Secondary proposal review for 2012-3
Proposal summary
This proposal aims to gain an understanding of how the Arabidopsis sugar transporter AtSWEET15 is
involved in pathogenicity of Pseudomonas syringae DC3000. The author’s main hypothesis is that an
effector protein from Pst Dc3000 binds to AtSWEET15 promoter to induce transcription of the
AtSWEET15 gene. But their hypothesis is confusing and may not be what they first state but instead is
that the effector does not initiate AtSWEET15 transcription by binding to the promoter but by
interfering with downstream factors in SWEET signaling. To investigate their hypothesis the author
proposes two objectives. The first objective is to identify Pst DC3000 effector(s) which increase the
transcript levels of AtSWEET15. The second objective would be to look for transcription factors in
Arabidopsis which induce AtSWEET transcription. For Aim 1 the authors propose to screen Pst Dc3000
mutants in the 60 published effectors for changes in AtSWEET15 expression. The author suspects that
one of the mutants will show an inability to increase AtSWEET15 expression. Next the authors propose
to gel mobility shift assays to examine whether the effector binds directly to the sweet promoter. The
authors then plan to examine the phenotypes of Arabidopsis AtSWEET15 overexpression and knockout
plants for changes in the infection pattern. In objective 2 the authors wish to examine Arabidopsis
transcription factors which control AtSWEET15 expression. The author’s hypothesis is that Pst Dc3000
effectors interact not with the SWEET15 promoter but with another element which alters the expression
of the SWEET gene. The Authors propose to find the transcription factor using a yeast two-hybrid assay
by using the promoter as a bait and looking for interaction between the bait and a transcription factor.
They also want to mutate any candidate transcription factors they find by mutating the transcription
factors by creating a T-DNA mutant knockout.
Proposal critique
Based on the work presented here I would not support funding this proposal. The research here does
not appear to provide really novel information. Both objectives in this proposal are possible to fail and
not provide any information which will be of benefit to the scientific community. Secondly this research
is very narrow in its scope and the influence it will have on the scientific community. Additionally the
proposal has some grammatical issues which make me question the care which would be taken with the
research if that much care is not placed in the proposal drafting process. Some of the objectives and
hypotheses were not well stated and are confusing. In objective 1 there is a good chance that none of
the known effectors will change the AtSWEET15 expression profile. The second objective of the proposal
suggests using a yeast two-hybrid screen by using the promoter sequence for AtSWEET15 as a bait. From
What I know of yeast two-hybrid interactions they screen the interaction of protein with protein and not
DNA to protein. This in of itself makes me choose to not fund this study, the authors should have
provided a better assay for looking at the interaction between protein and DNA such as a DNA pull down
assay. Due to their inappropriate methods there is no support for continued work in their second
objective.