2012-5 Proposal Review
“Molecular basis of pathogenicity and Cucurbit host specificity in the Erwinia tracheiphila genome”
Score: Fair
Summary
The investigator is proposing to analyze the molecular mechanisms by which the Erwinia
tracheiphila pathovars/subspecies “muskmelon” and “squash” are able to specifically cause disease in
their respective host species. Research on these pathogens is deemed necessary as they are important
pathogens of muskmelon and squash, and thus can have a large economic effect. An emphasis is placed
on the type III secretion system (T3SS), which is a common tool of mechanism for plant pathogens, as it
allows them to transfer effectors to plant cells, which can act in increasing pathogen virulence.
In order to study the specificity of these pathogens’ virulence and pathogenicity, the author first
proposes to sequence the genomes of the two E. tracheiphila strains through the use of next-generation
Illumina sequencing which can be provided by the DNA facility at Iowa State University, the institution
of the investigator (Aim 1). Following Illumina sequencing and assembly of draft genome(s), the author
proposes to compare the sequences of the two strains to one another, along with comparing them to E.
amylovora, another plant pathogenic species of Eriwinia, and non-pathogenic species such as E. bilingiae
(Aim 2). This will be done in order to identify potential strain specific genes that may confer hostspecific virulence/pathogenicity. The investigator then wishes to create knockout mutant strains for at
least some of the strain-specific genes identified in Aim 2, and hypothesizes that the newly sequenced
strains of E. tracheiphila will have T3SS’s, and knockouts of T3SS genes will be created as well, if found
in the genome(s). The proposed studies will then conclude with pathogen-inoculation assays using these
developed mutants on both the strain-specific hosts, along with the non-host Nicotiana tabacum (Aim 3).
The author will be looking for common disease symptoms found in the strain-specific host plants, and a
hypersensitive response (HR) in N. tabacum, along with measuring transcripts of one salicylic acid
defense related transcript (PR-1).
Scientific Merit
The presented proposal seeks to sequence the genomes of two important agricultural pathogens,
so the development of a quality draft genome will be extremely beneficial to the study of these bacteria.
However, while modern sequencing technology makes the task of sequencing the genome of two bacterial
species relatively easy (compared with past endeavors), it would seem that the time and man power
involved in this venture are not adequate to accomplish this within the proposed timeline of ~2 years.
Additionally, the comparison of entire genomes to find unique genes may be non-trivial, and thus more
difficult than anticipated. Because of these things, the final two aims of this proposal seem unlikely to be
accomplished in this timeline.
Unfortunately, even with the assumption of success in the first aim, which is relatively
straightforward in terms of approach and results, the final two aims do not seem to be developed well
enough to be thoroughly study this phenomenon of host specificity between members of the same species.
While genome comparisons may identify unique genes, these may not necessarily be involved in
virulence or pathogenicity, and thus mutant strains may not show any difference in infection of the host
plant. Additionally, specificity can be conferred by SNPs, or other small changes in a shared
virulence/pathogenicity gene, and this possibility is not addressed for study by the author. Finally,
knockouts of the T3SS’s, if they are present in the newly sequenced strains, do not really advance the
knowledge of this field greatly, at least as it is presented. The investigator only wishes to knock out the
T3SS to see if this has any effect on pathogenicity. This in and of itself would not produce a lot of new
information about the pathogenicity of these types of plant pathogens.
Broader Impact
The sequencing of the two E. tracheiphila genomes will be quite beneficial to the scientific
community along as to agriculture, as these two strains are important pathogens of economically relevant
crops (muskmelon and squash). Thus, fully sequenced genomes will allow for much broader studies of
how these pathogens confer pathogenicity and virulence, and may open opportunities for control of their
disease.
While the impact of sequencing these bacterial genomes may be quite large, the impact of the last
two aims is much less so. Comparison of the new genomes to other pathogenic and non-pathogenic
Erwinia may provide insight into potential genes involved in host specificity, but the approach suggested
by the author would probably not satisfy the amount of study that would be necessary to tease out the
reasons why one strain infects one particular plant species but not another. In line with the reasons
mentioned above, the author makes no note of analyzing how particular bacterial pathogenicity or
virulence factors which may be shared by the two E. tracheiphila strains can still be specific to different
plant species (i.e. small mutations make a particular effector interact with a plant protein that differs by a
few critical amino acids in the respective host plants.
Conclusions
Overall, this proposal seems to lack the sufficient detail to be considered a strong candidate for
funding. Additionally, a lack of focus throughout the proposal made it at times difficult to follow the
author’s train of thought. While the proposed area of study is certainly important, and genomic sequences
of the aforementioned strains of plant pathogenic E. tracheiphila would greatly aid the study of these
pathogens by many others, the proposal as a whole seems to have a number of gaps in structure and
depth. The studies proposed by the investigator seem to be reasonable and relatively basic
experimentally, but it does not seem that the approach has been developed such that the phenomenon in
question can be thoroughly answered.