Dr. Rakesh Punia Assistant Scientist CCS HAU Hisar

1. PRIMING FOR ENHANCED DEFENSE DURING
PLANT-PATHOGEN INTERACTION
Dr. Rakesh Punia
Assistant Scientist
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CONTENTS
Priming
Induced Resistance in the context of Priming
Priming techniques
Molecular mechanism of Defense priming
Chromatin modification
Application of priming in plant-pathogen interaction
Advantages and Limitations of Priming
Conclusions
Future prospects

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Priming is the phenomenon that enables cells to respond to
much lower levels of a stimulus in a more rapid and robust
manner than non-primed cells.
Cell priming involves accumulation of signaling
components that are not used until challenge exposure to
stress.
PRIMING

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6. Induced Systemic Resistance (ISR)
 Most of the Bio-priming agent
follow ISR.
Jasmonic acid (JA) and/or Ethylene
(ET) mediated.
 Lox gene activation is
characteristic.
(Perzalli et al., 2011)
Induced Resistance in context of Priming
Systemic Acquired Resistance (SAR)
 Chemical priming agent follow this
pathway.
Salicylic acid (SA) mediated.
 Pathogenesis Related protein 1
(PR1)expression is characteristic.
(Perzalli et al., 2011, Conrath et al., 2015)
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Priming is accompanied by Induced resistance as:

7. PRIMING TECHNIQUES
 Hydro priming: Achieved by soaking seeds in water.
 Osmo priming: By adding osmotica like mannitol, poly ethylene
glycerol (PEG).
 Solid matrix priming: Matrix carriers like calcinated clay, vermiculite.
 Chemo priming: By Benzothiadiazole (BTH), β-amino butyric acid
(BABA), Azealic acid, Pipecolic acid.
 Halo priming: Using salts of sulphate, chloride, nitrate.
 Bio-priming: By Trichoderma spp., Plant growth promoting
Rhizobacteria (PGPR), Plant extracts.
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8. MOLECULAR MECHANISMS OF DEFENSE PRIMING
Elevated
Levels of
Pattern-
Recognition
Receptors
Dormant
Mitogen-
Activated
Protein
Kinases
Chromatin
Modification
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(Conrath et al., 2015)

9. Mechanism of Defense Priming
NAIVE
PRIMED
PRIMED and
CHALLENGED
Priming for enhanced defense
Challenging stimulus
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(Conrath et al., 2015)

10. • Priming for enhanced defense comprises spotting of
diverse PRRs to the plasma membrane thereby enhancing
the plant’s responsiveness to different Pathogen
Associated Molecular Patterns (PAMPs) and Damage
Associated Molecular Patterns (DAMPs).
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Enhanced level of Pattern Recognition Receptors
(PRRs)
PAMPs and DAMPs
flg22
Peptidoglycon
EF-Tu
Chitin
Chitosan
Oligogalacturonic acid (OGs)
PRRs
FLS 2 and BAK1 ------
LYM ------
EFR ------
CERK 1 ------
CBPK ------
WAK ------
BACTERIAL
FUNGAL
DAMPs
Plant cell
surface

11. Tateda et al., 2014
FLS2 and BAK1 protein level after BTH treatment on the leaves of A. thaliana.
Arabidopsis
thaliana
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12. Arabidopsis
thaliana
(Tateda et al., 2014)
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13. A.Thaliana
A.thaliana
(Tateda et al., 2014)
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flg22- Pseudomonas syringae pv maculicola, Chitin-Perenospora parasitica
RLU-relative light unit
1µM flg22, 4h Measure ROS

14. Dormant Mitogen-Activated Protein
Kinases
Defense responses are regulated by a complex signaling network
that includes MAPKs cascades after plant recognition of
pathogens by PRRs .
MAPKs can control the synthesis and/or signaling of defense
hormones, reprogram gene expression through phosphorylation
of target proteins, including enzymes and transcription factors.
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15. PLANT MAPKs CASCADES
MAPKs(20)
Thr-Glu-Tyr (TEY)
activation motifs
(Group A,B,C)
Thr- Asp-Tyr (TDY)
activation
motifs(Group D)
MAPKKs/MEKs(10)
Sequence
homology
Group(A,B,C,D)
MAPKKKs (60)
MEKK-like kinases. Raf-like kinases
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Meng et al., 2013

16. PLANT MAPKs CASCADES
MAPKKKs
MEKK-like kinases
MEKK1, MAPKKK3
( MAPKKKs upstream of
MAPKKs in plant MAPK
cascades)
(
Raf-like members
MAPKKs/MEKs
Group A
MKK1, MKK2, MKK6
(MKK1 and MKK2 act upstream
of MPK4 in response to cold,
salinity, and pathogens )
Group B
MKK3
(Upstream of MPK6 in
regulating JA signalling).
Group C
MKK4 and MKK5
( upstream of MPK3 and
MPK6)
Group D
MKK7, MKK8, MKK9
(MKK7 or MKK9 activates
MPK3 and MPK6)
MAPKs
Group A
MAPK3 and MAPK6
(Ortholog of SAMK,SIMK, SIPK
and WIPK , involved in biotic and
abiotic stresses response,
growth- development )
Group B
MAPK4 and MAPK11
(Pathogen defense and abiotic
stress responses, cell division)
14Meng et al., 2013

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Meng et al., 2013

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19. BTH induces accumulation of MPK3 and MPK6 transcripts and proteins but does not
elicit dual TEY motif phosphorylation.
Beckers et al., 2009
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BTH treated
BTH untreated
BTH treated
BTH untreated

20. Attenuation of Priming for Potentiated
Defense Gene Activation in MPK-Deficient
Plants.
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Beckers et al., 2009
Dual TEY Phosphorylation Is Enhanced
in BTH-Primed Leaves after Dip
Inoculation with P. syringae pv.
maculicola.
Stress
(P. s pv. maculicola)
min

21. Attenuation of BTH-IR, SAR, and Infection-Induced Dual TEY Phosphorylation in mpk
Mutants.
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Beckers et al., 2009
P.syringae pv. maculicola, 2h
Mock
BTH treated

22. Rewiring Mitogen-Activated Protein Kinase Cascade by Positive
Feedback Confers Potato Blight Resistance
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Schematic representation of mechanism
of immune responses in transgenic
potato plants
Chihiro Yamamizo, Kuchimura, Kobayashi, Katou, Kazuhito
Plant Physiology, 2006, Vol. 140, pp. 681–692,
1 2 43 5 (h)
/StMEK1

23. Transgenic potato plants indicate elevation of MAPK activity and up-regulation of defense-
related genes during compatible P. infestans-potato interactions
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Chihiro et al., 2006

24. Transgenic potato plants harboring PVS3::StMEK1DD show resistance to P. infestans 24
Chihiro et al., 2006

25. CHROMATIN MODIFICATION
Chromatin consists of DNA and histone proteins and
pivotal to eukaryotic gene regulation .
DNA (methylation) and histones (methylation, acetylation)
both are modified during gene regulation .
Acetylation of histone lysine residues slacks the interaction
and loosens the ionic DNA-histone interaction, and
provides docking sites for trancription activators (gene
loading).
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26. Transcript abundance and histone modifications after priming and potentiated activation
WRKY29 transcription factor gene.
A. thaliana 100mM BTH/WP
72h
Primed plant Leaves collection
P.Syringae pv. maculicola
3h
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Chromatin modification acts as a memory for systemic
acquired resistance in the plant stress response
Michal Jaskiewicz, Conrath & Christoph
EMBO reports (2011) 12, 50–55

27. Application of Defense Priming in Plant-
Pathogen Interaction
Chemical agent
• BABA
• BTH (Boost R)
• Probenazole
• Acibenzolar–Smethyl
(Actigard)
• Fosetyl Al
• Metalaxyl (Ridomil)
• Strobilurin (Azoxystrobin)
• Pipecolic acid
• Azealic acid
Bio-priming agent
• Trichoderma asperellum
(Remedier R)
• P. fluorescens + T. harzianum
(PB-3)
• Bacillus subtilis (Taegro R)
• Trichoderma fertile
(TrichoPlusTM)
• Mycorrhiza (MycoGrow TM)
• Chitosan
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28. Oligogalacturonic Acid and Chitosan Reduce Stomatal Aperture by Inducing
the Evolution of Reactive Oxygen Species from Guard Cells of Tomato
Lee , Hyunjung Choi,and SuJeoung Suh
Effects of OGA on stomatal opening in tomato leaf epidermis
OGA 5µg/ml, Catalase 3mg/ml, EGTA 2mM
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29. Effect of chitosan on stomatal opening in tomato leaf epidermis
Control Chitosan Chitosan + Cat Chitosan + Asc
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Lee et al., 1999
Chitosan-induced production of H2O2 by guard cells of tomato leaf (Fluores. DCF-DA microscpy)
3 mg/mL catalase , 10 mM ascorbic acid, 1ooµg/ml Chitosan

30. M. Perazzolli , B.Roatti , E. Bozza a and I. Pertot
T39 @ 8g/L, BTH @ 0.5g/l, Cu (OH)2 @ 1.42g/l 30
Local effect
Systemic effect
Post inoculationPre inoculation
L=Local effect S =Systemic effect

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Perazzolli et al., 2011
Pre inoculation
Post inoculation
L=Local effect
S =Systemic effect

32. Reduction of Disease Progression in BTH-Treated Plants.
Germination rate, formation of appressoria, frequency of penetration,
and formation of mature primary and secondary haustoria were
determined for BTH-treated (0.3 mM) and control plants (set to
100%). Inoculation was performed 4 days after chemical treatment,
and the development of 300 conidia was monitored
Phenotypic Expression of BTH (@ 30g/ha) lnduced Resistance
against E. graminis tritici .
35% symptoms reduction and 18% increase in yield w.r.t.
control. Plants were photographed 2 months after treatment.
Control BTH treated
Jorn Gorlach, Sandra Volrath and John Ryals
The Plant Cell, Vol. 8, 629-643, April 1996
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BTH treated

33. BTH-Mediated Resistance Responses.
(A) Infection 10 DPI with E. g. tritici. BTH (0.5 mM) and control.
(B) Autofluorescence of a BTH-mediated HR of an attacked cell 48 hr PI
Infection sites in control (C) and BTH-treated (D) leaves 48hr PI
Infection sites in control (E) and BTH-treated (F) leaves 72hrPI.
Induction of WCI Genes by BTH Treatment.
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Gorlach et al., 1996

34. Plant growth-promoting rhizobacteria mediate induced systemic resistance in rice against
bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae
Chithrashree , Udayashankar and C. Srinivas
Figures in parentheses represent percentage protection offered.
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35. Chithrashree et al., 2011
Untreated Untreated + Xoo Treated Treated + Xoo
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Limitations
 Priming compounds should be
applied before pathogen infection.
 Priming compounds do not tend to
be highly specific, which can be an
advantage or a disadvantage
depending on the situation.
 A major limitation of chemical
priming compounds is their dose
application because improper dose
may cause phytotoxicity and can
exhaust the plant itself.
Advantages and Limitations of Defense Priming
Advantages
 An ecofriendly approach of
disease management.
 Reduces the application rate
and frequency of chemical
pesticides.
 Biopriming agents also helps in
Plant growth and development
so maintain the quality of
product without energy cost of
plant.

37. Priming for enhanced defense accompanies by SAR and ISR.
 Molecular mechanisms in the primed innate immune includes elevated
levels of PRRs and dormant cellular signaling enzymes (MPKs), transcription
coactivator function, and histones modifications in defense gene promoters
which provide stress memory.
We expect the emerging knowledge will increasingly translate defense
priming to practice, thereby improving sustainable agriculture .
We believe that priming compounds will have an impact on future
agricultural practices by providing the farmer with new options for disease
management in respect to both application rate and frequency.
CONCLUSION
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38. FUTURE PROSPECTS
Development of functional genomic tools for enhanced resistance -
interactions between defense signaling and other plant processes.
Designing the new tools and techniques that can be used for
identification of the priming activators.
Deciphering the impact of endophytes (beneficial microbes that
live inside a plant) in priming and induced immunity.
Development and identification of new priming activators for the
management of disease.
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