Characterizing the Mimicry of Plant Peptides by Effectors from the Soybean Cyst
Nematode, Heterodera glycines
A Grant Proposal
PLP692
11/08/2010
Plant-Protein Mimicry of H. glycines Effectors
Project Summary
Sedentary plant-parasitic nematodes possess an unusual ability to modify and maintain host
plant tissue in a prolonged state of disease. The destructive soybean cyst nematode,
Heterodera glycines, establishes and maintains a feeding site (syncytium) characterized by a
state of high metabolic activity and a transcriptional profile distinct from other plant tissues.
These changes are produced by secretion of effectors that reprogram the host cells. Recently,
HgCLE2, an effector with homology to CLAVATA3/ESR from Arabidopsis, was shown to
functionally mimic its host archetype in planta. This was an unprecedented example of plant
protein mimicry by an animal gene involved in parasitism. CLV3 homologs have been
discovered in Heterodera schachtii and the potato cyst nematode, Globodera rostochiensis.
Twenty nine other plant-peptide homologs with parasitically-regulated expression patterns were
recently identified in H. glycines. This suggests H. glycines utilizes other secreted proteins
(HgPECs for H. glycines plant-like effector candidates) which provide important contributions to
successful parasitism by functional mimicry of host-peptides.
To test this hypothesis, the expression profile of the 29 HgPECs will be screened by in situ
hybridization and those candidates showing effector-like expression patterns will be assayed for
secretion by immunolocalization (Objective #1). Secreted HgPECs will be tested for virulence
contributions by assaying susceptibility of Arabidopsis over expressing each effector and also
expressing siRNAs against each effector (Objective #2). To demonstrate functional mimicry of
host-protein homologs, secreted HgPECs will be evaluated for their ability to rescue the
phenotype of Arabidopsis knockout mutants (Objective #3). Finally, secreted HgPECs will be
used in yeast-two-hybrid assays or in transient promoter activation assays to determine host
interactor molecules (Objective #4).
This research presents an imperative follow-up to the HgCLE2 case by determining whether
plant-peptide mimicry is an isolated phenomenon or part of a major strategy of phytonematode
parasitism. By characterizing functional mimics of host proteins, it will identify key weaknesses
in plant architecture exploited by H. glycines. Except the transient promoter activation studies in
Objective #4, every technique here proposed has been used for characterization of other
nematode effectors, suggesting a high probability of success in the project’s technical details.
The broader impacts of this research will be a major advance in our understanding of plantnematode interactions and possible identification of useful starting points for engineering broad
and durable resistance to cyst nematodes. This activity will provide research opportunities for
one post-doctoral researcher and one graduate student, as well as several undergraduate
students.
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Plant-Protein Mimicry of H. glycines Effectors
Project Description
Introduction
In contrast to the relatively rapid exploitation of host tissue by most biotrophic plant pathogens,
sedentary plant-parasitic nematodes possess an unusual ability to modify and maintain host
tissue in a prolonged state of disease. This type of complex, highly specialized pathogenicity
demonstrates a substantial ability of the nematode to manipulate host genes in its favor
(Sijmons, 1994), and to maintain a long-term equilibrium of disease. The soybean cyst
nematode, Heterodera glycines, is the most destructive soybean (Glycine max) pathogen
worldwide (Gelin et al., 2006), and its damage resulted in an estimated yield reduction of
8,314,480 tonnes from 2003-2005 in the U.S. alone (Wrather, 2006). H. glycines hatch in the
soil and migrate into host roots to feed (Sijmons, 1994), where they establish elaborate,
multinucleated feeding sites (syncytium) at the vasculature (Davis et al., 2000; Hewezi et al.,
2010). The nematode maintains syncytial cells in a prolonged state of high metabolic activity
(Golinowski et al., 1996; Sobczak et al., 1997) and a unique transcriptional profile (Sobczak et
al., 1997; Szakasits et al., 2009), securing nourishment for the remainder of its sedentary lifecycle (Sijmons, 1994; Davis et al., 2008). Premature removal or killing of the parasite results in
degradation of the syncytia, suggesting a continuous, nematode-derived signal maintains
syncytial cells in a uniquely differentiated state (Szakasits et al., 2009). Such a signal likely
passes through the stylet, or mouth-spear, which is used to breach the cell wall at the onset of
syncytial formation (Hussey, 1989). A number of recent studies point to the importance of a
stylet-delivered effector cocktail in successful parasitism (reviewed in Davis et al., 2004; Davis
et al., 2008; Bellafiore and Briggs, 2010).
Of particular interest among the validated effectors of H. glycines are two similar plant-peptide
mimics, HgCLE1 and HgCLE2, with domains homologous to the Arabidopsis CLAVATA3/ESRlike (CLV3) protein (Wang et al., 2001; Gao et al., 2003; Olsen and Skriver, 2003; Wang et al.,
2005; Wang et al., 2010). In Arabidopsis, CLV3 restricts the size of stem cell populations in
shoot and floral meristems (Fletcher et al., 1999) suggesting that the nematode manipulates
plant developmental pathways to alter the differentiation state of root cells and establish the
syncytium (Wang et al., 2005; Mitchum et al., 2008; Wang et al., 2010). Transgenic overexpression of HgCLE2 caused shoot apical meristem differentiation and a CLV3 overexpression phenotype in Columbia-0 (Col-0) Arabidopsis thaliana (hereafter, Arabidopsis), and
rescued the clv3-1 mutant phenotype, indicating similar in planta function of the nematode
effector to its plant archetype (Wang et al., 2005; Wang et al., 2010). The discovery and
characterization of HgCLE2 marked an unprecedented example of plant protein mimicry by an
animal gene involved in parasitism (Mitchum et al., 2008). More recently, CLV3 homologs have
been discovered in the sugar beet cyst nematode, Heterodera schachtii, and in the potato cyst
nematode, Globodera rostochiensis, suggesting plant-protein mimicry may be a common
mechanism of cyst nematode parasitism (Mitchum et al., 2008). It is not known, however,
whether CLV3 imitation is an isolated case of host-peptide mimicry by cyst nematodes, or
whether it is merely one part of a broad strategy utilizing mimicry of numerous plant proteins for
host manipulation. Because functional mimicry of plant peptides is a viable strategy for H.
glycines in the case of HgCLE2 (Wang et al., 2005; Wang et al., 2010), it is plausible, perhaps
even likely, that other examples of plant-peptide-mimicry by the parasite exist, but none have
yet been confirmed.
A recent analysis of large-scale microarray and expressed-sequence-tag data has produced a
pool of candidate H. glycines effectors based on differential expression during the parasitic
stages and a number of other factors (Elling et al., 2007; Elling et al., 2009). Among this rich
3
Plant-Protein Mimicry of H. glycines Effectors
source of material for further investigation, 29 sequences were found to have homology to plant
genes (HgPECs for H. glycines plant-homolog effector candidates; Elling et al., 2009). At least
4 of the HgPEC relatives in plants point to a likely function in protein translation, and at least 5
are related to transcription initiation or DNA modification (Elling et al., 2009), making the list of
29 HgPECs an attractive lineup of candidate effectors that may have a substantial role in
reprogramming of host cells. Two of the most promising HgPECs share high homology with
Arabidopsis histone deacetylase 2 or a potato protein induced in giant cells by the root-knot
nematode Meloidogyne (Elling et al., 2007; Elling et al., 2009).
Given our background in molecular plant pathology, this lab is well positioned to probe the list of
HgPECs for functional plant-peptide mimics. In research we seek outcomes that offer a
substantial advance in the general body of scientific knowledge and a reasonable future
probability of offering a viable solution to a significant problem. Therefore, our long-term
objectives are 1) to characterize the molecular basis for successful parasitism by H. glycines
and 2) to use this knowledge to develop strategies for more durable resistance than is currently
available in soybean cultivars. By identifying and characterizing plant-peptide mimics of H.
glycines, susceptible links in the host’s molecular architecture will be discovered. Therefore,
this study fits nicely with our first long-term objective. In addition, the project is likely to result in
identification of several starting points for future proof-of-concept studies on durable resistance.
Ultimately, this project should contribute to both long-term objectives of the lab, and should
result in several new leads for continued investigation after the conclusion of this project.
Hypothesis and Objectives
In addition to the characterized HgCLE effectors, H. glycines utilizes other secreted proteins,
here referred to as HgPECs, that provide important contributions to successful parasitism by
functional mimicry of host-peptides; these effectors are likely to be involved in reprogramming of
the host cell by manipulating gene expression, developmental or cell-signaling pathways. This
hypothesis will be challenged by pursuit of the following 4 research objectives:
Objective #1: To determine whether H. glycines utilizes other effector mimics of host-peptides,
the 29 HgPECs will be tested for parasitism-specific activation and effector-like expression
patterns. Effector candidates will be assayed for secretion.
Objective #2: To assay host-protein mimics for important contributions to successful parasitism,
secreted HgPECs will be screened for affects on susceptibility of Arabidopsis to H. schachtii, a
close relative of H. glycines that can parasitize Arabidopsis.
Objective #3: To demonstrate functional mimicry of host-protein homologs, secreted HgPECs
will be evaluated for their ability to rescue the phenotype of their plant-homolog knockout
mutants.
Objective #4: To elucidate the mechanism and manner in which host-peptide-mimicking
effectors reprogram the host cell, secreted HgPECs will be employed in association assays to
discover interacting components and manipulated pathways within the host cell.
Rationale and Significance
Characterization of the HgCLE effectors marks a major milestone in phytonematode research
because it is the only known example of plant-peptide mimicry in animal systems for the
4
Plant-Protein Mimicry of H. glycines Effectors
purpose of parasitism (Mitchum et al., 2008). It is not known, however, whether CLV3 imitation
is an isolated case, or one of many examples of plant-peptide mimicry. Because of the potential
for finding more host targets manipulated during H. glycines infection, filling this knowledge gap
is imperative. Because functional mimicry of plant peptides is a viable strategy for H. glycines in
this instance, it is plausible, perhaps even likely, that other plant peptides are imitated by the
parasite’s effectors as well. This, combined with the identification of 29 HgPECs with
parasitism-associated expression patterns (Elling et al., 2009), suggests the search for
additional plant-peptide mimics in the arsenal of H. glycines effectors may be a fruitful endeavor
indeed. By linking parasitism to the activity of plant-peptide mimics, an association is made
between parasitism-promoting conditions and whole pathways targeted for manipulation within
the host. Therefore, characterization of even one HgPEC should provide an economical ratio of
research effort to knowledge gained. This research will almost certainly have correlations in
other cyst nematode pathosystems, and it is possible that host targets of HgPECs could be
important objects for manipulation by other plant pathogens as well. There may also be
correlation to nematode parasites of animal systems.
Experimental Approach
Objective #1: To determine whether H. glycines utilizes other effector mimics of host-peptides,
the 29 HgPECs will be tested for parasitism-specific activation and effector-like expression
patterns. Effector candidates will be assayed for secretion.
The 29 HgPECs were found to be differentially expressed during parasitic stages (Elling et al.,
2009) in whole-nematode microarray experiments (Elling et al., 2007). Taken together,
expression patterns and sequence homology to plant genes suggest they are likely candidates
for secretion and functional mimicry of host proteins in planta. Because nematode effectors are
expressed exclusively in the esophageal gland cells and secreted through the stylet (Davis et
al., 2004; Davis et al., 2008), tissue-specific expression patterns can be informative in the
search for candidate effectors. To assess the transcription pattern of the HgPECs, in situ
hybridizations (John et al., 1969; Buongiorno-Nardelli and Amaldi, 1970) will be performed on
preparasitic and parasitic stages of H. glycines. This assay has been successfully utilized to
observe expression patterns of H. schachtii effectors (Hewezi et al., 2008; Hewezi et al., 2010).
Plant-protein homologs will be considered effector candidates 1) if transcription is verified to be
upregulated at the onset of parasitism, and 2) if transcripts are localized primarily in the
esophageal gland cells. Because all 29 HgPECs have already shown parasitism-associated
expression patterns in whole-nematode microarray experiments (Elling et al., 2007; Elling et al.,
2009), most of them are expected to fulfill the criteria for effector candidates.
HgPECs exhibiting the expression profile described above are highly likely to be secreted
effectors, but confirmation will be necessary. Given that no method of phytonematode
transformation currently exists, successful expression of epitope-tagged or reporter::effector
fusions in transgenic nematodes is unlikely. Therefore, detection of the peptide in its natural
form will be necessary. Due to its high specificity and reliable results, immunolocalization is the
preferred method for visualizing subcellular localizations of pathogen-secreted proteins. This
technique has been used to confirm secretion of the HgCLE2 effector (Wang et al., 2010). A
unique, high-quality, monoclonal antibody will be generated for each HgPEC to be tested for
secretion. Due to the high cost associated with generating such antibodies, a long list of
HgPECs matching the expression profile described above will have to be pared. HgPECs will
be prioritized for immunolocalization assays according the following three criteria:
1) The presence of secretion signal peptides.
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Plant-Protein Mimicry of H. glycines Effectors
Secretion signal peptides (Muesch et al., 1990) are thought to be important markers of
putative effectors (Elling et al., 2009), and have been found in previously characterized
effectors (Wang et al., 2005; Hewezi et al., 2008; Hewezi et al., 2010; Wang et al.,
2010). Putative, N-terminal secretion signals have already been identified in 3 of the 29
HgPECs (Elling et al., 2009), despite a protocol that removed the 5’ sequence from
many genes. Cloning the genomic sequence of candidate effectors will likely result in
the identification of secretion signal peptides in additional genes. Preference will be
given to such genes for immunolocalization assays, but this criterion will not be an
absolute requirement because of the potential for HgPECs with unknown secretion
signals to be overlooked (Elling et al., 2009).
2) The absence of transmembrane domains.
Functional transmembrane domains result in imbedding of the protein in nematode cell
membranes, preventing secretion.
Therefore HgPECs found to contain known
transmembrane domains will be automatically discarded.
3) Plant homologs with functions that could contribute to host cell reprogramming.
Of genes meeting the first two criteria, priority for immunolocalization testing will be
given to the four HgPECs with plant homologs associated with translation (probesets
HgAffx.15039.1, HgAffx.18043.1, HgAffx.4738.1 and HgAffx.5700.1) and the five genes
with plant homologs implicated in DNA modification or transcription (probesets
HgAffx.18770.1, HgAffx.19046.1, HgAffx.19783.1, HgAffx.19967.1, HgAffx.23921.1) due
to their potentially important role in reprogramming the host cell for syncytial formation.
Using these criteria to eliminate unlikely HgPECs and prioritize remaining candidates, it is
anticipated that at least 5, but not more than 10, of the 29 genes will be tested for secretion with
immunolocalization assays.
After demonstration of HgPEC secretion through immunolocalization, it may be desirable to
confirm the in planta location of the candidate effectors. Therefore, secreted HgPEC::GFP
fusions will be transiently expressed in onion epidermal cells as further demonstration of the
localization patterns within plant cells. This method has been used successfully to show
localization of at least two H. schachtii effectors (Hewezi et al., 2008; Hewezi et al., 2010).
Objective #2: To assay host-protein mimics for important contributions to successful parasitism,
secreted HgPECs will be screened for affects on Arabidopsis susceptibility to the close relative
of H. glycines, H. schachtii.
It is reasonable to expect potent H. glycines effectors may produce obvious morphological
changes when expressed in planta. Indeed, the utility of stable expression of nematode
effectors in transgenic Arabidopsis has been repeatedly demonstrated, both for assessment of
effector-induced morphological changes in the host (Wang et al., 2005; Hewezi et al., 2008;
Hewezi et al., 2010; Wang et al., 2010), and in tests for increased susceptibility to H. schachtii
(Hewezi et al., 2008; Hewezi et al., 2010), a close relative of H. glycines that is capable of
infecting Arabidopsis. Effectors with a demonstrated contribution to parasitism are of
substantial interest due to their importance in successful infection. Therefore, secreted HgPECs
will be stably expressed in Arabidopsis, and these plants will be assessed for morphological
and/or developmental changes induced by the effector. Plants expressing HgPECs will also be
screened for increased susceptibility to H. schachtii as described (Hewezi et al., 2010). At least
1 of the secreted HgPECs is likely to produce morphological changes and increase
6
Plant-Protein Mimicry of H. glycines Effectors
susceptibility when stably expressed. If no changes in susceptibility are observed for a given
HgPEC, it may be necessary to generate transgenic soybean with stable expression of the
protein in order to conduct parasitism assays with H. glycines. Because of the difficulty in
transformation of soybean, however, this approach will not be utilized unless absolutely
necessary.
Small, interfering ribonucleic acids (siRNA; Hamilton and Baulcombe, 1999) expressed in
Arabidopsis cells are capable of reducing effector gene expression in four species (Meloidogyne
spp.) of parasitizing root-knot nematodes, probably by nematode uptake of siRNAs generated
by the host’s Dicer complex (Huang et al., 2006). Because no loss-of-function mutants of H.
schachtii have been generated, knockdown of HgPEC homologs in H. schachtii via plantproduced siRNA will be used as an alternative for additional assessment of the contribution of
secreted HgPECs to parasitism.
It will be necessary to use the more pliable H.
schachtii/Arabidopsis pathosystem for these experiments due to the greater ease of Arabidopsis
transformation. This approach should result in corroboration of the data obtained by over
expressing HgPECs in Arabidopsis, and could result in identification of viable targets for plantderived siRNAs for engineered resistance against H. glycines.
Objective #3: To demonstrate functional mimicry of host-protein homologs, secreted HgPECs
will be evaluated for their ability to rescue the phenotype of their plant-homolog knockout
mutants.
Mutant complementation has long been regarded as the ideal manner in which to confirm gene
function, and recently, HgCLE2 was shown to rescue the phenotype of the clv3-1 Arabidopsis
mutant (Wang et al., 2005), providing convincing evidence that HgCLE2 is indeed a functional
mimic of its plant CLV3 sequence homolog. Of the 29 HgPECs, 14 of them are most closely
related to Arabidopsis sequences, but 7 others have closest homologs in rice (Oryza sativa;
Elling et al., 2009). The list of HgPECs awaits comparison against the recently released
soybean (Glycine max) genome (Schmutz et al.), however, and many are likely to have even
closer homologs in the actual H. glycines host. Because of its genetic tractability and the vast
library of publicly available mutants (indexed at The Arabidopsis Information Resource;
http://www.arabidopsis.org/index.jsp), complementation of Arabidopsis mutants with secreted
HgPECs will be attempted in preference to complementation in other plant species. Mutant
plants will be crossed with the transgenics over expressing the HgPECs from experiments
planned for Objective #2. These plants will be carefully compared to Col-0. It is likely that one
or more HgPEC will show functional mimicry of its plant homolog by full or partial mutant
complementation. If complementation is not observed for any tested HgPEC, clues of the
effector’s in planta function will be determined by pursuing the experiments described in
Objective #4 below.
Objective #4: To elucidate the mechanism and manner in which host-peptide-mimicking
effectors reprogram the host cell, secreted HgPECs will be employed in association assays to
discover interacting components and manipulated pathways within the host cell.
True understanding of the molecular mechanisms underlying nematode parasitism can be
achieved by dissecting the pathways and characterizing the effector-host molecule interactions
that enable successful disease. Effectors that operate via functional mimicry of host proteins
must, by definition, share at least some of the molecular interaction partners in the host. A wellcharacterized host protein homolog could make the search for effector targets or regulators
fairly straightforward; CLV3 mimicry of HgCLE2 (Olsen and Skriver, 2003; Wang et al., 2005;
Wang et al., 2010), for example, was confirmed after the CLV3 regulatory pathway in
7
Plant-Protein Mimicry of H. glycines Effectors
Arabidopsis had been characterized (Fletcher et al., 1999). Conversely, poorly characterized or
unknown host protein homologs leave much room for investigation into effector-associating
proteins. Fortunately, many excellent tools for nematode effector characterization already exist.
Yeast-two-hybrid (Y2H) assays (Fields and Song, 1989) have previously revealed effector
interactions with host proteins (Hewezi et al., 2008; Hewezi et al., 2010), and will be used for
initial screening for HgPEC-interactors. HgPECs will be used as bait and screened against
soybean cDNA libraries. Historic performance of Y2H systems suggests false positives must be
distinguished from true interactions, so results will be verified by immunoprecipitation. Extracts
of transgenic Arabidopsis expressing both an HgPEC and FLAG-tagged (Hopp et al., 1988)
candidate molecular partners from soybean will be purified on columns with the antibodies
generated against specific HgPECs (described in Objective #1). Column fractions will be
subjected to Western blots (Burnette, 1981), and the presence of the soybean-derived
interactors will be verified with FLAG-epitope antibodies. This approach should eliminate false
positives detected in the Y2H assays.
A second possible function of plant-peptide homolog effectors is direct transcriptional activation
of host target genes, especially given the 5 HgPECs implicated in DNA modification and
transcriptional regulation among the 29 potential effectors. HgPECs suspected to be involved in
direct activation of host gene transcription will be screened against a soybean promoter trap
using the yeast-one-hybrid (Y1H) system (Li and Herskowitz, 1993). Like the Y2H proteinprotein interaction assays, false positives generated by the Y1H assays will be eliminated by
reconstitution of the effector-promoter pair in an Agrobacterium tumefaciens-mediated transient
assay in Nicotiana benthamiana. Co-delivery of the HgPEC coding sequence and the candidate
soybean target promoter should result in greater activation of a downstream β-glucuronidase
(GUS; Jefferson et al., 1986) reporter gene than when the promoter::GUS fusion is delivered
alone. This assay is a rapid, reliable method for analyzing promoter activation of Transcription
Activator-Like effectors from Xanthomonas spp. (Römer et al., 2009).
Future Directions
Successful identification of plant-peptide-like H. glycines effectors that induce morphological
changes in planta and provide a demonstrable contribution to virulence by interacting with
defined host molecules will open an array of fruitful possibilities for future research projects. For
each effector so characterized, at least 2 major opportunities for additional inquiry will be
available. First, it will be important to delineate and characterize the functional domains of each
plant-peptide-like effector, especially if their respective plant homologs are not well
characterized. Specific details will vary with the type of effector being investigated, but rational
mutagenesis approaches of functional domains are likely to be a productive approach.
The second important avenue for future research is in the application of knowledge obtained
from this study toward the design and testing of a broader and more durable resistance
mechanism against cyst nematodes than what is currently available to growers. Although
development of marketable agricultural products is not the primary goal of this lab, we are
interested in conducting proof-of-concept studies for viable H. glycines resistance strategies that
may be developed as a result of this project.
8
Plant-Protein Mimicry of H. glycines Effectors
Broader Impacts
The broader impacts resulting from this research will be a substantial advance in understanding
of molecular plant-nematode interactions and additional approaches for future engineering of
broad and durable resistance against H. glycines. This activity will provide exceptional research
opportunities for one post-doctoral researcher and one graduate student, as well as 2 or 3
undergraduate students. All researchers will be evaluated and selected strictly on merit and
potential, without regard for race, gender or ethnicity. Results will be presented annually as
posters or oral presentations at appropriate research meetings. In addition, informational
presentations will be offered to growers on an annual basis for the duration of the project. A
summary of the research methods and outcomes described herein may also be developed for
local high school biology courses if instructor interest exists.
1.
2.
3.
4.
Time Table for Proposed Objectives
Objectives
2011
2012
2013
Assess candidate
effector expression
and secretion
patterns; clone
genomic sequences
Screen effectors for
contribution to
parasitism success
Complement mutant
plants with effectors
to confirm functional
mimicry
Employ effectors in
association assays to
discover interacting
components from
host cells
2014
9
Plant-Protein Mimicry of H. glycines Effectors
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12
Plant-Protein Mimicry of H. glycines Effectors
Budget Justification
The total budget request is $500,000.00. This includes a minimal salary of $37,500.00/year for
a post-doctoral researcher and associated benefits. This person will conduct all
experimentation for roughly 2/3 of the effector candidates studied. In addition, one graduate
student will be supported with a minimal salary of $18,000.00/year, plus benefits and tuition
costs. This person will conduct research on the remainder of the effector candidates to be
examined. The total personnel expenses requested amount to $230,313.00 for the duration of
the project.
A request for $74,182.00 for the purchase of laboratory supplies such as enzymes, reagents,
oligonucleotides, etc. is also submitted. This represents roughly $12,000 per researcher in
annual laboratory supplies for the first year, plus a 3% increase for the second and third years.
In addition to the cost of laboratory supplies, a total of $40,000.00 is request for the generation
of up to 10 high quality, monoclonal antibodies against candidate effectors. This is the largest
single-item cost of materials for the project, but it will be utilized for the core work of the
research. It is an essential expense for the project.
For publication costs, $5,075 is requested. This should be sufficient for roughly 3 publications,
which is about half the number anticipated for this project.
13
Worksheet for Project Budget - Cumulative Budget
A
Senior/Key Personnel
B
Other Personnel
Year 1
Year 1
Year 2
Year 2
Year 3
Year 3
Year 4
Year 4
Year 5
Year 5
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
Funds Requested
Cost-Sharing /
Matching Funds
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$65,469
$0
$67,433
$0
$69,456
$0
$0
$0
$0
$0
$65,469
$0
$67,433
$0
$69,456
$0
$0
$0
Total Number other Personnel
4.00
Total Salary, Wages and Fringe Benefits
4.00
4.00
0.00
Total
Total
Total
Funds Requested
Cost-Sharing /
Matching Funds
Cost of Project (all
sources)
$0
$0
$0
$202,358
$0
$202,358
0.00
$0
$0
$202,358
$0
$202,358
C
Equipment
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
D
Travel
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
Domestic
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
Foreign
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
E
Participant/Trainee Support Costs
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
1
Tuition/Fees/Health Insurance
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
2
Stipends
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
3
Travel
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
4
Subsistence
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
5
Other
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
6
Number of Participants/Trainees
F
Other Direct Costs
$40,898
$0
$64,553
$0
$37,761
$0
$0
$0
$0
$0
$143,212
$0
$143,212
1
Materials and Supplies
$24,000
$0
$24,720
$0
$25,462
$0
$0
$0
$0
$0
$74,182
$0
$74,182
2
Publication Costs
$0
$0
$2,500
$0
$2,575
$0
$0
$0
$0
$0
$5,075
$0
$5,075
3
Consultant Services
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
4
ADP/Computer Services
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
5
Subawards/Consortium/Contractual Costs
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
6
Equipment or Facility Rental/User Fees
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
7
Alterations and Renovations
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
8
Graduate Student Tuition
$8,898
$0
$9,333
$0
$9,725
$0
$0
$0
$0
$0
$27,955
$0
$27,955
9
Monoclonal antibody generation
$8,000
$0
$28,000
$0
$0
$0
$0
$0
$0
$0
$36,000
$0
$36,000
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$106,367
$0
$131,986
$0
$107,217
$0
$0
$0
$0
$0
$345,570
$0
$345,570
$46,785
$0
$58,873
$0
$46,796
$0
$0
$0
$0
$0
$152,455
$0
$152,455
$153,152
$0
$190,859
$0
$154,014
$0
$0
$0
$0
$0
$498,025
$0
$498,025
Percent
Requested
Percent CostShared
100.00%
0.00%
10
0.00
0
G
Direct Costs
H
Indirect Costs
I
Total Direct and Indirect Costs
J
Fee
Rate
48.00%
Base Amount > >
If this is an NIH Modular application, this is your modular amount
each year (must be in multiple of $25,000) >>
Salary
Fringe
$
97,469
0.00
$
-
0.00
$
122,653
0.00
$
-
0.00
$
97,493
0.00
$
-
0.00
$
-
0.00
$
-
0.00
$
-
0.00
$
-
$106,367
$131,986
$107,217
$0
$0
$345,570
$55,500
$9,969
$57,165
$10,268
$58,880
$10,576
$0
$0
$0
$0
$171,545
$30,813
100.00%