infect Arabidopsis, the wild‐type strain could not infect Arabidopsis
Flagellin is required for NHO1 induction and resistance to P.s. tabaci (b) Disease symptoms of Arabidopsis plants (Col‐0) 7 days after inoculation with the wild‐type (WT) and fliCmutant strains of P.s. tabaci . (c) Bacterial growth of the wild‐type (WT) and fliCmutant P.s. tabaci strains on Arabidopsis plants (Col‐0).
But Pathogen Effector Molecules Can Suppress Innate Resistance(Li et al. 2005 PNAS)
P. syringae pv tomato strain DC3000 makes flagellin, and NHO1 is induced, but DC3000 injects a series of effector molecules into the plant using a Type III Secretion System (TTSS), and the effector molecules rapidly suppress NHO1induction.
DC3000 strains with defects in the TTSS lose the ability to suppress NHO1.
DC3000 strains produce many effector molecules, including HopS1, HopAI1, HopAF1, HopT1‐1, HopT1‐2, HopAA1‐1, HopF2, HopC, and AvrPto that block the NHO1 induction by flg22.
Illustrates that flagellin‐induced innate immunity is important for nonhost resistance and shows that virulence factors (effectors) can suppress the innate immunity response.
Yersinia pestis, the black plague bacterium
A common mechanism for injecting bacterial effectormolecules into host cells.
Part of the HRP (Hypersensitive Reaction and Pathogenicity (harpins)) complex identified by plant pathologists in 1980s.
Genes encoding TTSS are clustered on the bacterial chromosome (forming “pathogenicity islands”) and often are tightly linked with genes encoding the effector molecules.
TTSS required for pathogenicity and for avirulence function in many bacteria.http://webdeptos.uma.es/gen
etica/tipoiii/Research.html
Type III Secretion Systems
Effector Molecules Are Also Produced by Other PathogensEffector molecules are also called pathogenicity factors, and include:
Host selective toxins (HSTs) from fungi (eg ToxA, Victorin)
Small secreted peptide effectors from fungi (NIP1, Cladosporium fulvum (Cf) peptides, RXLR effectors from Phytophthora infestans)
Suppressors of post‐transcriptional gene silencing from viruses
Many of these effector molecules can be recognized by plant R‐genes and act as avirulence factors
The Fungal Haustorium:
Characteristic feature of biotrophic fungi that likely moves effectors into plant
cells
Hemileia vatatrix (coffee leaf rust)
Erisyphe graminis (barley powdery mildew)
The haustorium is intracellular
Does not penetrate the plant cell membrane
The hyphae connecting HMCs is intercellular
R
The recently discovered biotrophic interfacialcomplex (BIC) in Magnaporthe oryzae translocates
effectors into rice cells and may be a common structure in necrotrophic fungi
Understanding Effector Function Offers a Key to Understanding Pathogenicity
Effectors are pathogen molecules that manipulate host cell structure or host defence responses and thereby facilitate infection and/or trigger defense responses (effector-triggered immunity, ETI)
Unlike the terms “avirulence”, “elicitor”, “toxin”, and “virulence”, the term effector is neutral and does not imply a negative or positive impact on the outcome of the disease interaction
Plant effectors have been described in bacteria, oomycetes, fungi, and nematodes
Encoded by genes in pathogen genomes but function in (inside) plant cells
Fast evolving and a target of natural selection as a result of arms-race coevolution between hosts and parasites
Model for the Evolution of Gene‐for‐Gene Interactions in Plants (Bent and Mackey 2008, Ann Rev Phytopathology)
(a) Recognition of pathogen‐associated molecular patterns (such as bacterial flagellin) by extracellular LRRs triggers basal immunity, which includes signaling cascades that activate genes involved in basal (innate) immunity.
(b) Pathogenic bacteria use the type III secretion system to deliver multiple effector proteins that target host proteins and suppress basal immune responses. Fungi produce similar effector proteins with the same purpose.
(c) Plant resistance proteins (R‐gene products, such as a NB‐LRR proteins) recognize effector activity and restore resistance through strong effector‐triggered immune responses.
(d) Pathogen avoids R gene‐mediated defenses by modifying or eliminating the effector(s) that triggers those defenses. This state resembles that shown in (b) except the pathogen has had to alter or lose an effector protein, or deploy an additional effector protein.
Model for the Evolution of Gene‐for‐Gene Interactions in Plants (Bent and Mackey 2008, Ann Rev Phytopathology)
The evolution of effectors is influenced by how they are perceived by Resistance proteins (NBS‐LRR proteins). An effector contributes to virulence only if recognition by R proteins is avoided. Mutation to avoid recognition (ievia point mutations that change effector 3D structure) or deletion of the corresponding gene is a viable option for effectors that are directly recognized.
The fitness penalty that the loss of an effector causes, either in virulence or in overall pathogen fitness, should be an important factor in determining the durability of resistance that functions through recognition of that effector.
Selection operating on effectors and R‐proteins can be positive, negative, diversifying, purifying, or balancing
Coevolutionary scenarios involve Red Queen dynamics, trench warfare vs. arms race processes.
Model for the Evolution of Gene‐for‐Gene Interactions in Plants (Bent and Mackey 2008, Ann Rev Phytopathology)
Host-Specific Toxins (HSTs) Are Also Effectors
A substance produced by a pathogenic microbe that is toxic to the hosts of that pathogen and shows little or no toxicity against non-susceptible plants. HSTs are determinants of host range or specificity.
In general, all known HSTs are produced by fungi. Most HSTs are produced by two genera, Cochliobolus (also known by the anamorphnames Helminthosporium or Bipolaris) and Alternaria.
The Receptor‐Toxin Model of HST Interactions
Pathogen Host genotypegeno. S- ssT + -
(susc) (res)t - -
(res) (res)
PathogenTox allele
toxin present
Host susceptibleToxin affects target
Host resistantNo target for toxin
Susceptible allele
receptor present
Plant cell
Host resistantNo toxin produced
PathogenTox- allele
No toxin
resistance allele
no receptor present
Plant cell
Host resistantNo toxin produced
Susceptible allele
receptor present
Plant cell
S-
Plant receptor (target): product of Sensitivity allele (NB-LRR)
resistance allele
no receptor present
Plant cell
ss
Pathogen toxin: product of Tox allele (effector)
PathogenTox allele
toxin present
T
PathogenTox- allele
No toxin
t
Connections between Avr and HST GFG systems
R- rr
A- - +
aa + +
Host
Path
ogen
S- ss
T- + -
tt - -
Host
Path
ogenG
enet
ic b
asis
Avr gene-for-gene interaction HST gene-for-gene interaction
Lack of recognition = virulenceA resistant plant (R) interacts with an
Avr factor of a biotroph to induce a HR and host cell death that prevents
further pathogen propagation.“Effector-triggered immunity”
Recognition = virulenceA toxin sensitive plant (S) interacts with
the toxin of a necrotroph to induce a HR and host cell death that allows
further pathogen propagation.“Effector-triggered susceptibility”
Apoptosis: Programmed cell deathhttp://en.wikipedia.org/wiki/Apoptosis
• Differs from necrotic cell death that results from cellular damage
• Is needed for normal development of most metazoans• Probably important for preventing cancer and for removing
diseased (infected) cells• Has characteristic morphological, biochemical, and molecular
features, including:– TUNEL positive cells– Protease (caspase) activation– Cell shrinkage, chromatin condensation– Formation of DNA ladders
Terminal deoxynucleotidyl Transferase Biotin‐dUTP Nick End Labeling (TUNEL assay)
The Hypersensitive ResponseFirst observed in rusts on grasses ~100 years ago by Marshall WardPlant cell dies soon after penetration, prevents spread of pathogen to other cells
HR is initiated by recognition of an elicitor produced by the pathogen
Recognition of elicitor begins a cascade of biochemical reactionsOxidative burst (superoxide, hydrogen peroxide)Cell membrane changes permeability and cell wall strengthenedAntimicrobial substances produced, phytoalexins, PR proteins
Result is a hostile environment for the pathogen and plant cell death
Hypersensitive reaction in Arabidopsis thaliana infected with Phytophthora
Often only one cell affected, not noticed and does not lead to observable symptoms
Operates for biotrophic and hemi‐biotrophic fungi, and for bacteria and viruses
This is the resistance involved in Gene‐for‐Gene interactions, the type often selected by plant breeders
Hypersensitive Response = Apoptosis (?)
There are many similarities between the HR and apoptosis (programmed cell death or PCD)
For biotrophic pathogens, PCD leads to death of the pathogen
For necrotrophic pathogens, PCD opens up a food source that can be exploited
Thus there is a trade‐off for the host in using PCD to prevent pathogen spread among host tissues
A necrotrophic pathogen that produces an effector that activates PCD (called a host‐specific toxin) can use the host’s own defenses to promote further pathogen infection.
The literature for this coursehttp://www.path.ethz.ch/education/courses