Evaluation of “Epigenomic landscape changes caused by generalist aphid feeding” (2012-2)
By: Matt Hunt (Primary Reviewer)
Overall Score: Good
Summary of Objectives and Approaches:
The primary goal of this project is to understand how herbivory from insects can affect the
epigenome of plant hosts. This study would specifically examine the effect of the green peach aphid
(Myzus persicae) feeding on Arabidopsis thaliana (Arabidopsis) genomic methylation, histone
modifications, and small RNA expression. A microarray study carried out by De Vos et al. in 2005
compared gene expression between aphid infested and non-aphid infested Arabidopsis plants. This
study identified ~2100 differentially expressed genes between the two conditions. This project seeks to
correlate the methylation status, histone modification status, and small RNA expression profiles seen in
their experiments with the differential gene expression list from the De Vos et al. 2005 study.
As a first step for this project Arabidopsis plants will be infested with the green peach aphid and
mock infested (with brushes) different plants in three biological replicates. RNA extracted from these
plants will be checked via QRT-PCR for differential expression as compared to the De Vos et al. 2005
study. If the chosen set of genes show similar results to the previous study, the project will go forward.
The first objective is to identify correlations between differentially expressed genes in the De
Vos et al. 2005 study and differential methylation status of these genes in Arabidopsis plants. To do this
genomic DNA will be extracted from the above mentioned three biological replicates and subjected to
bisulfite sequencing with next generation Illumina sequencing. This data will be mapped to the
Arabidopsis genome and correlated with the differential expression gene list from De Vos et al., 2005.
The second objective is to identify correlations between differentially expressed genes in the De
Vos et al. 2005 study and differential histone modification status of these genes in Arabidopsis plants.
To accomplish this objective, extracted nuclei from the same three biological replicates will be subjected
to ChIP-seq using antibodies specific for “…trimethylation of histone H3 on lysine residues 4 and 27
(H3K4me3 and H3K27me3)… [and]… lysine residue 9 on histone H3 (H3K9ac)”. These reads will be
mapped to the Arabidopsis genome and a 5 Kb region upstream of all gene models will be evaluated for
differential histone modification status. This list of genes with differential histone modification status
will be compared with the De Vos et al. 2005 list.
The third objective is to identify correlations between differentially expressed genes in the De
Vos et al. 2005 study and differential small RNA expression of these genes in Arabidopsis plants. To
accomplish this objective, small RNAs will be extracted from the same three biological replicates and
subjected to Illumina next gene sequencing. These small RNAs will be mapped to the genome and
potential silencing targets evaluated. The list of potential targets will be compared with the De Vos et
al. 2005 study list of differentially expressed genes.
Strengths of the scientific merit of the proposal:
The authors have identified an underserved area of research in the area of plant biotic stress.
They make clear that very little research has been done on the epigenetic effects of feeding insects on
plants from previous studies. The approach of studying the methylation status, histone modification
status, and small RNA expression levels in comparison to gene expression levels is certainly unique, and
may give some insights to the effect of insect herbivory on the plant epigenome. The methodology
proposed in this study could be carried out as described in this proposal, with some modifications. The
timeline proposed for the research objectives appears reasonable for the personnel attached to the
proposed project.
Weaknesses of the scientific merit of the proposal:
One of the primary weaknesses of this proposal is the reliance on data collected from another
lab (described in De Vos et al. 2005). The authors would be better served if they carried out their own
RNA seq experiments comparing the two stated conditions (aphid-infested and non aphid-infested).
Correlating data across experiments that were carried out in different labs, under different conditions,
in different years can be dangerous, as the possibility of false positives and false negatives cannot be
well controlled.
Another weakness of the proposal is the experimental design. They state that three biological
replicates will be carried out for this project. They do not state clearly how these replicates will be set
up or randomized. This number of replicates is probably too low for the types of downstream analyses
they propose for their project. With a low replication count, they run a real danger of a low signal to
noise ratio, which can drastically effect downstream statistical analysis and therefore the conclusions of
the study.
The third objective may be the weakest of the three, as the connection between small RNA
sequences and their proposed silencing targets has to be verified experimentally before making any
conclusions about their individual effects. The correlation between a list of differentially expressed
small RNAs and a differential gene list from another study would be tenuous at best.
Strengths and weaknesses of broader impacts:
According to the author of the proposal there has been very little research carried out in the
area of effect of herbivory on the epigenome of plants. This area of research has the potential to impact
plant resistance to herbivory. However, they did not make clear how this research would impact the
broader research community. These objectives would each create a list of potential markers of insect
herbivory in Arabidopsis. These lists, however, have the potential to be large and uninformative to the
broader research community considering the limitations in experimental design. A clearer description
by the author of how this research would impact the broader research community would help increase
the impact of this proposal.
Summary:
The proposed area of research has the potential for high impact science in the area of plant
herbivory. However, the experimental design and reliance on outside data sources makes this proposal
somewhat less effective. The simplistic experimental design, and a lower number of replicates, will
make downstream statistical analysis difficult. The reliance on outside data in a different format than
those proposed here will make correlations tenuous at best. If the authors had proposed to carry out
their own expression analysis through next-generation sequencing, these correlations would have been
much stronger, and may have led to high impact publications.