This document summarizes a seminar presentation on phytopathogen-induced changes to plant methylomes. It discusses various methods for profiling genome-wide DNA methylation in plants and provides examples of plant methylome changes induced by different pathogens, including bacteria, fungi, viruses, cyst nematodes, and rhizobium. It also describes a case study that analyzed differential DNA methylation and gene expression changes in Arabidopsis roots infected with the cyst nematode Heterodera schachtii using methylation profiling and RNA-sequencing. The study found H. schachtii preferentially induces hypomethylation and targets specific transposable elements and genes for differential methylation.

1. 03/23/18 Dept. of Plant Biotechnology 1
Phytopathogen induced changes
to the plant methylomes
Phytopathogen induced changes
to the plant methylomes
Seminar (0+1)
Roshni.M,Roshni.M,
II PhD Plant Biotechnology,II PhD Plant Biotechnology,
PALB6078PALB6078

2. Introduction
DNA methylation and demethylation
Methods for genome wide DNA methylation
Sequenced plant methylomes
Pathogen induced hypo and hypermethylation
Plant DNA methylation and viral infection
Plant methylome changes due to
• Bacteria, fungi, cyst nematode and rhizobium
Case study
Conclusion and future prospects
OutlineOutline
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IntroductionIntroduction
Methylome- DNA methylation in
genomic scale/ single cell level
Methylome- DNA methylation in
genomic scale/ single cell level
Genome-wide DNA methylation
Histone modification
Histone variant deposition
Nucleosomal positioning
(Banerjee and Roychoudhury, 2017)
Epigenome comprises of

4. DNA Methylation and Demethylation
(Law and Jacobsen, 2010)
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5. Gene body methylationTransposable elements and Repeats
Du et al., 2015
In Arabidopsis- four DNA demethylases of the DNA glycosylase family
have been identified: demeter(dme), demeter-like 2(dml2), dml3 and
repressor of silencing1(ros1)
(Zhu, 2009)
Gene body methylation-CG
•Functions in suppression of
aberrant transcription,
•Regulation of alternative
splicing,
•Fine-tuning the expression of
moderately expressed genes
Non-CG- CHH and CHG
•Heterochromatin regions
for silencing transposable
elements and repeats
Functional importanceFunctional importance
Regulski et al., 2013
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6. Methods for genome-wide profiling of DNA methylationMethods for genome-wide profiling of DNA methylation
Step2: Detection
Methylation sensitive
restriction enzymes
Affinity enrichment /
Immunoprecipitatio
n
Bisulphite
conversion
Step1: Differentiation of methylated and unmethylated DNA
• Enzyme based approach
• Early method of detection
• Lacks sensitivity and
• Low resolution
Microarray/ tiling array/ NGS platform
• Proteins-specifically
bind to 5-meC
• Monoclonal antibody
specific against
5-meC
• Chemical treatment
• Denatured DNA with
sodium bisulfite
• Deaminates unmeC residues
to uracil
• Followed by PCR
amplification
• Methylation data at single
base-pair resolution
MethylC-seq (Whole Genome Bisulphite
Sequencing)
Gene body methylation from TE methylation ?
A B C
Kim et al., 201403/23/18 Dept. of Plant Biotechnology 6

7. (Mattia Pelizzola, 2011)
Methylation levels in 23 eukaryotic organismsMethylation levels in 23 eukaryotic organisms
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8. (Kyung Do Kim , 2014)
Species Sequenced methylome
Reference genome Other genotypes
A. thaliana Columbia-0 142 accessions
B. distachyon Bd21 -
Glycine max - Heinong 44, LD00-
2817P, LDX01-1-
65
Oryza sativa ssp. japonica Nipponbare Dianjingyou1
O. sativa ssp. indica 93-11 IR64
Oryza rufipogon - 105327
Oryza nivara - 105426
Solanum lycopersicum - Ailsa Craig
Zea mays B73 Mo17
Sequenced methylomes at the single base-pair level
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9. Plants respond to biotic and abiotic stresses by
modulating their epigenomes
Epigenetic interplay between plant host and
pathogen
Hewezi et al., 2017
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10. Maize plants -Robertson’s Mutator
DNA methylation and transposonsDNA methylation and transposons
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11. • Each pathosystem has unique hypomethylome
signatures
• Bacterial pathogens largely impacts defense and stress-
related genes
• Plant-parasitic cyst nematodes impacts a variety of
hormonal and developmental process-related genes
(Hewezi et al., 2018)
Pathogen induced hyper- and hypomethylationPathogen induced hyper- and hypomethylation
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12. Plant DNA methylation and viral infectionPlant DNA methylation and viral infection
Virology provided the first glance of plant DNA methylation changes
upon infection
FIRST REPORT -DNA methylation inhibited accumulation of Tomato
golden mosaic virus (TGMV) in tobacco protoplasts
(Brough et al., 1992)
Potato spindle tuber viroid (PSTVd) sequences integrated into the
tobacco genome became fully methylated, and this was dépendent on
viroid replication
(Wassenegger et al., 1994)
RdDM as an antiviral mechanism was the demonstration that infection
with an RNA virus directs de novo DNA methylation
(Jones et al., 1998, 1999)
DNA methylation is also important for limiting infections by plant DNA virusesDNA methylation is also important for limiting infections by plant DNA viruses
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13. drb3 and dcl4 mutants were hyper-susceptible to these
viruses
(Raja et al., 2014)
Geminiviruses can inhibit DNA methylation-mediated
TGS by suppressing the expression of met1 and cmt3
both in local and systemic fashion
(Rodrıguez et al., 2013)
Host methylome changes during viral infection have not
yet been characterized on a genome-wide scale
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14. Plant methylome changes induced by bacteriaPlant methylome changes induced by bacteria
Arabidopsis plants exposed to the strains of Pseudomonas
syringae-induce active hypomethylation, specifically in the
peri/centromeric regions, as early as 1-day post-infection
Arabidopsis plants exposed to the strains of Pseudomonas
syringae-induce active hypomethylation, specifically in the
peri/centromeric regions, as early as 1-day post-infection
Pavet et al., 2006
First report
But, limited by low resolution and non-quantitative analysis
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Mutants-more resistant to the bacterial
pathogen Pseudomonas syringae pv.
Tomato
Many pathogen-responsive genes-
constitutively expressed
Pathogen-induced overproduction of 21-
nt Small RNAs from TEs–trans regulation
of defense genes
Upon avirulent pathogen infection–
genome wide hypermethylation
Absence of chlorotic lesions compared
with control
Mutations in the CG –(met1) and the non-CG methyltransferases (drm1, dmr2, and
cmt3-ddc) -genome- wide hypomethylation and pleiotropic developmental defects
Dowen et al., 2012

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Activation of the PRR FLAGELLIN SENSING 2 (FLS2) induces
hypomethylation at the retrotransposon AtSN1
Ros1- transcriptional reactivation of TEs during
antibacterial defense responses
(Yu et al., 2013)
Eg. rmg1- disease resistance gene contains two helitron-
related repeats in its promoter region (AtREP4 and AtREP11)
Biological relevance of DNA methylation in antibacterial defenseBiological relevance of DNA methylation in antibacterial defense
Global loss of DNA methylation may activate plant
immune system
Global loss of DNA methylation may activate plant
immune system

16. Plant methylome changes induced by fungiPlant methylome changes induced by fungi
Hemibiotrophic fungal pathogen
The triple hypermethylation
mutant, rdd (ros1 dml2 dml3)-
enhanced susceptibility to the
hemibiotrophic pathogen
Fusarium oxysporum
Enhanced susceptibility response
was due to the downregulation
of a significant number of stress-
related genes that have TEs in
their promoters
DNA demethylases target promoter transposable elements to positively regulate
stress responsive genes in Arabidopsis
03/23/18 Dept. of Plant Biotechnology 16Le et al., 2014
Hypothetical model

17. hypo-methylated mutant-
hyper-methylated mutant-
nrpe1
ros1
Biotrophic pathogen Necrotrophic pathogen
Resistant Susceptible
Susceptible Resistant
nrpe1
ros1
Cell wall defense and SA-dependent gene
expression
Repressed sensitivity of jasmonic acid (JA)-inducible gene
expression
(Lopez et al., 2016
Infection by biotrophic or necrotrophic fungiInfection by biotrophic or necrotrophic fungi
Biotrophic pathogen Hyaloperonospora arabidopsidis (Hpa), Necrotrophic pathogen-
Plectosphaerella cucumerina, A. brassicicola
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18. Majority of nrpe1 and ros1
dependent defense genes are
regulated in trans by DNA
methylation.
Lopez et al., 2016
Global transcriptome analysis of nrpe1 and ros1- after Hpa infection- 49% of the
pathogenesis-related transcriptome is influenced by NRPE1 and ROS1-controlled
DNA methylation.
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Effect of DNA methylation on the immune response is largely
mediated through a trans-acting mechanism, and only limited
numbers of stress- and defense-related genes are directly regulated
by localized differential DNA methylation via cis-regulatory
mechanisms
Revealed the contrasting impacts of hyper- and hypomethylation on
immune responses.

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• Cyst and root-knot nematodes -plant
biotrophic pathogens
• Differentially terminated cells in the vascular
root tissues redifferentiate into a syncytium
cell type- involves changes in the
expression of thousands of genes
simultaneously
• Widespread hypomethylation of protein-
coding genes and transposable elements
(TEs) (adjacent to protein-coding genes)
• Extensive methylome studies in Soybean
roots infected with soybean cyst nematode
and beet cyst nematode in Arabidopsis roots
Plant methylome changes induced by plant parasitic cyst nematodesPlant methylome changes induced by plant parasitic cyst nematodes
(Hewezi and Baum , 2015)

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The Differentially Methylated Regions (DMRs) were found
to overlap for a significant number of Differentially
Expressed Genes (DEGs)
Eg: Genes encoding PR proteins- lipoxgensae 1 and
glutaredoxin480 – negative regulators of defensin gene
PDF1.2
Hewezi et al., 2017
Rhizobium-induced reprogramming of plant DNA methylationRhizobium-induced reprogramming of plant DNA methylation
• Reprogramming of DNA methylation was found to be vital for
Rhizobium-induced root nodules in Medicago truncatula
• Showed spatiotemporal expression patterns in the developing
nodules
• Remarkable upregulation of Mtdme in the differentiation zone of
fully developed nodules
• Upregulation of genes, particularly those encoding nodule specific
cysteine-rich (NCR) proteins
Mtdme knockdown-induced hypermethylation was associated with
suppressed expression of a significant number of genes functioning in
nodule differentiation
(Satge et al., 2016)

21. • Epigenetic recombinant inbred lines
(epiRILs) of A thaliana
• Within-species variation in functional
traits can be created
• Methylation variation of genetically
identical individuals increased
productivity (biomass by 40%) stability
(pathogen and competitor resistance)
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22. Case study
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23. Objective :
To determine the differential DNA methylation associated with gene
expression changes in the syncytium during compatibility of the
interaction between Arabidopsis and H. schachtii
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24. Nematode Inoculation, Tissue Collection, and MethylCseq Library construction
Identification of DMRs and Mapping to Annotated Genomic Features
RNA-seq Library Construction and Sequencing
Small RNA-seq Library Construction and Sequencing
Nematode Infection Assay
Work flowWork flow
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25. Materials and methods
Nematode Inoculation, Tissue Collection, and MethylCseq Library Construction
Arabidopsis thaliana seeds (ecotype Columbia-0) - surface sterilized and planted on modified
Knop’smedium (at 24°C under a photoperiod of 16 h of light and 8 h of dark)
Ten-day-old seedlings - inoculated with about 200 surface-sterilized second-stage juveniles of
H. schachtii per seedling.
5 and 10 d post inoculation, root tissues were collected from both infected and non infected
control plants
Four treatments- three biological samples- 12 samples- MethylCseq Library
Paired-end sequencing of 100-basepair reads -Illumina HiSEquation 2500 system.
Identification of DMRs and Mapping to Annotated Genomic Features
High-quality MethylC-seq reads were mapped to the Arabidopsis reference
genome(TAIR10) (Bismark)-identify differentially methylated cytosines-methylKit
package
Cytosines were called – if covered by a minimum of 10 reads.
Three methylation call files - sequence contexts were generated- for each
treatment
Hyper- and hypo-DMRs in CG,CHG, and CHHcontexts identified using a 200-
basepair non-overlapping window (min. methylation difference of 25% using a
FDR cut-off of 0.01)
DMRs allocated various annotated features of the Arabidopsis genome-
Bioconductor packages GenomicRanges03/23/18 Dept. of Plant Biotechnology
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26. • These results indicate that in all sequence contexts H.
schachtii induces hypomethylation to a much higher degree
than hypermethylation
• Dynamic changes in both methylation patterns and activity
during nematode parasitism
Classification of differential DNA methylation induced by H. schachtii in Arabidopsis roots
Each methylation context is preferentially linked to
specific genic regions
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27. H. schachtii targets various TE families for differential methylation
 Indicate that H. schachtii preferentially
targets specific TE families for differential
DNA methylation with influence observed
for methylation sequence context
 CHH hypermethylation occurred
preferentially in the TEs that were located
within 1 kb upstream or downstream from
the nearest genes
 All hypermethylated TEs in CG or CHG
contexts, respectively, were located >8 kb
away from the nearest genes
H. schachtii preferentially induces differential CHH methylation in class I and II TEs located
nearby genes
Indicate differential CHH methylation of class I and II transposons
was abundant in the TEs that were relatively close to genes,
whereas differential methylation in the CG and CHG contexts was
abundant in the TEs that were relatively distant from genes
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28. RNA-seq Library Construction and Sequencing
mRNA was isolated – magnetic mRNA Isolation kit
RNA-seq libraries were generated from 250 ng mRNA using the NEBnext mRNA library prep
master mix
Single-end sequencing of 100-basepairp reads was performed using Illumina HiSEquation 2500
platform
Aligned to the Arabidopsis reference genome (TAIR10) using the splice-aware software
package
DEGs between infected and non-infected samples were determined using the edgeR package
DEGs were assigned to GO terms using the AgriGO databasea
H. schachtii-induced differential DNA methylation impacts transcript abundance
• Significant enrichment for DMGs among the DEGs DNA
methylation of the DEGs
• Few genes maintained their methylation patterns during
disease progression
• Majority of these 262 genes were hypomethylated-
exhibited significantly higher expression than the
hypermethylated genes
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29. Differential methylation of TEs impacts the expression of nearby genes
Hypomethylation in TEs is associated with low expression of adjacent genesHypomethylation in TEs is associated with low expression of adjacent genes
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30. Nematode Infection Assay
Seeds of Arabidopsis wild-type (Col-0) and T-DNA insertional mutants of 10
DMGs planted in 12-well tissue culture plates
(Knop’s medium using randomized complete block design with 20 replicates)
10d inoculated with about 250 surface-sterilized second-stage juvenile
nematodes of H. schachtii per plant.
Three weeks post-inoculation, the number of fourth-stage juveniles/females
per root system was scored and used to determine susceptibility levels in each
mutant
Overlaps between syncytium DEGs and DMGs
Differential methylation-mediated gene expression
changes in the syncytium may contribute to the
compatibility of the interaction between Arabidopsis and
H. schachtii
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31. Small RNA-seq Library Construction and Sequencing
Total RNA was isolated using TRIzol
Then, 1 mg of total RNA was used to prepare small RNA libraries -Illumina TruSeq
Small RNA library preparation protocol
Small RNA-seq libraries were pooled and compared to reference genome
Known tRNAs, miRNAs, ncRNAs, rRNAs, snoRNAs, and snRNAs -removed
Remaining siRNAs were further processed to assess their positions relative to
DMRs overlapping with protein-coding genes and TEs.
Association between siRNAs and DNA methylation
These data suggest that high abundance of the 24-nt siRNA class-
associated with hypermethylation of TEs, gene promoters, and to a
lesser extent gene body regions
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32. ConclusionConclusion
• Integrate genomics and epigenomics together with the generation of high
yielding trait
• Future generation of agronomically improved trans-‘epigenic’ lines
• Interfaces for comparative epigenomics
Spaceflight experiments
Epigenetic change in Arabidopsis thaliana in response to spaceflight - differential cytosine
DNA methylation of plants
• Comprehensive analyses of methylome patterns between
resistant and susceptible near-isogenic lines
• Identifying pathogen effector proteins
• Spatial and temporal localization of the activity of different
components upon pathogen infection
• Novel experimental approaches that allow clear distinctions
between localized and systemic changes
Future prospects
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