4. Terminologies
• Elicitor: The signaling molecules.
• Effector: Typical proteins that are delivered outside the microbe.
• Pathotype: population of a parasite species in which all
individuals have a pathogenicity or parasitic ability in common.
• Biotype: progeny developed by variant having similar heredity.
• PRR-Transmembrane Protein Recognition Receptors.
• PAMP/DAMP:Pathogen Associated Molecular Patterns.
• PTI: PAMP-Triggered Immunity
• ETI-Effector Triggered Immunity.
• NBS-LRR-Nucleotide Binding Luicine rich Repeat regions.
• MAPK:Mitogen Activated Protein Kinase
4
6. Biffin(1901)
Demonstrated Genetic basis of
Disease resistance.
Studies conducted on Wheat rust.
Obtained 3:1 Mendelian ratio by crossing
Rivet x Red king.
“Resistance is Heritable”
Resistance and susceptibility are independent
of other plant characters.
6
7. H H Flor(1952)
Genetic factors of both plant and pathogen
are required for the successful defence
response of plant.
Wheeler rule-1: Incompatible Reaction
Found in Biotrophs.
Avr-R recognises each other and their
speificity and interaction gives resistence to
host.
Wheeler rule-2: Compatible reaction
Avr-r produce specific compounds ,which
interact
each other and produce the
susceptible response.
7
8. Rules of Flor’s Hypothesis(1952)
Virulence gene
Resistance Gene
Incompatibility
Avr1
Compatibility
avr1
Avr1
avr1
R
R
R
s
R1
R
S
r1
S
S
Given by Wheeler(1975)
8
10. Results From Flor’s Crosses
• 25 resistance alleles distributed across 5 loci were identified
• Locus # Alleles
K1
L11
M6
N3
P4
• For every resistance allele found in the plant, a corresponding
virulence 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. 10
12. Each
race specific r genes has only a
limited life span
BOOM YEARS
Priestley(1970)
BUST YEARS
13
13. Types of Genetic Resistance
•
Qualitative Resistance
• Distinct classes of resistance
and susceptible plants
• Controlled by one or a few
genes
• Also called “Vertical”
resistance
•
Quantitative Resistance
• Continuous variation among
genotypes
• Many loci
• Also called “Horizontal”
resistance
14
14. Genetics of Resistance
Mechanisms of resistance:
1.Disease escape
2.Disease endurance.
3.True resistance
Classification Based on:
1.Number of Genes
2.Biotype reaction
3.Population/Line concept
4.Evolutionary concept.
5.Specificity.
15
17. Perception
How pathogen and host recognize each other.?
The gene-for-gene hypothesis between host and pathogen for
triggering race-specific resistance.
Elicitor – receptor model
I- incompatible ,C- compatible
18
18. Perception of elicitor signals/ receptors
for elicitor signals in plant cell
membrane
• Salicylic acid, Jasmonic acid
and Ethylene
• Protein kinase as receptor sites
• LRR-type receptors
• Lectins as receptors
• Resistance gene product as
receptors
19
19. Secondary messengers(signaling)
•
Molecules that relay signals from receptors on the cell surface to
target molecules.
Earl Wilbur Sutherland, discovered secondary messengers, won the
1971 Nobel Prize in Physiology or Medicine.
Functions:
• They greatly amplify the strength of the signal.
• Component of signal transduction cascades.
• Secondary messengers.
• Calcium ion
• Anion channels in signal transduction
• Phosphorylation and Phospholipids signaling system
• Mitogen-activated protein kinase signaling cascade
20
20. Role of Mitogen-activated protein kinase cascade
ACS- 1-aminocyclopropane-1-carboxylic acid synthase,
(Hamel
et al., 2012)
22
23. Mechanisms of plant defense
• Hypersensitive response
• Production of reactive oxygen species
• Production of antimicrobial metabolites
• Defense signal transduction
• Synthesis of enzymes (e.g. - chitinases, glucanases)
(Nurnberger et al.,2006)
25
25. Signaling systems
• Alkalization and cytoplasmic acidification
• Reactive oxygen species
• Nitric oxide in signal transduction
• Salicylic acid signaling system
• Jasmonate signaling pathway
• Ethylene dependent signaling pathway
• Fatty acids as systemic signal molecules
27
27. Domain
Function
Gene
LRR
Protein-protein interaction.
Its major determinants of recognition specificity
Pi-ta, Cf 4,9,5
NBS
To bind ATP r GTP.
Race specificity functions of R gene
L6
TIR
Race specificity functions of R gene
L6
CC
Involved in recognition of avr gene product
RPW 8
Schematic representation of domains found in plant LRR R proteins. Domains are not drawn to scale. TIR Toll/interleukin-1
receptor, CC coiled coil, NB nucleotide binding, ARC1/2 APAF1, R protein and CED4, LRR leucine rich repeat, SD solanaceous
domain, BED BEAF/DREAF zinc finger domain, TM transmembrane, Kin kinase, WRKY WRKY transcription factor
29
(Wladimir et al., 2008)
28. Sources of Resistance
• Primary Gene Pool
•
Other breeding programs
•
Landraces
•
Germplasm collections (GRIN)
• Wild Relatives
•
Tomato: Lycopersicon genus
•
Wheat: Agropyron genus
•
Aegilops tauschii, Triticum monococum
• Mutant Transformations: powdery mildew in Barley
30
29. Identification of R genes
Candidate gene identification by readily designed
PCR primers.
R gene molecular isolation
2 methods:
1.Map based identification.
2.Transposon tagging
33
30. Map based isolation of R genes
Genetic
marker 1
Genetic
marker 2
R gene
M3
M4
34
31. Map based isolation of R genes
5. Transform a susceptible genotype
with a single cosmid clone
Disease reaction
R
R
S
S
Types of problems encountered
- A ‘LONGER WALK’ than expected - lack of recombination
- An unknown ‘HOLE IN THE BAC CLONE’ RPM1
35
- Which gene is it ? PTO
33. Map Based Cloning of Pi9
Include recent R gene cloning paper in r
37
34. Cloned rice resistant genes
Include bullet points of Whole presentation
Then conclude
38
35. Transposon Tagging of R Genes
with genetic selection
Tomato Cf-9 : Avr9
Tobacco N gene
30 C
22 C
TMV
nn
NN
Transposon
inactivation
of R gene
39
36. R gene expression
Very low and constitutive
Occasionally low level induction following infection
but only in the vicinity of the pathogen
Rarely expressed only in resistant genotype
- Rice Xa27 – Xanthomonas oryzae pv. oryzae
Susceptible alleles 10 bp and a 25 bp insertion in the
promoter.
(Gu et al, (2005) Science 435: 1122-1125)
40
37. How do R proteins function ?
Three examples
Direct pathogen molecule recognition
Indirect pathogen molecule recognition
41
38. Direct recognition Tomato Pto - AvrPto
Pseudomonas syringae
Susceptible
host
AvrPto
or AvrPtoB
Resistant
Pto host
cell wall
plasma
membrane
pto
Pto
AvrPto and AvrPtoB
effectors bind to
unknown host target
Prf
Enhanced
pathogen
virulence
Adapted from Jones and Dangl (2006)
Nature
HR
Less pathogen
proliferation
42
39. Indirect recognition Arabidopsis RPM1- AvrRpm1
Pseudomonas syringae
AvrRpm1
or AvrB
Susceptible
host
No RPM1
NDR1
RIN4 P
P P
AvrRpm1
effector bind to
host target RIN4
and other targets
Enhanced pathogen
virulence
Adapted for Jones and Dangl (2006) Nature
NDR1
Resistant
RPM1 host
RIN4 P
P P
RPM1
HR
Less pathogen
proliferation
43
40. R Protiens Guard The Virulence Target
Avr
Susceptible response
to favour pathogen
growth and development
Virulence
Target
B. INCOMPATIBLE INTERACTION
Resistance by guarding
Avr
Virulence
Target
R3
AvrB/
AvrRpm1
RIN4
RPM1
Avr2
Rcr3
Cf-2
Resistance
response
44
41. Approaches for Host Plant
Resistant Breeding
Breeding for Specific Resistance
Individual Major Genes.
Breeding for Quantitative traits.
Multilines.
Marker assisted back crossing.
Pyramiding.
45
FIG. 1. L6 gene DNA probes detect RFLP markers linked to the L and M loci. (A) Map of the L6 region and location of DNA probes. Thelocation of the transcribed region and the two 480-bp direct repeats are indicated with arrows. H, HindIII; Bg, Bgl II; RI, EcoRI; B, BamHI; S,Sph I; X, Xba I. (B) RFLP pattern detected in the parents, Forge (F) and Hoshangabad (H), and some of the 52 test-cross progeny using XbaI-digested DNA and probe LU-2. The presence of the L6 and M resistance genes was determined by inoculation with the appropriate rust strains.The absence of these genes is indicated by a blank square. The positions of the DNA fragments that provide the four RFLP alleles LU2-2A/B (Mlinked) and LU-2-1A/B (L linked) are indicated. In each case, the A allele is derived from Forge and the B allele is from Hoshangabad.by probe
After evaluation of numerous physiological, biochemical and genetic experiments,
Genetically defined relationships be-tween salicylic acid (SA)-dependent andethylene–jasmonic acid (ET–JA)-depend-ent defense responses in Arabidopsis.The SA-dependent response is deployedagainst a biotrophic pathogen that ob-tains nutrients from living cells, whereasthe ET–JA response is activated bynecrotrophic pathogens that kill plant tis-sue. Resistance to necrotrophs (but notthe biotroph) is compromised in anArabidopsis mutant (coi1) that does notrespond to JA. Expression of a transgene(NahG) that degrades SA compromisesresistance to the biotroph but not thenecrotrophs. These pathways appear tobe mutually inhibitory.
RLK-receptor like protien kinase.pdf1.2 gene-a pathogen related gene act as a defensin in arabidopsis.wound related kinase 1.PRR_pattern recognition receptorsTtss-type 3 secretion system
Superoxidedismutase,catalase,ascorbicparoxidase.
Fig. 2 Physical map of chromosome 7D in wheat showing markersthat are flanking Lr34/Yr18 and contained within the deletion bin7DS-4 which consists of approximately 39% of the short arm ofchromosome 7D (on the left). The genetic map of the Lr34/Yr18region shows microsatellite markers that flank the resistance genes.Numbers on the left hand side denote genetic distance incentiMorgans. Resistance to powdery mildew (Pm) cosegregatedwith leaf rust and stripe rust resistance. A–D denote confidenceintervals for QTLs for rust resistance previously assigned to thisregion: A QTL for leaf rust and stripe rust identified by Suenagaet al. (2003), B QTL for leaf rust QLr.str-7DS described bySchnurbusch et al. (2004), C QTL for leaf rust QLrP.sfr-7DSidentified by Schnurbusch et al. (2004), D QTL for stripe rustQYr.sgi-7D identified by Ramburan et al. (2004)
Identification in pib gene isolation.
Aa sequence is-nbslrr.Duplicated kinase 1a 2a 3a motifs,8cysten residues are clustered in the middle of lrr.
Contigs –gel readings. Pi9 was introgressed from wild rice Oryzaminuta to cultivated rice line 75-1-127. Tightly linked markers (pBV14, pB8 and pBA14) wereidentified using RAPD and bulk segregant analysis. pB8 was used to screen both BAC and cDNA libraries. A contig was constructed usingpositive BAC clones. Candidate genes, identified from the sequenced contig, were used for transformation. Transgenic lines were evaluatedfor resistance to rice blast.
Cladosporumfulvum-blue mold
Pto-psuedomons syringe pv.tomato
Prr-transmembrain pattern recognition receptorsRlps –arabidopsis receptor like proteinsRIN4-A 211 AMINO ACID ACYLATEd,and plasma membrane associate protien,is type 3 effector gaurded by nbs-lrr.AvrRpm1 or avrb are the 2 unrelated protiens
Fig. 3. Strategy for engineering broad-spectrum resistance by induction of Avr/Rtransgene combinations. A pathogen Avr gene is expressed in plant cells as atransgene, under the control of a plant promoter that is induced by a range ofpathogens. A corresponding R gene (either endogenous or a transgene) is alsoexpressed. Upon pathogen attack, the pathogen-responsive promoter is activated,the Avr gene is expressed, and the Avr protein interacts with the R protein toinduce the HR and other defense responses. Note that this system can be activatedby any pathogen (or spurious stimulus) that is capable of activating the promoterof the Avr gene.
Fig. 4. Regulation of defense gene expression by NPR1. NPR1 has an importantrole in ‘local’ resistance triggered by some R genes, as well as two types of sys-temic resistance: Systemic acquired resistance (SAR), which is induced by nectro-tizing pathogens on foliar tissue and which acts through salicylic acid (SA); andinduced systemic resistance (ISR), which is induced by soil-dwelling bacteria andacts through jasmonic acid (JA) and ethylene (ET) [5]. These cascades trigger anas-yet unknown posttranslational modification of NPR1 (depicted by a red star).NPR1 then translocates from the cytoplasm to the nucleus [53]. Once inside thenucleus, NPR1 interacts with TGA transcription factors, and perhaps other pro-teins, to regulate transcription of defense-associated genes [54,55].