Understanding the interaction between RNA-directed DNA methylation and DNA demethylases and its role in Fusarium oxysporum disease response in Arabidopsis thaliana
Date
2016
Authors
Lee, Joanne
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Abstract
RNA-directed DNA methylation (RdDM), directed by 24 nt small
interfering RNA (siRNA), is a
plant-specific de novo cytosine methylation pathway responsible
for natural suppression of
repeat sequences such as transposons and centromeric repeats.
RdDM can be counteracted by
DNA demethylation, which in Arabidopsis is catalyzed by four DNA
demethylases; Demeter
(DME), Repressor of silencing (ROS1) and Demeter-like 2 and 3
(DML2, DML3). Early studies in
our laboratory have suggested DNA demethylases and RdDM work
together to regulate plant
defense-related genes and mediate Arabidopsis resistance against
the fungal pathogen
Fusarium oxysporum. How these two pathways interact to regulate
defense-related genes
remained unclear. The aim of this study is to investigate how
RdDM and DNA demethylases play
a role in Fusarium resistance by analysing changes in small RNA
(sRNA) accumulation, DNA
methylation, and gene expression in wild-type Arabidopsis (Col-0)
and the DNA demethylase
mutant rdd (ros1 dml2 dml3) with and without Fusarium infection.
Whole-genome sRNA deep sequencing plus northern blot
hybridisation detected a large
number of differentially accumulated (DA) siRNAs Col-0 and rdd
plants. A large majority of these
DA 24 nt siRNAs occurred in gene bodies, the 3kb flanking regions
or transposable elements
(TEs). These results indicate that DNA demethylases are involved
in the accumulation of RdDM
associated siRNAs, and that RdDM and DNA demethylases function
co-ordinately to regulate
gene expression through targeting TE sequences.
Comparative analysis of gene expression and DNA methylation
between Col-0 and rdd, using
whole genome mRNA and bisulfite sequencing, supported by qRT-PCR
analysis and existing DNA
methylation data, provided evidence that DA siRNAs participate in
DNA demethylase mediated
regulation of nearby defense related gene targets. These analyses
revealed;
i) genes differentially expressed (DE) between Col-0 and rdd are
enriched for stress
response functions;
ii) siRNAs and DNA methylation co-exist near transcription start
sites (TSS) of the DE
genes in the rdd background but are absent in Col-0; and
iii) the new methylation in rdd spreads towards the TSS, and is
immediately adjacent
to the end of TEs that are methylated in Col-0 as well as in rdd.
These features suggest that TEs in the promoters are the target
of RdDM and have the potential
to induce spread of RdDM to adjacent sequences near TSS resulting
in gene repression. DNA
demethylases are required to counter this effect ensuring active
gene expression.
In contrast to the Col-0 vs rdd comparison, of which a large
proportion of DE genes have
differential siRNA and DNA methylation, a very small proportion
of the Fusarium responsive
genes are associated with differential siRNA and DNA methylation
between mock treated and
Fusarium infected plants. This suggests the Fusarium responsive
expression pattern is not
directly controlled by RdDM changes. I propose the Fusarium
response is determined by the
intrinsic property of cis-elements present in stress response
gene promoters, but their
accessibility by transcription factors can be inhibited by DNA
methylation. DNA demethylases
therefore are required to maintain a low-level methylation state
to regulate gene transcription
activity.
Previous studies state the DME demethylase is expressed and
functional only in developing
seed, and therefore Ros1, DML2 and DML3 should account for the
bulk of demethylase activity
in vegetative tissues. However, my mRNA sequencing data and our
previous microarray
expression data both indicated a high level of DME expression in
vegetative tissues. This
suggested the rdd mutant may retain some DME DNA demethylase
activity in vegetative tissues,
which would affect the interpretation of our data. To address
this issue, I generated DME
knockdown transgenic lines in the rdd background using hairpin
RNA technology, and assayed
the transgenic plants for Fusarium resistance. These
ros1-dml2-dml3-hpDME plants exhibited
enhanced Fusarium susceptibility than rdd, suggesting DME
contributes to DNA demethylase
activity in vegetative tissues. These transgenic materials will
be valuable materials for future
functional studies of DNA demethylases in Arabidopsis.
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sRNA, Arabidopsis, Fusarium oxysporum, RNA-directed DNA methylation
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