REVIEW PAPER 2012-4
Explorations of DNA methylation and transgenerational effector-triggered immunity in
Arabidopsis thaliana against its intimate parasitic oomycete, Hyaloperonospora
arabidopsidis
This is an excellent grant proposal
Objectives and approaches
1. Test for transgenerational resistance.
To test for this, two Arabidopsis thaliana accessions Columbia (Col) and Niederzenze (Nd) will
be infected with two isolates of Hyaloperonospora arabidopsidis, Maks9 and Emoy2. Pathogene
plus mock-inoculated plants will be allowed to set seed for 3 generations and then analyzed for
resistance. Similar tests will be carried out on plants derived from DNA methylation mutants
(ddc) and transgenic (DDC overexpression) lines.
2. Test for transgenerational priming of ETI.
This will be tested in two parts. First, ATR13-Maks9 and –Emoy2 alleles will be subcloned into
pEDV (Effector Detector Vector) and introduced into Pst DC3000 expressing the operon of
Photorhabdus luminescens (Pst-LUX) for bacterial growth assays on leaves from Columbia and
Niederzenze P0 and P1 generations. Second, pEDV-ATR13-Maks9 and –Emoy2 will be
introduced into weakly virulent Pst DC3000 ΔCEL (Pst-ΔCEL) and inoculated onto leaves from
Columbia and Niederzenze P0 and P1 generations. The amount of bioluminescence emitted by
Pst-LUX and the amount of electrolyte leakage in the presence of Pst-ΔCEL will be measured
and used for scoring ETI.
3. Map DNA methylation changes at the RPP13 locus and quantify resulting expression.
To investigate DNA methylation changes at the RPP13 locus, pathogen and mock infected leaf
genomic DNA (gDNA) will be treated with sodium bisulfite for conversion of unmethylated
cytosines to uracil. This will be followed by amplification, cloning and sequencing of promoter
and genic region of RPP13 using PCR. Also total RNA from pathogen and mock infected leaf
tissue will be collected and RRP13 expression levels will be analyzed using quantitative realtime PCR (qRT-PCR). Results from both experiments will be compared across Arabidopsis
plants.
The proposal had a good abstract and introduction of the study. This study complements a
previous study by Luna et al. 2012. The author of the proposed study has done a good job
explaining how his/her study ties in with the study carried out by Luna et al. 2012. This presents
greater impacts and provides a broader understanding of the previous and current research.
The objectives were well delineated. Expectations from each objective, possible shortcomings
and alternative approaches were clearly stated and discussed. The approaches for each objective
were well elaborated and backed up with appropriate references. The author had a good number
of references to back up his/her study and this gave the study more validity and credibility.
As much as the objectives in this study look independent of each other, they seem connected.
Therefore, the author runs a risk of not being able to achieve his/her objectives if one of them
fails. The author did not show his/her competence to carry out the proposed study. Whether
he/she is trained in this field or has prior experience is not stated.
Results from this study apply to plants, pathogens but can also be extended to human and animal
disease research hence having broader impacts. Good layout of timeline, budget including what
items are needed and justification on why the items are needed. These all guarantee a successful
study. If the anticipated results from this study are achieved, novel findings such as evidence for
transgenerational priming of ETI will be obtained. Overall, I scored this as an excellent proposal.
It was well written, easy to understand and follow.
REVIEW PAPER 2010-2
Investigating RNA silencing as a method of controlling Fusarium verticillioides
infections
Score for this proposal – Good
Objectives and approaches
1. Identify how siRNAs enter cells of Fusarium verticillioides.
Fusarium verticillioides strain M‐3125 that has been modified to express green fluorescent
protein (GFP) will be exposed to small double stranded RNAs (dsRNA) on potato dextrose agar
(PDA). Controls will be exposed to buffer without dsRNA. Changes in gene expression due to
this exposure will be analyzed using suppression subtraction hybridization (SSH). Collected
DNA from the fungus will be sequenced to identify differentially expressed genes. Restriction
enzyme‐mediated integration (REMI) will be used to make Fusarium verticillioides mutants that
do not take up dsRNA and therefore do not undergo RNA silencing. Similar tests will be carried
out on these mutants.
2. Identify target genes that are effective in controlling the growth of Fusarium
verticillioides.
Genes involved in metabolism and growth of this fungus will be retrieved from the genomic
sequence database of Fusarium verticillioides. These genes will be compared with those
expressed when Fusarium verticillioides was grown on PDA in the presence of ground and
sterilized corn.
The objectives were clearly and concisely outlined. Approaches and expectations for each
objective were clearly stated and discussed. The proposal had a well explained timeline and
future directions. The author indicated his/her past training in this field which shows that he/she
is competent to carry out this study.
On the other hand, the author did not include any pitfalls in the approaches or alternative
approaches for the pitfalls. He/she also had few references to support the proposed study. This
makes it hard to believe that this study will be successful. The limited number of references
could be an indicator of author’s limited knowledge concerning other studies that have been
previously carried out in this field. This could potentially result in proposing a study that has
narrower impacts. Additionally, there was no justification for the money requested to be spent on
items listed in the budget.
I scored this proposal as good because it gave a good general picture of what the author hopes to
achieve from the proposed study. This study is capable of having good impact on plant-pathogen
related studies. Nevertheless, the proposal had a weak project summary and introduction. There
was a lack of connection between most statements. This plus the many grammatical errors and
wrong sentence construction made it hard to follow and understand what information the author
was trying to relay.