Project Summary
Xanthomonas bacteria as a genus are able to infect over 300 species of plants. Despite this
broad host range, each species and pathovar has high host and tissue specificity. In
general, nonhost resistance is the most common form of plant resistance to pathogens; a
more complete understanding of nonhost resistance has the potential to open new avenues
of resistance in host plants. A better understanding of the nonhost response of Hordeum
vulgare (barley) to Xanthomonas oryzae pv. oryzicola will lead to insights into strategies to
enhance host resistance to X. oryzae pv. oryzicola and other bacterial pathogens employing
a Type Three Secretion System.
Objective 1 will determine the molecular trigger of an HR-like response to X. oryzae pv.
oryzicola by barley using a bioinformatics approach to identify candidate effectors followed
by a screen of individual effectors delivered by non-pathogens to determine any elicitors of
HR. Objective 2 uses RNA sequencing to compare the transcriptional response of barley to
infection with host and nonhost Xanthomonas pathogens to identify candidate host genes
that regulate the nonhost defense response. The top genes’ role in defense will be verified
through RNAi/overexpression analysis. One of the major strengths of this proposal lies in
the screening of individual effectors, which addresses the issue of redundant effectors in
natural pathogens.
Upon completion of this project I expect to have identified the molecular trigger of the HRlike response to X. oryzae pv. oryzicola by barley and have developed a list of genes and
regulators differentially expressed in barley upon infection with host and nonhost
pathogens. The results of this study will provide candidate gene targets for manipulation
in host genomes to prevent colonization of rice by X. oryzae pv. oryzicola. In addition, they
will also provide a more detailed framework for interactions between nonhosts and
bacterial pathogens.
As an avenue to increase awareness of plant/bacterial interactions, I will collaborate with
area high school science teachers to develop laboratory activities they can take back to
their classrooms. This strategy is particularly effective because after training one or two
teachers in the summer, they will return to their classrooms and influence more than 100
students per year. Ideally, engaging students in the process of science and enabling them
to experience the sense of discovery that makes science so exciting to all of us; the best and
brightest of our students will chose to pursue degrees and careers in STEM fields.
Project Description
INTRODUCTION
All cereal crops belong to the family Poacea. Pathogens of grasses are often virulent on
only a few species meaning most grasses are resistant to most of the diseases of other
grasses; and importantly, this nonhost resistance is not easily overcome. The maize gene
Rxo1, a gene coding for an NBS-LRR, recoginizes Xanthomonas oryzae pv. oryzicola and
initiates a defense response. Interestingly, X. oryzea pv. oryzicola is not a pathogen of
maize, it is the causal agent of bacterial streak disease of rice. Studies have shown that
when transferred to rice, Rxo1 can recognize X. oryzea pv. oryzicola and initiate a defense
response(Zhao, 2005). This is more impressive when considered with the fact that no
known simply inherited resistance genes to this pathogen are known in rice.
The Avr gene in X. oryzea pv. oryzicola has been identified and is referred to as AvrRxo1.
When maize containing Rxo1 is infiltrated with X. oryzea pv. oryzicola containing AvrRxo1, a
hypersensitive response (HR) is elicited; this response is not present when infiltrated with
X. oryzae pv. oryzae which lacks AvrRxo1. When AvrRxo1 is transformed into X. oryzae pv.
oryzae and then infiltrated into maize containing Rxo1, an HR is elicited. When AvrRxo1 is
transformed into X. oryzae pv. oryzae lacking a functional type three secretion system, no
HR is observed in infiltrated maize containing Rxo1 (Zhao, 2004). These experiments
effectively demonstrate that R genes from one cereal species can be transferred to another
cereal species and retain their function.
In other experiments X. campestris has been used to deliver candidate effectors of fungi
that are suspected of inhibiting the host cell death response. X. oryzae pv. oryzicola is
typically used as a cell death elicitor in these experiments. One issue with this system is the
inability to estimate the ratio of effector delivery to elicitor delivery; by identifying the HRlike symptom elicitor in X. oryzea pv. oryzicola and transferring it to X. campestris under the
same promoter as the effectors, the ratio of effector to elicitor can be more accurately
estimated. If an X. oryzae pv. oryzicola mutant containing a knockout of this elicitor was
able to infect barley instead of induce HR-like symptoms, it could be considered an Avr
gene since it’s presence is resulting in avirulence of the pathogen. This knowledge could
lead to identification of the associated R gene in barley that could be used as a source of
resistance to X. oryzae pv. oryzicola in rice.
Nonhost resistance is defined as the resistance of a plant species to a specific pathogen
(Heath, 2000). This is the most common type of resistance displayed by plants since most
plants are resistant to most potential pathogens. X. oryzae pv. oryzicola is a natural
pathogen of rice while maize and barley both exhibit nonhost resistance to this pathogen;
specifically rice exhibits water-soaking while maize (Zhao 2004) and barley (Figure 1)
exhibit HR-like responses. While there is no known natural resistance to X. oryzae pv.
oryzicola in rice, nonhost resistance has been effectively transferred via expression of the
maize Rxo1 gene in genetically transformed rice (Zhao 2005) .
In my barley/Xanthomonas system, X. oryzae pv. oryzicola elicits an HR-like response when
syringe infiltrated into ten day old seedlings. This phenotype is not present when type
three secretion system(T3SS) deficient X. oryzae mutants are infiltrated. This result is
consistent with an effector triggered HR-like defense response by the plant to the pathogen
since effectors are delivered from the bacterial cell to the plant cell via the T3SS.
Plants have a complex system of defense. We hypothesize that the defense response
induced by pathogens on host plants are different than the defense response induced by
pathogens on nonhost plants; specifically the expression of genes involved in Salycilic
Acid(SA) signaling, Jasmonic Acid(JA) signaling and Ethylene(ET) signaling will be
compared. Knowing what genes are differentially expressed will likely provide clues to the
mechanism(s) of nonhost resistance (Zellerhoff 2010; Bart et. al. 2012). A better
understanding of nonhost resistance can be exploited as a source of potential R genes and
inform decision making when determining how to breed for resistance and/or implement
pest management systems. (Zellerhoff 2010)
Because of my extensive experience working with barley and my close collaboration with
Dr. Bogdanove who studies Xanthomonas interactions with rice, I am well positioned to
merge these two model systems to identify specifically the effector or effectors responsible
for HR-like symptoms elicited by X. oryzae pv. oryzicola in barley but not in rice.
HYPOTHESIS AND OBJECTIVES
The long-range goal of this project is to better understand the interaction between
Xanthomonas species and their plant hosts and nonhosts. The short-term goal of this
application is to identify the X. oryzae pv. oryzicola effector or effectors that elicit an HRlike response in barley and compare the transcriptional response of barley to infection with
host and nonhost pathogens. The central hypothesis of the proposed research X. oryzae
pv. oryzicola is thought to have an effector that elicits plant defense and prevents
colonization and disease in nonhosts. This study will 1) identify the effector(s) that result
in nonhost resistance and 2) identify candidate host genes that regulate the nonhost
defense response.
To accomplish the objectives of this application, I will pursue two specific aims:
I. Determine the molecular trigger(s) of an HR-like response to X. oryzae pv.
oryzicola by H. vulgare.
II. Compare the transcriptional response of barley to infection with host and nonhost
Xanthomonas pathogens to identify candidate host genes that regulate the
nonhost defense response.
My expectations are that, at the conclusion of the proposed period of support, I will have
determined: 1) how the HR-like response of barley to X. oryzae pv. oryzicola is triggered,
and 2) barley genes, differentially expressed when exposed to host or nonhost pathogens,
that play a role in nonhost defense. This framework of knowledge on Xanthomonas
nonhost interactions will provide a pool of candidate R genes that, in this case specifically,
could be transgenically expressed in rice which has no known natural resistance to X.
oryzae pv. oryzicola.
RATIONALE AND SIGNIFICANCE
The rationale behind the proposed research is that Xanthomonas serves as an excellent
model system for bacterial pathogens employing the T3SS; in addition, rice and barley are
both important food crops. A more detailed understanding of the HR-like response elicited
by X. oryzae pv. oryzicola in barley but not in rice could shed light on differences between
host and nonhost interactions and provide candidate resistance genes in barley for
transformation into rice. In addition, the wealth of information available through RNA
sequencing, given appropriate experimental design, has the potential to elucidate genes
and/or regulator elements involved in defense that have so far gone undetected.
Ultimately the results of this study could be applied to plant breeding/engineering projects
to confer robust nonhost resistance traits to plants susceptible to bacterial pathogens.
EXPERIMENTAL APPROACH
Specific Aim #1: Determine the molecular trigger(s) of an HR-like response to X. oryzae
pv. oryzicola by H. vulgare.
Introduction.
The objective of this aim is to identify the effector(s) that elicit a defense response in
nonhosts by X. oryzae pv. oryzicola . When X. oryzae pv. oryzicola is infiltrated into barley
leaves, an HR-like response is elicited (Figure 1). When T3SS deficient strains of X. oryzae
pv. oryzicola are infiltrated into barley leaves, no HR-like response is elicited. Because the
HR-like response is T3SS dependent, we hypothesize that it is due to a secreted effector and
not a pathogen associated molecular pattern (PAMP).
The rationale behind this approach is that X. oryzae pv. oryzicola is thought to have an
effector that elicits plant defense and prevents colonization and disease in nonhosts. This
study will effectively identify the effector(s) that result in nonhost resistance.
Figure 1. 10 day old barley (Mla6) was syringe infiltrated with X. oryzae pv. oryzicola (OD 0.02). This image
was taken 3 days after infiltration.
It is possible that AvrRxo1, the X. oryzae pv. oryzicola effector that elicits HR in maize, is
responsible for elicitation of HR in Barley. To test this AvrRxo1 will be transformed into X.
campestris and delivered via T3SS into barley. In addition, other candidate effectors from
X. oryzae pv. oryzicola will be screened using the same method. Xanthamonas bacteria
generally contain genes for around 15 effectors (Kay 2009); this seems to be a reasonable
number of effectors for this experiment.
Experimental Design.
To test this, we will use the published X. oryzae pv. oryzicola genome to identify putative
effectors based on the presence of a T3SS signal peptide; this seems reasonable since the
HR-like response is dependent upon a functional T3SS. A potential problem with this
approach is an incomplete set of putative effectors because there is not a known consensus
sequence for the T3SS signal peptide(Bart et al. 2012). To address this, we will also
consider genes with homology to known effectors, and genes with hrp-box sequences.
Even with this broad genomic screen, it is unlikely that all effectors will be identified.
Those putative effectors will be transformed into Xanthomonas campestris pv. Raphani
strain 756C (this strain has no phenotype when infiltrated by itself into barley) and a T3SS
mutant strain of X. campestris. These transformants will then be infiltrated into barley
leaves and screened for the ability to elicit an HR-like response. Each transformant will be
syringe infiltrated in three different spots (tip, middle, and base) on a barley leaf. Three
leaves will be infiltrated per replicate and three replications will be done. Untransformed
X. campestris will serve as a negative control (no HR) and X. oryzae pv. oryzicola will serve
as a positive control (HR). An effector will be considered to have elicited HR if the legion
extends beyond the impression ring of the syringe into the watersoaked region of the leaf
(resulting from syringe infiltration, watersoaking edge defined by black lines in Figure 1).
This result should be repeatable on other leaves within an experiment and across
replicates.
Those effectors eliciting HR in barley when delivered by X. campestris will be knocked out
in X. oryzae pv. oryzicola by making in-frame deletion mutants; this strategy avoids
interruption the function of other genes in the operon. These knockout strains will then be
infiltrated in barley leaves to determine if they are sufficient or essential for eliciting HRlike symptoms. Like in the first screen, each knockout will be infiltrated three times per
leaf. Three leaves per knockout will be infiltrated per replicate and three replications of
each knockout will be performed. Wild type X. oryzae pv. oryzicola will be used as a
positive control and T3SS deficient X. oryzae pv. oryzicola will be used as a negative control.
If the effector is knocked out in X. oryzae pv. oryzicola resulting in no HR-like phenotype;
and the effector is transformed into X. campestris resulting in an HR-like phenotype, this
would be strong evidence that this effector is the causal agent of the HR-like phenotype.
The successful identification of AvrRxo1 as a single effector capable of eliciting HR in maize
suggests single effectors may also elicit HR in barley.
Because AvrRxo1 is capable of eliciting HR in maize, all effectors identified from X. oryzae
pv. oryzicola that elicit HR-like symptoms in barley will be screened in maize and rice. The
knockout strains that lose the ability to induce HR will infiltrated into maize leaves and rice
leaves using the same experimental design described above. Maize leaves will be screened
for reduced HR while rice leaves will be screened for reduced pathogenicity (lower
accumulation of pathogen populations in the leaves and/or reduced disease phenotypes).
I expect at least one effector to cause HR-like symptoms when delivered into barley cells
via X. campestris T3SS. Limitations to this approach include no guarantee of all effectors
being identified and included in the screen; given the definitions of putative effectors
above. To address this, I will initially screen 15 effectors. If no HR is observed I am
prepared to add more genes and expand my definition of “effector candidates.”
Once the effector(s) responsible for eliciting HR-like symptoms in Barley is identified, I will
determine the protein family to which each belongs. Software tools like the Phyre2 server
(http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) are available to predict
protein structures if the amino acid sequence does not align well to existing protein
structures. It is also important to determine the degree of conservation of identified
effectors across Xanthomonas strains. To this end, I will BLAST the amino acid sequence of
the identified effector(s) against all sequenced strains of Xanthomonas.
Overall expectations for Specific Aim #1.
I expect to have determined the X. oryzae pv. oryzicola effector(s) that elicit an HR-like
response in barley. I will also have tested the effect of this effector in maize and rice. Once
this work is done, the family of proteins each effector belongs to will be identified. I will
also screen the genomes of other sequenced xanthomonads for the presence of these
effectors. This information will improve our understanding of nonhost resistance and
enable the transformation of this effector into non-pathogenic strains for use as a cell death
elicitor.
Specific Aim #2: Compare the transcriptional response of barley to infection with host
and nonhost Xanthomonas pathogens to identify candidate host genes that regulate
the nonhost defense response.
Introduction.
The objective of this aim is to identify differences in host transcriptional responses when
challenged with host vs. nonhost bacterial pathogens. The rationale for this approach is
that nonhosts effectively mount defense responses that prevent growth and colonization of
the pathogen. Understanding specifically how barley recognizes and defends itself from
nonhost pathogens enables application of that knowledge to compatible interactions. RNA
sequencing will generate expression data for every gene under a specific set of conditions.
This can be used to look for genes (individual or clusters) specifically upregulated or down
regulated upon infiltration with nonhost bacteria as compared to mock infiltrated, or host
infiltrated plants. With this data we can address, among others, the following questions, 1)
What genes are commonly differentially expressed under host and nonhost infiltration? 2)
What genes are uniquely differentially expressed under host and nonhost infiltration? 3)
What GO terms are associated with these two sets of genes?
Once the top differentially expressed genes have been identified, their involvement in
defense can be verified through gene silencing or overexpression experiments. Several
protocols using Barley stripe mosaic virus to knockdown or overexpress genes have been
developed. Virus-induced gene silencing (VIGS) can knockdown expression of genes; this
has been shown to be effective in many systems (Lee et. al. 2012). Virus-induced
overexpression (VOX) is able to induce gene expression using a protocol nearly identical to
VIGS with a different infectious vector. This BSMV system has been used to confirm
function of genes predicted to be involved in defense (Delventhal 2010); therefore, it
should serve to confirm genes predicted in this study as well.
Experimental Design.
To test this 10 day old barley (CI16151) plants will be vacuum infiltrated with strains X.
oryzae pv. oryzicola (host), X. campestris (nonhost), or mock infiltrated (control). Three
biological replicates will be conducted for each sample. At time points 0, 4, 8, 24, and 48
hours after inoculation, tissue will be harvested and snap frozen in liquid nitrogen. After
harvest RNA will be extracted from the 3rd leaf. cDNA will be generated from these RNA
samples using oligoT primers to avoid amplification of bacterial transcripts. These samples
will be submitted for high throughput sequencing. The advantage to RNAseq over
microarrays in this case is the ability to pick up on gene changes in expression of genes that
may not be on a microarray but are involved in defense.
I expect to identify genes that are differentially regulated (induced in a host interaction
while remaining unchanged or being suppressed in a nonhost interaction; or vice versa)
and genes that are not differentially regulated. The difficulty is identifying what genes are
involved in pathogenicity. Given the importance of SA, JA and ET signaling in plant
defenses, differential expression of genes in these pathways under host and nonhost
pathogen interactions would be very interesting. In addition, any genes that accumulate to
very high or very low levels under one condition and not another would be interesting. A
potential problem with this approach is the huge number of differentially expressed genes
likely to be returned from this study. It is possible that a few genes will stand out above the
rest (either induced or suppressed); if this is not the case, my PhD student will employ an
increasingly stringent statistical analysis to limit our pool of genes to those most likely to
be involved in the nonhost defense response.
For genes that are upregulated in nonhost infiltrated plants as compared to host infiltrated
plants, I will use VOX to overexpress the gene before infiltration with a host bacterial
strain. I would expect to see decreased disease sympotoms if indeed this gene is involved
in defense. For genes that are downregulated in nonhost infiltrated plants as compared to
host infiltrated plants, I will use VIGS to knockdown the gene before infiltration with a host
bacterial strain. I would expect to see decreased disease symptoms is this gene is involved
in defense. I propose to test the top three induced genes and the top three suppressed
genes to verify their role in nonhost defense.
Overall expectations for Specific Aim #2.
I expect to identify genes and regulatory elements that are differentially expressed by
barley (CI16151) when infected with host or nonhost bacterial pathogens. Once identified,
these genes will serve as candidates for further investigation (eg. silencing, over
expression) into differences between host and nonhost interactions.
TIMETABLE OF THE WORK PLAN
Year 1:
Objective 1: Identify Type 3 Secretion System effector candidate genes; clone candidate
genes into T3SS expression vector.
Objective 2: Conduct RNAseq infiltrations, extract RNA, submit to RNA sequencing facility.
Year 2:
Objective 1: Screen candidate effectors for elicitation of HR; begin production of
Xanthomonas knockout strains.
Objective 2: Identify top candidate defense genes using RNAseq data; design
RNAi/overexpression constructs for verification in the lab.
Year 3:
Objective 1: Complete production of knockout strains; screen knockouts for altered
infection phenotype. Identify protein family to which verified effectors belong; BLAST
sequenced Xanthomonads for conservation of verified effector.
Objective 2: Confirm candidate defense genes identified from RNAseq data through
RNAi/overexpression.
FUTURE DIRECTIONS
While this study addresses several basic science objectives, specifically identifying
molecular triggers of HR and identifying putative nonhost defense genes; the end goal is
field application. Once the mechanism of recognition of Xanthomonas species by is
identified, we would like to confer robust resistance to X. oryzae pv. oryzae to rice. This
would require identification not only the effector eliciting HR (proposed in this study) but
also the identification of interacting plant protein(s) that sense the effector and elicit HR
(R-protein). Data generated by the RNAseq experiment proposed in this study coupled
with verification of defense involvement by RNAi/overexpression would help to identify
candidate R-genes. This would be followed by the production of stable transgenic lines
expressing the R-protein.
BROADER IMPACTS
In order to encourage young people to pursue degrees in STEM fields, particularly in plant
pathology and microbiology, I will collaborate with area high school science teachers to
develop laboratory activities they can take back to their classrooms. This strategy is
particularly effective because after training one or two teachers in the summer, they will
return to their classrooms and influence more than 100 students per year. This project not
only has the potential to educate students about plant/bacterial interactions, but also
expose them to processes integral to research such as how to formulate a testable
hypothesis, how to design an experiment, the importance of controls, sterile technique and
more. By engaging students in the process of science and enabling them to experience the
sense of discovery that makes science so exciting to all of us in the field; I hope to spark a
curiosity that leads the best and brightest of our students to pursue degrees and careers in
STEM.
REFERENCES
Bart, R., Cohn, M., Kassen, A., McCallum, E. J., Shybut, M., Petriello, A., Krasileva, K.,
Dahlbeck, D., Medina, C., Alicai, T., Kumar, L., Moreira, L. M., Rodrigues Neto, J.,
Verdier, V., Santana, M. A., Kositcharoenkul, N., Vanderschuren, H., Gruissem, W.,
Bernal, A., and Staskawicz, B. J. (2012). High-throughput genomic sequencing of cassava
bacterial blight strains identifies conserved effectors to target for durable resistance. PNAS.
109: E1972–E1979.
Delventhal, R., Zellerhoff, N., Schaffrath, U. (2011). Barley stripe mosaic virus-induced
gene silencing (BSMV-IGS) as a tool for functional analysis of barley genes potentially
involved in nonhost resistance. Plant Signaling & Behavior. 6: 867-869
Heath, M. (2000). Nonhost resistance and nonspecific plant defenses. Current Opinion in
Plant Biology. 3: 315-319
Kay, S., Bonas, U. (2009) How Xanthomonas type III effectors manipulate the host plant.
Current Opinion in Microbiology. 12: 37-43
Lee, W., Hammond-Kosack, K., Kanyuka, K. (2012). Barley Stripe Mosaic Virus-Mediated
Tools for Investigating Gene Function in Cereal Plants and Their Pathogens: Virus-Induced
Gene Silencing, Host-Mediated Gene Silencing, and Virus-Mediated Overexpression of
Heterologous Protein. American Society of Plant Biologists. 160: 582-590.
Mysore, K., Ryu, C. (2004). Nonhost resistance: how much do we know? TRENDS in Plant
Science. 2: 97-104
Oh, K., Lee, S., Chung, E., Park, J., Hun Yu, S., Ryu, C., Choi, D. (2006). Insight into Types I
and II nonhost resistance using expression patterns of defense-related genes in tobacco.
Planta. 223: 1101–1107
Zellerhoff, N., Himmelbach, A., Dong, W., Bieri, S., Schaffrath, U., Schweizer, P. (2010).
Nonhost Resistance of Barley to Different Fungal Pathogens Is Associated with Largely
Distinct, Quantitative Transcriptional Responses. Plant Physiology. 152: 2053–2066
Zhao, B., Ardales, E., Raymundo, A., Bai, J., Trick, H., Leach, J., Hulbert, S. (2004). The
avrRxo1 Gene from the Rice Pathogen Xanthomonas oryzae pv. oryzicola Confers a Nonhost
Defense Reaction on Maize with Resistance Gene Rxo1. MPMI. 17: 771–779.
Zhao, B., Lin, X., Poland, J., Trick, H., Leach, J., Hulbert, S. (2005). A maize resistance
gene functions against bacterial streak disease in rice. PNAS. 102: 15383-15388.
BUDGET JUSTIFICATION
Total Budget Request: $478,245
Salaries/Benefits/Tuition: Total: $175,293
Student A (1/2 time research assistant) $1,667/month: Student A will be a wet lab student
pursuing a graduate degree in genetics, plant biology, plant pathology, microbiology or a
related field. This student will take coursework associated with lab techniques involved in
the proposed research (eg. Molecular genetics, bacteriology, bioinformatics, etc.). This
student will also conduct the experiments proposed including clone putative effectors,
bacterial infiltrations, phenotype plants for HR, create kncokouts, collect RNA for the
RNAseq experiment, and RNAi/overexpression verification of candidate defense genes.
Additionally this student will contribute a large portion of the writing for publication of the
results.
Student B (1/2 time research assistant) $1,667/month: Student B will be pursuing a
graduate degree in bioinformatics, computational biology or a related field. This student
will take coursework associated with the computational analysis required to interpret
RNAseq data (eg. Graduate level statistics, bioinformatics, programming, etc.). This student
will work to identify putative effectors based on available sequence data from
Xanthomonas strains. This student will also analyze the RNAseq data to identify top
candidate nonhost defense genes. This student will contribute a large portion of the
writing for publication of the results.
Travel support costs: Total: $1,600
$800 each for these students to present their findings at professional society meetings has
been included. This is intended to cover registration, lodging and food while at the
meeting, and travel expenses to and from the meeting.
Materials and supplies: Total: $80,363
This request is for general laboratory reagents, enzymes, oligonucleotides, etc. This
equates to about $13,000 for each of two researcher assistants over the course of three
years with a 3% increase each year.
RNA sequencing facility services: Total: $49,500
This represents the largest single service charge outside of salaries. The RNA sequencing
experiment is required to compare the transcriptional response of different treatments. 3
treatments * 3 treatments/replication * 5 timepoints/treatment = 45 samples *
$1,100/sample = $49,500
Publication Costs: Total: $5,000
This is intended to cover the cost of a first authored paper by each of the graduate
assistants.
Research Experience for Teachers: Total: $22,500
1 teacher/summer * $5,000/teacher * 3 summers = $15,000.
$500/summer for classroom supplies * 3 summers = $1,500.
$2,000/summer for reagents and consumables * 3 summers = $6,000
Project Budget Worksheet - Iowa State University of Science and Technology
Eff. 7-1-12
Program Sponsor
Title
PI
Period of Performance
1/1/2013-12/31/2017
Deadline
Year 1
Year 2
Year 3
Total
Salary
Monthly
Calendar
Months
Academic
Months
Summer
Months
$0
$0
$0
$0
1
$0
0.00
0.00
0.00
$0
$0
$0
$0
2
$0
0.00
0.00
0.00
$0
$0
$0
$0
3
$0
0.00
0.00
0.00
$0
$0
$0
$0
4
$0
0.00
0.00
0.00
$0
$0
$0
$0
5
$0
0.00
0.00
0.00
$0
$0
$0
$0
6
$0
0.00
0.00
0.00
$0
$0
$0
$0
7
$0
0.00
0.00
0.00
$0
$0
$0
$0
8
9
$0
$0
0.00
0.00
Calendar
Months
0.00
0.00
0.00
0.00
Number of
persons
$0
$0
$0
$0
$0
$0
$0
$0
$45,008
$46,358
$47,749
$139,115
A
Key Personnel
B
Other Personnel
Monthly
1 Post Doc
0.00
0.00
$0
0.00
0.00
$0
$0
$0
$0
3 Research Asst-Halftime
$1,667
$0
12.00
2.00
$40,008
$41,208
$42,444
$123,661
4 Research Asst-Halftime
$0
0.00
0.00
$0
$0
$0
$0
5 Hourly Undergraduate student
$0
0.00
0.00
$0
$0
$0
$0
6 Hourly Undergraduate student
$0
0.00
0.00
$0
$0
$0
$0
7 P&S
$0
0.00
0.00
$0
$0
$0
$0
8 P&S
$0
0.00
0.00
$0
$0
$0
$0
9 Secretarial/Clerical
$0
0.00
0.00
$0
$0
$0
$0
10 Secretarial/Clerical
$0
0.00
0.00
$0
$0
$0
11 Non-Student Hourly
$2,500
2.00
1.00
$5,000
$5,150
$5,305
12 Non-Student Hourly
$0
0.00
0.00
$0
$0
$0
$45,008
$46,358
$47,749
$139,115
Rate
$5,161
$5,316
$5,475
$15,952
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
0
30.5%
$0
$0
$0
$0
Post Doc
22.0%
$0
$0
$0
Post Doc
22.0%
$0
$0
$0
$0
Research Asst-Halftime
12.9%
$5,161
$5,316
$5,475
$15,952
Research Asst-Halftime
12.9%
$0
$0
$0
$0
Hourly Undergraduate student
4.6%
$0
$0
$0
$0
Hourly Undergraduate student
4.6%
$0
$0
$0
$0
P&S
37.0%
$0
$0
$0
P&S
37.0%
$0
$0
$0
Secretarial/Clerical
49.7%
$0
$0
$0
$0
Secretarial/Clerical
49.7%
$0
$0
$0
$0
Non-Student Hourly
12.0%
$0
$0
$0
$0
Non-Student Hourly
12.0%
$0
$0
$0
$0
$50,169
$51,674
$53,224
$155,067
$0
$0
$0
$0
Travel
$0
$0
$1,600
$1,600
1. Domestic Travel
2. Foreign Travel
$0
$0
$0
$0
$800
$800
$800
$800
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$0
$89,424
$41,158
$47,460
$26,000
$0
$0
$49,500
$0
$0
$0
$0
$11,424
$0
$2,500
$0
$26,780
$0
$0
$0
$0
$0
$0
$0
$11,878
$0
$2,500
$0
$27,583
$5,000
$0
$0
$0
$0
$0
$0
$12,377
$0
$2,500
$0
$178,043
$80,363
$5,000
$0
$49,500
$0
$0
$0
$0
$35,680
$0
$7,500
$0
Subtotal: Total Direct Costs (TDC)
$139,593
$92,832
$102,285
$334,710
Subtotal: Modified Total Direct Costs
$128,169
$80,954
$89,907
$299,030
$61,521
$38,858
$43,156
$143,535
$61,521
$38,858
$43,156
$201,114
$131,690
$145,440
2 Post Doc
$0
$0
$0
Check
$0.00
$0
$139,115.23
Subtotal: Salaries and Wages
C
Fringe Benefits
Subtotal: Salaries, Wages, and Benefits
Equipment (List Item and $ amount for each item > $5k)
D
$139,115.23
$0
$15,952.23
1
2
E
F
Participant Support Cost
See notes below
1. Stipend
2. Travel
3. Subsistence
4. Other
G
Other Direct Costs
1
2
3
4
5
6
7
8
9
10
Materials and Supplies
Publication cost
Computing support
Instrumentation facility
Subcontractor1 - Subject to IDC (first $25,000) See notes below
NOT subject to IDC (Amount over $25,000)
Subcontractor2 - Subject to IDC (first $25,000) See notes below
NOT subject to IDC (Amount over $25,000)
Tuition - Non-Engineering
(Click on "Tuition" sheet)
Tuition - Engineering
(Click on "Tuition" sheet)
Other
RET Supplies
Other
$334,709.98
[ MTDC = TDC - Tuition - Equipment - Participant Support Cost ]
H
Indirect Costs
IDC on MTDC
Rate
48.0%
[ IDC = MTDC * Indirect Rate ]
I
Total Project Costs
[ Total = TDC + IDC ]
$478,245
$478,244.60