This document summarizes a study on the molecular mechanisms of plant defense responses to the tomato powdery mildew fungus Oidium neolycopersici. The study investigated three monogenic genes (Ol-1, ol-2, and Ol-4) that confer resistance to the fungus via different mechanisms. It found that reactive oxygen species and callose accumulation were associated with resistances from both dominant and recessive Ol genes. cDNA-AFLP profiling identified different expression classes of genes, with Class III genes specifically upregulated only during incompatible interactions. The study provides insights into the molecular interactions and defense signaling pathways involved in the plant-pathogen system.

1. The molecular basis of plant resistance and
defense responses to pathogens: Current status
Sruthi.N

2. Introduction
 Many plant-associated microbes are pathogens that impair plant
growth and reproduction
 Pathogens may proliferate in intercellular spaces (the apoplast)
after entering through stomata or hydathodes (bacteria), enter
plant epidermal cells, or extend hyphae onto the plant cells
(fungi)
 Innate immune receptors in plants detect the presence of
microbial pathogens and trigger defense responses to terminate
or restrict pathogen growth

3. Elicitors of defence responses
 Any substance that has the capability of activating defense
responses in plants
 Include components of the cell surface as well as excreted
metabolites
Elicitors
General Race specific
a) Oligosaccharide elicitors a)avr gene products
b) Protein/peptide elicitors
(Ebel et al.,1998)

4. Perception of elicitor signals
 Binding proteins:
 Oligosaccharide-binding sites:
-Specific glucan-binding sites on soybean root plasma
membranes
-high-affinity binding sites for chitin fragments in tomato, rice
 Glycopeptide- and peptide-binding sites:
-Binding sites for peptidoglycans have been identified in wheat
plasma membranes

5. Plant defense to pathogens
• Plants respond to infection using a two-branched innate
immune system
-Recognition and response to molecules common to many
classes of microbes (basal disease resistance)
-Response to pathogen virulence factors
(Liu et al.,2008)

6. Basal defense
 Triggered by trans-membrane receptors that recognize
conserved molecules released by a variety of (unrelated)
microbes
 Include cell wall fragments, chitin or peptide motifs in bacterial
flagella - PAMPs or MAMPs
 PAMP- triggered immunity (PTI)
(Liu et al.,2008)

7. Secondary defense response
 Against virulence effector proteins produced by pathogens
 Effector –triggered immunity (ETI)
 Mediated by resistance (R) proteins
(Liu et al., 2008)

8. Phases of pathogen-triggered immunity
(Chisholm et al.,2006)

9. Concepts regarding pathogen recognition and defense
 Gene-for-gene resistance (Flor , 1947)
 Guard hypothesis (Beizen et al.,1998)

10. Gene-for-gene resistance
 For resistance to occur, complementary pairs of dominant genes,
one in the host and the other in the pathogen, are required
(incompatibility)
 A loss or alteration to either the plant resistance (R) gene or the
pathogen avirulence (Avr) gene leads to disease (compatibility)
(Hammond-Kosack et al., 1997)

11. Types of genetic interactions between plants and
pathogenic microbes
(Hammond-Kosack et al., 1997)

12. Plant disease resistance genes
 Encode proteins that recognize Avr-gene-dependent ligands
 Activate signaling cascade(s) that coordinate the initial plant
defense responses to impair pathogen ingress
 Capacity for rapid evolution of specificity
 Common feature of resistance proteins is a leucine-rich repeat
(Hammond-Kosack et al., 1997)

13. Classes of resistance proteins
(Chisholm et al.,2006)

14. Extracellular LRR class of R genes
 Have classic receptor-kinase formats - an extracelluar LRR, a
membrane spanning region and an intracellular protein kinase
domain
 Against pathogens that have an extracellular lifestyle
 Examples: rice Xa21 against Xanthomonas, cf genes of tomato
against Cladosporium fulvum.

15. NB-LRR R proteins
 Consists of four domains connected by linkers
 A leucine-rich repeat domain (LRR) fused to a nucleotide binding
(NB) domain
 NB-LRR core equipped with variable amino- and carboxy-terminal
domains
(Tameling et al., 2008)

16. NB-LRR subfamilies
 TIR NB-LRRs (Toll/interleukin-1 receptor like NB-LRRs)
 CC-NB-LRRs (Coiled Coil NB-LRRs)
 Solanaceous domain (SD)
 BED zinc finger DNA-binding domain
(Tameling et al.,2008)

17. Domains found in plant LRR R proteins
(Tameling et al.,2008)

18. Deviations from gene-for-gene concept
 One R gene may confer specificity to more than one ligand
- RPM1 in Arabidopsis confers resistance against P.syringae
expressing either avrRpm1 and avrB
 More than one R gene may exist for a given Avr gene
- Pto and Prf genes encode biochemically distinct components
of the same pathway
- Two genes at the Cf-2 locus furnish identical functions
(Bent, 1996)

19. Guard hypothesis
 Key points
d) An effector acting as a virulence factor has a target(s) in the host
f) By manipulating or altering this target(s) the effector contributes to
pathogen success in susceptible host genotypes
h) Effector perturbation of a host target generates a “pathogen induced
modified self” molecular pattern, which activates the corresponding
NB-LRR protein, leading to ETI
(Jones et al.,2006)

20. Guard hypothesis
(Jones et al.,2006)

21. Plant defense responses
 Hypersensitive response
 Production of reactive oxygen species
 Cell wall fortification
 Production of antimicrobial metabolites (phytoalexins)
 Defense signal transduction
 Synthesis of enzymes harmful to pathogen (eg. chitinases,
glucanases) (Nurnberger et al.,2006)

22. Types of activated defense responses
(A) Hypersensitive response in single lettuce mesophyll cells penetrated by haustoria of an incompatible isolate of
the biotrophic fungus Bremia lactucae B) H2O2 generation in lettuce cell walls, in the vicinity of the incompatible
bacterium P. syringae pv phaseolicola(C) Papillae formation. Papillae develop beneath the penetration peg (PP)
and germinating spore (S) of an avirulent isolate of the biotrophic fungusErysiphe graminis f sp hordei on barley
leaves expressing the Mlg gene.
(Hammond-Kosack et al., 1997)

23. Defense signaling pathways
SA-dependant signaling
 Effective against biotrophic pathogens
 Activated by the initiation of HR in plants
 Rise in SA levels dissociation of NPR1 oligomers to
monomers interaction with TGA-type transcription factors
activation of PR gene expression
 TGAs 2,5, and 6 and WRKY70 required for full expression of PR-1
(Glazebrook, 2005)

24. Defense signaling pathways
JA- and ET- dependent signaling
 Effective against necrotrophic pathogens
 Increase in JA levels and induction of effector genes (PDF1.2, VSP1)
 Induction of transcription factors ERF1, RAP2.6 and JIN1 which
activates many defense related genes
 Some JA regulated genes also regulated by ET (PDF1.2 , ERF1)
(Glazebrook, 2005)

25. Defense signaling pathways
JA- and ET- dependent signaling
 JA levels regulated by cellulose synthases
 JAR1 involved in conversion of JA to active form by conjugation with
amino acids like isoleucine
 In Arabidopsis, all activities of JA requires the function of CO11.
 Some responses to JA require the function of an MAP kinase
encoded by MPK4
(Glazebrook, 2005)

26. Defense signaling pathways
Cross-talk between SA and JA/ET signaling
 Helps the plant to minimize energy costs and create a flexible
signaling network that allows the plant to finely tune its defense
response to the invaders encountered
 Most reports indicate a mutually antagonistic interaction between
SA- and JA dependent signaling.
(Koornneef et al., 2008)

27. Molecular players in SA/JA crosstalk
 NPR1, required for transduction of SA signaling is a suppressor of
JA response
 Expression of GRX480 is SA inducible and dependent on NPR1
 Overexpression of GRX480 completely abolished MeJA-induced
PDF1.2 expression, but does not affect the induction of the JA-
responsive genes LOX2 and VSP2
(Koornneef et al., 2008)

28. Molecular players in SA/JA crosstalk
 Overexpression of WRKY70 caused enhanced expression of SA-
responsive PR genes and suppressed methyl jasmonate (MeJA)-
induced expression of PDF1.2
 MPK4 is a negative regulator of SA signaling and a positive
regulator of JA signaling in Arabidopsis
(Koornneef et al., 2008)

29. PR proteins
 Coded by host plants as a response to pathological or related
situations
 Accumulate not only locally in the place of infection, formed
systemically following infection by pathogens.
 Wide array of functions: hydrolases, transcription factors,
protease inhibitors etc.
(Scherer et
al.,2005)

30. Classes of PR proteins
(Scherer et al.,2005)

31. Non-Host Resistance
Two mechanisms
 In case of a potentially new host, pathogen’s effectors could be
ineffective, resulting in little or no supression of PTI, and failure
of pathogen growth
 One or more of the effector complement of the would-be
pathogen could be recognized by the NB-LRR proteins of plants
other than it’s coadapted host , resulting in ETI
 Arabidopsis resistance to non-adapted powdery mildew Blumeria
graminis f. sp. hordei
(Jones et al., 2006)

32. Arabidopsis Nonhost Resistance
to Bgh
(Ellis, 2006)

33. How R genes initiate defense signaling pathways
 R proteins recognize pathogen effectors in the cytoplasm
 Effector perception may result in altered intra and inter-molecular R
protein interactions, including oligomerization.
 Activated R proteins cycle into the nucleus and directly bind
transcriptional repressors of innate immunity, resulting in
transcriptional reprogramming of the plant cell
 Studies conducted for N, MLA,RPS4, and RX
(Liu et al., 2008)

34. Nuclear activity of MLA immune receptors
(Liu et al., 2008)

36. Objective of the study
 Study of the interaction of tomato plants with tomato powdery
mildew fungus, Oidium neolycopersici
 The monogenic genes Ol-1, ol-2 and Ol-4 confer resistance to
tomato powdery mildew Oidium neolycopersici via different
mechanisms
 Study of the molecular and biochemical mechanisms involved

37. Main interaction stages in compatible and incompatible
interactions

38. H2O2 and callose accumulation in compatible and
incompatible interactions

39. Different expression classes of the DE-TDF in cDNA AFLP
analysis

40. BLAST Results

41. RT-PCR

42. Conclusion of the study
 ROS, callose accumulation, and upregulation of DE-TDF
associated with resistances conferred by dominant and recessive
Ol genes
 cDNA – AFLP profiling clarified that 81% of upregulated DE-TDF
are common for both compatible and incompatible interactions
 Class III DE-TDF were up regulated only in incompatible
interactions and are specific for specific resistance genes
 DE-TDF profiles of NIL-OL-1 and NIL-OL-4 deviated much but
similarities were observed between NIL-OL-1 and F3-ol-2

43. Conclusion
An evolutionary arms race..

44. Discussion

45. THANK U…