Martha Ibore
PLP 692
2012-3: Proposal review
Role: Primary reviewer
Title: Pseudomonas syringae obtain sugar from Arabidopsis via type III effector AvrSweet15
induction
Score for the proposal: Fair
Summary of objectives and approaches
Main objective: To identify the type III effector of Pst.DC3000 (named AvrSweet15)
contributing to the increasing expression level of AtSWEET15 and whether it binds directly to the
promoter region of the AtSWEET15 gene.
Specific objective 1: Identify Pst.DC3000 that increases AtSWEET15 mRNA level.
Approaches to address objective 1 included:
a) Identification of effectors using mutant screens. 60 published Pst.DC3000 candidate
effectors will be used and AtSWEET15 mRNA levels by analyzed by qPCR after
inoculation with mutant strains.
b) Gel mobility shift assay (GMSA) will be used to find out if the effector (AvrSweet15)
doesn’t bind to the AtSWEET15 promoter or it activates transcription indirectly.
c) Genetic approach: Screening of AtSWEET15 mutants (knockout and overexpression lines
for Arabidopsis). Screening Pst.DC3000 ∆avrSWEET mutants all compared to wild type.
This will be followed by measurement of glucose accumulation levels after inoculation
and phenotyping experiments.
Specific objective 2: Determine the host transcription factors that directly interact with the
AtSWEET15 gene to induce its transcription.
Approaches:
a) Yeast-one-hybrid screens to identify the potential TF interacting with the promoter region
of AtSWEET15, as the bait. Identified protein will be named SWEET15-interacting
Protein (SWEEP)
b) GMSA to confirm the direct interaction between SWEEP and AtSWEET15 gene in vivo
c) Genetic approach to determine whether SWEEP functions in the same signaling pathway
as AvrSWEET15. Analysis of mutants of SWEEP will be done.
Strengths and weaknesses of the scientific merit of the proposal are shown in the table
below:
Strengths
Weaknesses
- Good and innovative research questions - Proposal has a lot of bad grammar and poor
Interesting hypotheses.
sentence construction and this makes clarity not
- Good analogy between Xanthomonas oryzae good and affected the flow of the entire proposal.
pv oryzae (Xoo) TAL effector and PstDC3000 One has to read a few times before understanding
effector function to be identified.
what some sentences really mean.
- Good justification for proposal (provide a - In the project summary, objective 2 is too wordy
model system for plant pathogen interaction and the message doesn’t stand out.
related to nutrients)
- Good choice of model organisms P. syringae - Don’t like the idea of having a timeline after each
and Arabidopsis both are widely studied
objective. It’s a bit confusing. I would rather have
one timeline at the end
- Good presentation of results from previous - He doesn’t clearly present alternative approaches
literature and good connection of proposed after limitations
study with related studies
- The limitations and anticipated problems are Two referencing systems used in the beginning text
well explained
pages 1 and 2 (numbering and then author names
later). Not consistent
- Acceptable rationale and significance, and
intellectual merit. Appropriate time allocation
of activities for the 3 years
Why I didn’t give this proposal an ‘excellent’ score
This proposal is very interesting with innovative ideas and previous literature is well utilized. I
didn’t score it as excellent because the content was rather difficult to understand due to many
mistakes with spellings and sentence construction which reduced the quality of the proposal. As
a result, the clarity and flow of the proposed research was greatly compromised. Although the
objectives in the project summary were good, they did not stand out as they should. The title of
the proposal also sounds more like an informative statement and not, a typical title. I would
rather have the proposal entitled “Elucidating the molecular mechanisms by which
Pseudomonas syringae (Pst.DC3000) obtains sugar from Arabidopsis” and then mention the
effector (AvrSWEET15) later in the text.