2010-3 Proposal Review
“Characterizing the Mimicry of Plant Peptides by Effectors from the Soybean Cyst Nematode, Heterodera
glycines”
Score: Very good
Summary
In this grant proposal, the investigator seeks to study potential effectors of the soybean cyst
nematode, Heterodera glycines (Hg). These Hg plant effector candidates (HgPECs) are of particular
interest because they have been shown to be homologous to plant proteins, suggesting that the nematode
is using mimicry in order to manipulate plant cells and produce a better environment for infection, mainly
through the production of a syncytium that allows the nematode to feed. In particular, the author wishes
to confirm that these proteins are indeed effectors that promote pathogenicity/virulence of the nematode,
along with potentially characterizing the actual function of the nematode proteins within infection.
To study these HgPECs, the investigator first will determine if the HgPEC expression is
appropriately localized within the nematode through the use of in situ hybridization (Aim 1). Proteins
that will be secreted through the nematode’s stylet are found in esophageal gland cells, and the presence
of HgPEC expression in these cells makes it possible for their delivery to the host plant cell.
Additionally, the author will then use specifically developed monoclonal antibodies to detect if the
HgPECs are actually secreted into the plant cell. The investigator will then screen any HgPECs that
appear to fit the criteria of nematode effectors by stably expressing them in Arabidopsis and challenging
those plants with H. schachtii to determine if the effectors affect the virulence of that nematode (Aim 2).
Next, Arabidopsis gene-knockout plants will be complemented by the appropriate HgPEC that appears to
be a homolog for the knocked-out gene (Aim 3). This will be done to determine if the HgPECs are
indeed acting in plant protein mimicry, and this would be held true if the HgPEC is able to rescue the
wild-type phenotype of the knockout Arabidopsis plants. Finally, the investigator will conclude this
project by evaluating the molecular interactions of the HgPECs (Aim 4). Protein interactions will be
tested first by using yeast two-hybrid, in which positive interactions will be verified through coimmunoprecipitation and Western blotting, and any potential of the HgPECs in direct transcriptional
regulation will be assessed through yeast one-hybrid, which will be confirmed through the use of
promoter:GUS assays.
Scientific Merit
Undoubtedly, the study of the soybean cyst nematode method(s) of infection are warranted due to
the extremely high economic/agricultural impact of this soybean pathogen. Additionally, research with
this pathogen could provide insight into the mechanisms of pathogenicity of other species of nematode.
The study of effectors are potential plant protein mimics will also further the understanding of how
proteins delivered to the host cell by a pathogen are able to manipulate the plant in order to promote better
infecting conditions. Though the methods suggested by the author here are not novel, the stepwise
sequence of identifying and functionally characterizing these HgPECs is logical, and appears to be
thoroughly developed. Additionally, the author has included multiple “back-up” experiments that can be
utilized if the initial experimentation proves to be difficult or non-feasible.
One major difficulty with this work is that it may be difficult to superficially describe the
phenotypes of these HgPECs (and their homologous plant counterparts), as their functions in
pathogenicity/virulence may be additive, such that the loss of one protein may not have a strongly
noticeable effect. In particular, the author wishes to complement Arabidopsis knockout plants (with
knockouts pertaining to the homologs of the HgPECs). Though this has been shown to be a potentially
successfully method there may be several issues. First, as suggested above, the Arabidopsis mutant may
not have a noticeable physical phenotype, and the infections of H. schachtii may not be noticeably
affected if the effector only has a subtle role in infection. Additionally, though it is acknowledged by the
author, these HgPECs, while homologous to proteins found in Arabidopsis and Oryza sativa (rice), are
used in nematode infection of soybean. While the homologous proteins may also be found in soybean,
there is the possibility that the HgPECs will not be functional, or will have reduced functionality within
the Arabidopsis system. Though soybean transformation may be difficult, it may be worth looking in to if
the Arabidopsis system does not seem to be fruitful.
Broader Impact
As mentioned above, this work could have a very broad impact on the plant pathology field, as
soybean cyst nematode is an extremely relevant pathogen of soybean. Furthermore, the study of their
effectors could give insight into means by which nematodes are able to successfully infect their host
plants, and may even be applicable to the use of effectors by pathogens in other systems. The thorough
approach laid out by the investigator suggests that these effectors will be studied in great enough depth
that future studies will be able to be carried out that perhaps look at individual HgPECs in much greater
detail in order to tease out their complete function in nematode infection (29 potential effectors is
probably too many to be intricately characterized within the work of this proposal).
While a potential setback is the use of Arabidopsis in evaluating the role of these HgPECs in
planta, if this approach turns out to be successful, then the effectors in question could have a strong
potential to be a common means of pathogenicity for many different nematodes that infect different
species of plant, and it is not outside the realm of possibility that other pathogens may attempt to
manipulate the same/similar pathways in order to increase their own pathogenicity/virulence.
Conclusions
Overall, I felt that this proposal was very well written and contains sufficient depth to be
considered for funding. While there may be a few issues experimentally that may have prevented a
higher score, the investigator appears to have thought through many of these problems and has provided a
number of secondary experiments that could be performed if the initial experiments fail. The author
seems to have good direction and focus, and their train of thought emphasizes their expertise in the
subject matter.