Branch of Biology which studies causal interaction between genes and their products, which bring the phenotype in to being.

2. Research supervisor:
Dr. l. vengadeshkumar,
INTERNAL mEMBER:
Dr. S. Sanjaygandhi,
EXTERNAL MEMBER:
Dr.T. sabesan,
Chair person :
Dr. d. john christopher,

3. Mechanism involved in
Epigenetics
3
Basic Concepts
2
Introduction
1
Epigenetic control of
plant immunity
4
Scientific Evidences
5
Conclusion
6
Outline

4. Introduction
 C.H. Waddington coined the term epigenetics – 1942
 To mean above or in addition to genetics to explain differentiation.
He defined as “ Branch of Biology which
studies causal interaction between genes and
their products, which bring the phenotype in
to being”.
Gupta et al. 2019

5. What does “epigenetics” mean?
Literally, epigenetics (epi - means
above, or on top), genetics.
Usually this means information coded
beyond the DNA sequence, such as in
covalent modifications to the DNA or
modifications to the chromatin
structure.
Practically, epigenetics describes
phenomena in which genetically
identical cells or organisms express
their genomes differently, causing
phenotypic differences.
Transcription
Epigenetic
Silencing
Genetically
identical cells or
individuals
Different epigenetic
modifications leading to
different expression
patterns
Gupta et al. 2019

6. Cloned cat: Genome is identical
Yet looks different from mother
Rainbow and Copycat
Normally asymmetric flower
becomes radially symmetric.
Wild-type toadflax flower and a Peloric
epimutant
What might be the reason ??
Gupta et al. 2019
EPIGENETICS

7. Genetics Epigenetics
mutations
alterations
Genetics vs. Epigenetics
Lakna et al. 2018

8. Chromatin
• Substance within a
chromosome consisting
of DNA and protein.
• Changes in chromatin
structure-associated
with DNA replication
and gene expression
Histone
• Protein - H2A,
H2B, H3 and H4
• A set of eight
proteins - Histone
Octamer.
Nucleosomes
• Section of DNA –
147 base pair.
• Fundamental sub
unit of chromatin.
• Wrapped around
a core of proteins.
Alain 2017

9. Nucleosomes : The site of epigenetic variation
Approximately 147 base pairs of
DNA wrapped around a histone
octamer
Alain 2017

10. 3. RNA
Interference
2. Histone
Modifications
1. DNA
Methylation
Gene
Expression
Mechanisms InvolvedIn Epigenetics
Barozai 2018

11. 1. DNA Methylation
Cytosine 5-methylcytosine
Methyltransferase
DNA can be covalently modified by cytosine
methylation.
TTCGCCGACTAA
Methyl-
cytosine
Enzymes Site of action
MET1- 5'-CG-3' sites
CMT3- 5'-CHG-3' sites
DRM 1, DRM 2- 5'-CHH-3' sites
Zhang 2006

12. Process of DNA Methylation
DNA methylation
De novo methylation
Methylation maintenance
• Transcriptional gene
silencing (TGS)
• Maintaining genome
integrity by silencing
transposable elements
(TEs).
• Mediated by RNA-directed
DNA methylation (RdDM)
pathway
• Stabilizes the methylated
sites - 24-nt siRNAs to
complement RNA
polymerase V transcripts
• CG - Methyltransferase1
(MET1)
• CHG - Chromomethylase3
(CMT3) and CMT2
• CHH - CMT2 or RdDM.

13. DNAMethylation Regulation in Plantsunder PathogenPressure
 Hypermethylation - RNA-directed DNA
methylation (RdDM)
 Hypomethylation - Repressor of Silencing 1
(ROS1)
 Both occur during pathogen attack can
regulate the expression of defense genes.
 Pathogen-induced DNA hypomethylation in
promoters of disease related genes, such as
NLR genes, can modify their expression
level and induce resistance responses.
 Hypo- and hypermethylation can both be
beneficial to plants under stress conditions.
Tirnaz and Batley 2019

14. Methylation can be propagatedduring DNA replication
MET1
5’ A T G C G T A C T
A T G C G T A C T
T A C G C A T G A
“MAINTENANCE”
METHYLATION
A T G C G T A C T
T A C G C A T G A
A T G C G T A C T
T A C G C A T G A
3’ T A C G C A T G A
A T G C G T A C T
T A C G C A T G A
BLUE = Gene
density
RED = Repetitive
element density
DNA methylation is densest at the
repetitive elements around the
centromere.
Kubota et al. 2013

15. Mass spectrometry
Methylated DNA
immune
precipitation
(MeDIP)
Methodto investigate
DNA methylation

16.  Addition of acetyl, methyl, and phosphate groups to the histone tails (N-terminal amino
acids).
 Alter the structure of chromatin, making genes accessible or inaccessible for transcription.
 Specific combinations of histone modifications (called histone code) control the
transcriptional status of a chromatin region.

17. Epigenetic modifications to the genome alter the spacing of
nucleosomes and the availability of genes for transcription.
Pearson et al. 2010

18. Histone modification and ATP dependent chromatinRemodelling
• High levelly expressed BRHIS1 complex binds to the histone variants -
obstructs monoubiquitinations.
• Suppress the expression of defense gene
Chen et al. 2016

19. Histone Modificationin Plant responseto Pathogen
Modification
category
Sub-
category
Name Gene locus
Mutant phenotype and biological
role
Reference
Histone
acetylation
Histone
deacetylase
(HDAC)
HDA19/At
HD
At4G38130 • Increases sensitivity to Alternaria
brassicicola and Pst DC3000;
• Down-regulates ET/JA pathway
genes (PDF1.2, VSP2, and ERF1),
• Enhances basal expression of SA-
responsive genes (PR1, PR4, and
PR5)
Zhou et al.
(2005), Ki
m et al.
(2008), Cho
i et al.
(2012)
HDA6/Axe
1
At5G63110 • Down-regulates expression of
ET/JA pathway genes (PDF1.2,
VSP2 ERF1)
Zhou et al.
(2005)
AtSRT2 At5G09230 • Increases resistance
to Pst DC3000.
Wang et al.
(2010)
HDT701 Os5G51830 • Increases resistance to rice blast in
RNAi plants
• Up-regulates mitogen-activated
protein kinases (MAPK6),
WRKY53
Ding et al.
(2012)
Histone
acetylase
HAC1 At1G79000 • Mutants deficient in priming the of
PTI
Singh et al.
(2014a)

20. Histone Modificationin Plant responseto Pathogen
Modification
category
Sub-category Name Gene locus
Mutant phenotype and biological
role
Reference
Histone
methylation
Histone
methytransferase
ATX1/SDG27 At2G31650 • Down-regulates expression of SA-
pathway genes (WRKY70 and
PR1)
• up-regulates expression of ET/JA
pathway genes (PDF1.2, VSP2)
Alvarez-
Venegas et
al. (2007)
SDG8/ASHH2/
EFS/LAZ2
At1G77300 • Increases sensitivity to Botrytis
cinerea; down-regulates expression
of ET/JA pathway genes.
• Increases sensitivity
to PST DC3000, down-regulates the
basal expression of R genes (LAZ5
and RPM1) and SA-inducible genes
(WRKY70 and PR1)
Berr et al.
(2010), Pal
ma et al.
(2010), De-
La-Pena et
al. (2012)
ASHR1 At2G17900 • Increases sensitivity
to Pst DC3000, down-regulates the
expression of SA-inducible genes
(WRKY70 and PR1)
De-La-
Pena et al.
(2012)
Histone
demethylase
FLD/RSI1 At3G10390 • Decreases resistance after systemic
acquired resistance (SAR)
induction, down-regulates
expression of SAR-inducible
WARY6 and WRKY29
Singh et al.
(2013, 201
4b)

21. Chromatin - remodelling factors in plant responses to pathogens
Modification
category
Sub-
category
Name
Gene
locus
Mutant phenotype and
biological role
Reference
Chromatin
remodeling
factors
SWI2-like
group
DDM1 At5G6675
0
• Increases resistance to Pst
DC3000 in mos1/snc1
background
• up-regulates the expression of
R gene SNC1
Li et al. (2010)
SWR1-like
group
PIE1/CHR
13
At3G1281
0
• Enhances resistance to
Pst DC3000
• up-regulates the expression of
SA-pathway genes
March-Diaz et
al. (2008)

22. Epigenetic Regulation of Disease Resistance in Rice
Hoang et al. 2018

23. 3. Non-coding RNAs or RNA Interference
 Not translated into proteins
 Regulate expression at the level of the gene.
 Involvement of Gene silencing
Non coding
RNAs
Regulatory
Non coding
RNAs
siRNAs
miRNAs
lncRNAs
Housekeeping
non-coding
RNAs
Wei et al. 2016

24. Mechanism of siRNA and miRNA
• miRNA - endogenous genes -
hairpin structures of 65–70 nt pri-
miRNA into pre-miRNA in the
nucleus.
• siRNA derived from the long
double stranded RNA molecule
was cut into a fragment of 21–25
nt by the Dicer enzyme.
Wei et al. 2016

25. Transcriptional Gene Silencing
• Discovered - Wassenegger et al.
(1994) in Tobacco -RNA-directed
DNA Methylation (RdDM)
Promoter region of a gene is
methylated
• Either dsRNA or siRNA directs
methylation of cytosine residues in
both the promoter region and ORF
Agrawal et al. 2003

26. Posttranscriptional gene silencing
Manjula et al. 2005

27. • RNA Directed DNA methylation(RdDM)
and RNAi - Homology Dependent Gene
Silencing (HDGS).
• RdDM act at transcriptional level -
Silencing the gene through DNA
methylation .
• RNAi involves cleavage of targeted mRNA
mediated by double stranded RNA.
• mRNA m6A modification- mRNA
methylation of adenosine.
• TFs - Transcription factors.
• TR - Transcriptional regulator
Yang 2020

28. Success stories of RNA interference
Using this RNAi technology deployed as a
GM traits against papaya ring spot viruses ‘
Rainbow Papaya’.
A similar technologies was
applied against squash mosaic
virus resistance and
commercialized successfully.
Silencing of hydrophobin gene in
Verticillium resulted in strong
resistance to V. dahliae in cotton.
HIGS targeted to a cellulose gene in a
Bremia lactucae resulted in high levels of
resistance to this pathogen in lettuce.

29. Barozai 2018

30. Epigenetic control of plant immunity
Protein / gene and
Function
Role in immunity Reference
SNI1 (At4g18470), putative
chromatin remodeller
exclusive from plants
Constitutive repression of the
SA pathway
Durrant et al. (2007); Mosher
et al. (2006)
SYD (At2g28290), Snf2-like
protein
Activation of JA/ET-sensitive
genes and resistance
against Botrytis cinerea
Walley et al. (2008)
DDM1 (At5g66750), Snf2-
like protein affecting DNA
Methylation
Maintenance of NBS-LRR
gene stability?
Stokes et al. (2002); Yi and
Richards (2007, 2009)

31. Effect of Plant chromatin componentson Immune response
Protein / gene and Function Role in immunity Reference
HDAC 19(At4g38130) Activation of resistance against Alternaria
brassicicola
Zhou et al. (2005)
HDA6(At5g63110) Activation of JA-sensitive genes
methyltransferase
Wu et al. (2008)
ATX1(At2g31650), putative
H3K4
Activation of WRKY70 and SA-sensitive
genes and basal resistance to Pseudomonas
syringae
Álvarez-Venegas et al.
(2006, 2007)
PIE1 (At3g12810), SEF
(At5g37055), and H2A.Z
(At1g52740 and At3g54560),
members of the Swr1-like
complex
Constitutive repression of the SA pathway March-Diaz et al.
(2008); March-Diaz
and Reyes (2009)
BRM (At2g46020), Snf2-like
protein
Constitutive repression of the SA pathway Bezhani et al. (2007)

32. Epigenetic Regulation in plant disease resistance
Crop Pathogen
Epigenetic
Regulation
Functions Reference
Arabidopsis Hyaloperonospora
arabidopsidis
RNA -directed
DNA
methylation
(RdDM)
• RdDM pathway provides the
machinery for different
transgenerational defense
responses
• Transgenerational SAR is marked
by increased acetylation of H3K9
at SA-inducible gene promoters
Luna and Ton
2012
Tomato Pseudomonas
syringae pv. tomato
DC3000
DNA
methylation
• Defective in Methyltransferase 1
(MET1) or methyltransferase
mutant gene - drm1, drm2 and
cmt3 (ddc) - increased resistance
– high level of gene expression.
Dowen et al.
2012

33. Hypomethylation of DNA during pathogen infection has been shown to influence
the defence-related gene expression. The rice R gene Xa21G, which was
demethylated chemically exhibited inherited resistance to Xanthomonas oryzae
pv. oryzae.
Crown gall tumour development in Arabidopsis demonstrated that ABA-dependent
drought stress defence regulates crown gall tumour formation is controlled by
DNA methylation.
Plants systematically utilize the siRNA-mediated methylation strategy as defence
mechanism towards various viruses, by methylating various viral genomic
components such as intergenic and transcribed region
Akimoto et al. 2007
Gohlke et al. 2013
Sharma et al. 2012

35. • Objectives: Blast Disease Resistance through expression of Pib resistance gene by
cytosine Methylation in rice.
• Hypothesis : That cytosine methylation at critical promoter regions of Pib instead
of inhibiting its induced expression upon the pathogen infection actually promotes
the reaction
• Loss of methylation by 5-azacytidine-treatment - down-regulation of Pib gene.

36. Confirmation for presence or absence of the Pib gene
Cytosine methylation map of the region I of the Pib gene
promoter before (A) and 24 h (B) and 96 h (C) after the
pathogen (Magnaporthe grisea) treatments in rice

37. Pib gene expression at different time

38. Resistanceor susceptibilityto Magnaporthe grisea
infectionby wild-typeand5-azaC-treatedplants

39. Summary
• Two cultivars containing the Pib gene, was not the result of a
general genotoxic effect of the 5-azaC treatments.
• But demethylation of the Pib gene by the chemical - reduction in
induced expression upon pathogen infection.

40. 2020
• Objective: To remove cytosine DNA methylation in advanced durum lines to test the
feasibility of generating a novel source of FHB resistance
• Eight advanced durum-breeding lines were treated with 5-methyl-azacytidine.
• The overall methylation levels (%) were compared using FASTmC method.
• The treated lines compared with the parental lines and FHB-susceptible checks

41. Seeds of eight advanced durum lines were treated with 5-azacytidine, allowed to
germinate, grow into plants, set seed and were propagated through to the M4 generation.
This was done to assure that the epigenetic modification is stable and heritable.
Visual score of FHB disease severity

42. Methylome level/percentageanalysis

43. Transcriptome analysis
The transcriptome analyses showed that distinct groups of genes were
activated at different stages (12 and 48 hpi) in the M4 line and the
susceptible parent in response to Fusarium infection.

44. Summary
• Generating a novel source of FHB resistance using DNA demethylation in
durum wheat seeds that may be useful for future breeding efforts.
• Treated with 5-azacytidine to remove methylation and allow the expression of
probable candidate genes.
• Methylome level/percentage analysis did not show a significant difference.
• Transcriptome analysis - significant differences between the parental and M4
line.
• The M4 line activated defense systems like PR proteins, transcription factors,
signaling, secondary metabolites, proteolysis, cell wall, oxidative stress and
hormone signaling.

45. Hypothesis: Methylation pathway mutated plant produce severe
symptom when infected by TRV viruses.
Objective: Investigate the influence of DNA methylation in the
control of viral proliferation and antiviral defense against tobacco
rattle virus (TRV) in Arabidopsis

46. Methylation-deficient Arabidopsismutantsare hypersusceptible to TRV
infection
Arabidopsis mutants
(related to methyl transferease)
Arabidopsis (wild type)
Less diseased
Severe
Diseased
(dcl2,dcl3)
TRV
Inoculation Inoculation

47. Summary
• TRV compromises the expression of DNA methylation genes in Arabidopsis.
• DNA (de) methylation is an important regulatory system controlling TRV
proliferation as the infection progresses.
• The expression of the disease resistance genes are largely influenced by the
methylation status of the plant.
• DNA methylation affects expression of SA-dependent PR1 gene in TRV-infected
plants.
• Tobacco rattle virus (TRV) causes changes in the expression of key transcriptional
gene silencing factors with RNA-directed DNA methylation activities that
coincide with changes in methylation at the whole genome level.

48. Objective: Studying the association of the sensitivity to phytopathogens with
differences in epigenomes of wheat plants from different harvest years.
Hypothesis: The role of DNA methylation as a factor of epigenetic regulation
leading to the formation of different gene expression patterns within the genetically
homogeneous plant material.

49. Dynamics of infection of rapidly and slowly germinating seedlings in the Podolyanka and Favoritka variety wheat
of different years of harvest
Podolyanka
Favoritka

50. Summary
 The association of individual disease resistance of plants with characteristics of DNA
methylation- individual epigenetic peculiarities.
 The plants possess both active, inducible and constitutive, passive or structural
mechanisms of the immunity associated with morphology and biochemical
composition of biological structures.
 The higher resistance to diseases is observed in fast germinating seedlings.
 Epigenetic distance in the Podolyanka variety is significantly higher than the plants of
the Favoritka variety.
 The role of DNA methylation as a factor of epigenetic regulation leading to the
formation of different gene expression patterns within the genetically homogeneous
plant material.

51. • DNA methylation in response to environmental conditions represents a potentially robust
mechanism to regulate gene expression networks.
• Profiling the DNA methylomes of plants exposed to bacterial pathogen, avirulent
bacteria, or salicylic acid (SA) hormone revealed numerous stress-induced differentially
methylated regions, many of which were intimately associated with differentially
expressed genes.

52. • Exposed mutant plants globally defective in maintenance of CG methylation (met1-3) or
non-CG methylation (ddc, drm1-2 drm2-2 cmt3-11).
• Both mutants – resistant
• Mutants partially impaired in CG (ddm1) or non-CG (rdr1, rdr2, rdr6, drd1, nrpd1a, and
dcl2/3/4) methylation displayed modest increases in Pst resistance

53. ProfilingDNA methylationduring different stressconditions revealsunique aspects of dynamic
differential methylation

54. Summary
• DNA methylation imparts persistent control over some defense genes during non
stressful conditions, in response to environmental stimuli, can change dynamically
to alter gene expression.
• The DMRs we identified were associated with protein coding genes, there were
also numerous transposons that were targeted by dynamic methylation in response
to SA.

55. • RNAi represents a robust and efficient tool that can be used in a highly targeted,
tissue specific manner to combat mycotoxigenic fungi infecting crop plants.
• Objective: Eliminating mycotoxin contamination, thereby improving food and
feed safety by using RNAi.

56. Interaction between a plant cell and fungal pathogen through plant RNAi- mediated host
induced gene silencing

57. Application of host induced silencing(through RNAi)
CROP PATHOGEN
TARGET
GENE(S)
FUNCTION REFERENCE
Zea mays Aspergillus
flavus
AflR Reduced AflR gene expression as
observed through semi-quantitative
RT_PCR; a 14-fold reduction in
aflatoxin contents (vs. control) in the
kernels derived from RNAi lines;
stunting and reduced kernel placement
phenotypes of transgenic plants
Masanga et al.
2015
Arachis
hypogaea
Aspergillus
flavus
aflS, aflR,
aflC, pes1,
aflep
4–20 fold expression of the hairpin
RNA expression in the RNAi lines;
100% reduction in aflatoxin B1 and B2
in the seeds
Arias et al.
2015
Musa sp. Fusarium
oxysporum f.
sp. cubense
(Foc)
Fusarium
transcription
factor 1
(0.9–8% of total RNAseq reads)
through DIG-labeled probes 7–25 fold
reduction in conidiophores count;
increased resistance (70–85%) to
Fusarium wilt
Ghag et al.
2014
Triticum
aestivum L.
Fusarium
graminearum
Chitin
synthase (Chs)
3b
1.4–4 fold reduction in Chs 3b
expression; 78–85% reduction in
deoxynivalenol (DON) contents
Cheng et al.
2015

58. Summary
RNA interference has shown promise as a technology for control of fungal
phytopathogens in food and feed crops as well as against a wide variety of other
plant pests that result in loss of crop value.
RNAi is used in conjunction with a precise genome editing tool to deliver an RNAi
cassette to a desired location in the genome, disease resistant plants without any T-
DNA backbone.
RNAi-based transgenic plants - reduction of disease incidence also reduce the
application of toxic synthetic pesticides
Positive impact on agro-economy, human health and ecosystem

60. Future Prospects
 Epigenetic mechanisms contribute to stress responses and memory in plants.
 Can be used to produce multiple diseases resistant cultivars
 Epigenetics can improve the resilience of food crops by priming them with disease
resistance.
 It may propose solutions to some of the greatest environmental challenges, such as
climate change and scarcity of clean water.
 Epigenetic modifications can be stably inherited for many generations provides new
opportunities to alter plant epigenomes using genome-editing approaches and identify
newly formed meiotically heritable epialleles that have major impacts on plant response
to pathogen infection