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Applications and Summary Statements indexed here:
http://www.niaid.nih.gov/ncn/grants/app/default.htm
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PI: ALFANO, JAMES R
Title: Suppression of innate immunity by an ADP-ribosyltransferase type III effector
Received: 03/07/2007
FOA: PA07-070
Competition ID:
FOA Title: RESEARCH PROJECT GRANT (PARENT R01)
1 R01 AI069146-01A2
Dual:
IPF: 578103
Organization: UNIVERSITY OF NEBRASKA LINCOLN
Former Number:
Department: Plant Science Initiative
IRG/SRG: HIBP
AIDS: N
Expedited: N
Subtotal Direct Costs
(excludes consortium F&A)
Year 1:
250,000
Year 2:
250,000
Year 3:
250,000
Year 4:
250,000
Year 5:
250,000
Animals: N
Humans: N
Clinical Trial: N
Exemption: 10
HESC: N
New Investigator: N
Senior/Key Personnel:
Organization:
Role Category:
James Alfano
University of Nebraska
PD/PI
Anna Block
University of Nebraska
Post Doctoral Associate
Thomas Elthon
University of Nebraska
Faculty
Byeong-ryool Jeong
University of Nebraska
Faculty
Joseph Barbieri
Medical College of Wisconsin
Consultant
Dorothee Staiger
University of Bielefeld
Consultant
Yuannan Xia
University of Nebraska
Consultant
Council: 10/2007
Accession Number: 2979402
APPLICATION FOR FEDERAL ASSISTANCE
SF 424 (R&R)
2. DATE SUBMITTED
Applicant Identifier
AlfanoNIHReSub
3. DATE RECEIVED BY STATE
State Application Identifier
1. * TYPE OF SUBMISSION
❍ Pre-application ❍ Application
● Changed/Corrected Application
4. Federal Identifier
AI069146
5. APPLICANT INFORMATION
* Legal Name: Board of Regents, Univ.of Nebraska, Univ. ofNebraska-Lincoln
Department: Office of Sponsored Programs
Division:
* Street1: 312 North 14th Street
Street2: Alexander Building, West
* City: Lincoln
County: Lancaster
Province:
* Country: USA: UNITED STATES
Person to be contacted on matters involving this application
Prefix:
* First Name:
Middle Name:
Nancy
* Phone Number: 402-472-3601
Fax Number: 402-471-9323
6. * EMPLOYER IDENTIFICATION NUMBER (EIN) or (TIN):
XXXXXXXXX
8. * TYPE OF APPLICATION:
● Resubmission
❍ Renewal
❍ New
❍ Continuation
❍ Revision
If Revision, mark appropriate box(es).
❍ A. Increase Award ❍ B. Decrease Award ❍ C. Increase Duration
❍ D. Decrease Duration ❍ E. Other (specify):
* Organizational DUNS:555456995
* State: NE: Nebraska
* ZIP / Postal Code:
68588-0430
* Last Name:
Becker
Email: [email protected]
Suffix:
7. * TYPE OF APPLICANT
H: Public/State Controlled Institution of Higher Education
Other (Specify):
Small Business Organization Type
❍ Women Owned
❍ Socially and Economically Disadvantaged
9. * NAME OF FEDERAL AGENCY:
National Institutes of Health
10. CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER:
TITLE:
* Is this application being submitted to other agencies? ❍ Yes ● No
What other Agencies?
11. * DESCRIPTIVE TITLE OF APPLICANT'S PROJECT:
Suppression of innate immunity by an ADP-ribosyltransferase type III effector
12. * AREAS AFFECTED BY PROJECT (cities, counties, states, etc.)
national
13. PROPOSED PROJECT:
14. CONGRESSIONAL DISTRICTS OF:
* Start Date
* Ending Date
a. * Applicant
b. * Project
07/01/2007
06/30/2012
01
NE-all
15. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION
Prefix:
* First Name:
Middle Name:
* Last Name:
Dr.
James
Robert
Alfano
Position/Title: Associate Professor
* Organization Name: University of Nebraska
Department: Plant Science Initiative
Division: Instit Agricultural Sciences
* Street1: 1901 Vine St.
Street2: The Beadle Center
* City: Lincoln
County: Lancaster
* State: NE: Nebraska
Province:
* Country: USA: UNITED STATES
* Phone Number: 402-472-0395
Fax Number: 402-472-0395
Tracking Number:
Funding Opportunity Number:
Suffix:
* ZIP / Postal Code:
68588-0660
* Email: [email protected]
Received Date: Time Zone: GMT-5
OMB Number: 4040-0001
Expiration Date: 04/30/2008
SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE
16. ESTIMATED PROJECT FUNDING
a. * Total Estimated Project Funding
$1,815,506.00
b. * Total Federal & Non-Federal Funds $1,815,506.00
c. * Estimated Program Income
$0.00
Page 2
17. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE ORDER 12372 PROCESS?
a. YES
❍ THIS PREAPPLICATION/APPLICATION WAS MADE AVAILABLE TO THE
STATE EXECUTIVE ORDER 12372 PROCESS FOR REVIEW ON:
DATE:
b. NO
● PROGRAM IS NOT COVERED BY E.O. 12372; OR
❍
PROGRAM HAS NOT BEEN SELECTED BY STATE FOR REVIEW
18. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein are true, complete
and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with any resulting terms if I accept an
award. I am aware that any false, fictitious, or fraudulent statements or claims may subject me to criminal, civil, or administrative penalties. (U.S.
Code, Title 18, Section 1001)
● * I agree
* The list of certifications and assurances, or an Internet site where you may obtain this list, is contained in the announcement or agency specific instructions.
19. Authorized Representative
Prefix:
* First Name:
Jeanne
* Position/Title: Director
Department: Office of Sponsored Programs
* Street1: 312 North 14th Street
* City: Lincoln
* Last Name:
Wicks
* Organization Name: Board of Regents, Univ.of Nebraska, Univ. ofNebraska-Lincoln
Division:
Street2: Alexander Building, West
County: Lancaster
* State: NE: Nebraska
Province:
* Country: USA: UNITED STATES
* ZIP / Postal Code:
685880430
* Phone Number: 402-472-3171
Fax Number: 402-472-9323
* Email: [email protected]
Middle Name:
* Signature of Authorized Representative
* Date Signed
Jeanne Wicks
03/07/2007
Suffix:
20. Pre-application File Name: Mime Type:
21. Attach an additional list of Project Congressional Districts if needed.
File Name: Mime Type:
Tracking Number:
Funding Opportunity Number:
Received Date: Time Zone: GMT-5
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
424 R&R and PHS-398 Specific
Table Of Contents
Page Numbers
SF 424 R&R Face Page--------------------------------------------------------------------------------------------------------------------
1
Table of Contents---------------------------------------------------------------------------------------------------------------------------
3
Research & Related Project/Performance Site Location(s)-------------------------------------------------------------------
4
Research & Related Other Project Information------------------------------------------------------------------------------------
5
Project Summary/Abstract (Description)-------------------------------------------------------------------
6
Public Health Relevance Statement (Narrative attachment)----------------------------------------
7
Facilities & Other Resources-------------------------------------------------------------------
8
Equipment-------------------------------------------------------------------
9
Research & Related Senior/Key Person----------------------------------------------------------------------------------------------
10
Biographical Sketches for each listed Senior/Key Person--------------------------------------------------
14
PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------------------------
33
PHS 398 Specific Modular Budget------------------------------------------------------------------------------------------------------
35
Personnel Justification-------------------------------------------------------------------
39
PHS 398 Specific Research Plan--------------------------------------------------------------------------------------------------------
40
Introduction to Revised/Supplemental Application---------------------------------------------------------
43
Specific Aims-------------------------------------------------------------------
45
Significance and Related R&D-------------------------------------------------------------------
46
Preliminary Studies/Phase I Final Report-------------------------------------------------------------------
50
Experimental/Research Design and Methods-------------------------------------------------------------------
58
Vertebrate Animals-------------------------------------------------------------------
69
Select Agent Research-------------------------------------------------------------------
70
Multiple PI Leadership Plan-------------------------------------------------------------------
71
Bibliography & References Cited-------------------------------------------------------------------
72
Consortium/Contractual Arrangements-------------------------------------------------------------------
83
Letters of Support-------------------------------------------------------------------
84
Resource Sharing Plan (Data Sharing and Model Organism Sharing)-------------------------------------
88
PHS 398 Checklist-------------------------------------------------------------------
Table of Contents
89
Page 3
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
RESEARCH & RELATED Project/Performance Site Location(s)
Project/Performance Site Primary Location
Organization Name: University of Nebraska
* Street1: 14th and R Streets
Street2:
* City: Lincoln
County: Lancaster
* State: NE: Nebraska
Province:
* Country: USA: UNITED
STATES
* Zip / Postal Code:
68588-0430
File Name
Mime Type
Additional Location(s)
Performance Sites
Tracking Number:
Page 4
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
RESEARCH & RELATED Other Project Information
❍
Yes
●
No
❍
Yes
❍
No
3
4
❍
Yes
●
No
❍
Yes
❍
No
3. * Is proprietary/privileged information ❍ Yes
●
No
1. * Are Human Subjects Involved?
1.a. If YES to Human Subjects
Is the IRB review Pending?
IRB Approval Date:
Exemption Number:
1
2
5
6
Human Subject Assurance Number
2. * Are Vertebrate Animals Used?
2.a. If YES to Vertebrate Animals
Is the IACUC review Pending?
IACUC Approval Date:
Animal Welfare Assurance Number
included in the application?
4.a. * Does this project have an actual or potential impact on
❍
●
Yes
No
the environment?
4.b. If yes, please explain:
4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or
environmental impact statement (EIS) been performed?
❍
❍
Yes
No
4.d. If yes, please explain:
5.a. * Does this project involve activities outside the U.S. or
❍
Yes
●
No
partnership with International Collaborators?
5.b. If yes, identify countries:
5.c. Optional Explanation:
6. * Project Summary/Abstract
9596-Project_Summary.pdf
Mime Type: application/pdf
7. * Project Narrative
9737-Project_Narrative.pdf
Mime Type: application/pdf
8. Bibliography & References Cited
2522-Bibliography_and_References_Cited.pdf
Mime Type: application/pdf
9. Facilities & Other Resources
7861-Facilities_&_Other_Resources.pdf Mime Type: application/pdf
10. Equipment
9825-AlfanoEquipt.pdf
Tracking Number:
Other Information
Mime Type: application/pdf
Page 5
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Project Summary
The eukaryotic innate immune system represents an important barrier that pathogens need to
circumvent in order to cause disease. Several components of this system are conserved in
eukaryotes. Recently, bacterial pathogen effectors that are injected into host cells by type III
protein secretion systems (TTSSs) have been shown to be capable of suppressing innate
immunity in eukaryotes. The bacterial plant pathogen Pseudomonas syringae is dependent on a
TTSS to cause disease on plants. The P. s. pv. tomato DC3000 effector gene hopU1 resembles
ADP ribosyltransferases (ADP-RTs) genes. These genes encode some of the best understood
toxins in bacterial pathogens of animals (e. g., cholera toxin). Preliminary data within this
proposal show that HopU1 is an active ADP-RT and that it ADP-riboslylates several plant
proteins. Mass spectrometry determined that chloroplast and glycine-rich RNA-binding proteins
acted as in vitro substrates for HopU1. These are novel substrates for ADP-RTs. Moreover,
HopU1 has the ability to suppress several responses of the plant innate immune system in a
manner that is dependent on its ADP-RT active site. An Arabidopsis mutant lacking one HopU1
substrate, AtGRP7, displayed enhanced susceptibility to P. syringae suggesting that it is a
component of innate immunity. AtGRP7 is a glycine-rich RNA-binding protein, which suggests
this pathogen targets proteins involved in RNA metabolism to suppress innate immunity. The
central hypothesis of the proposed experiments is that AtGRP7 and perhaps other targets of the
HopU1 ADP-RT type III effector are components of innate immunity. Several of the experiments
seek to elucidate the role AtGRP7 plays in innate immunity using biochemical and molecular
biological approaches. The P. syringae-Arabidopsis pathosystem is an excellent model to study
the innate immune system because of the resources available, the similarities between innate
immune systems between eukaryotes, and the cost efficient research. These experiments will
contribute to a fundamental understanding of the molecular mechanism of bacterial
pathogenesis and innate immunity.
The Specific Aims are the following:(1) Determine the molecular consequence of ADPribosylation on the function of AtGRP7 and elucidate the role this protein plays in innate
immunity; (2) Identify additional substrates of HopU1 and verify their involvement in innate
immunity; (3) Analyze the affect that HopU1 has on host-microbe interactions.
Project Description
Page 6
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Project Narrative
Identifying the eukaryotic targets for the P. syringae HopU1 ADP-ribosyltransferase will
contribute to our understanding of bacterial pathogenesis and will likely reveal important
components of the innate immune system. One HopU1 target belongs to a large group of
proteins called glycine-rich RNA binding proteins, which are not well understood, and this
research will likely increase our understanding of these proteins. Because there are
considerable similarities between the innate immune systems in plants and mammals we expect
that our findings will be relevant to the mission of the NIH and be broadly interesting to
researchers studying molecular mechanisms of bacterial pathogenesis and innate immunity.
Public Health Relevance Statement
Page 7
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
FACILITIES & OTHER RESOURCES
The Alfano lab is housed in the Beadle Center at the University of Nebraska. The lab
consists of two 500 sq. ft. rooms in the Beadle Center and is a state-of-the art research facility
with several resources that deserve mention. Within the Beadle Center are several resource
centers including a Genomics Core Facility that has high throughput DNA sequencing and
microarray readers, and Proteomics Core Facility. Also housed in the Proteomics Core Facility
is a Biacore System 200 for protein-protein interaction studies. We have access to a wellequipped Microscopy Core Facility that has confocal, TEM, and SEM microscopes. The
confocal microscope is equipped with several filters optimal for viewing GFP, YFP and other
commonly used fluorescent compounds. There is a plant transformation facility at this university
that will transform most of the major crop species as well as Arabdopsis with a gene of interest
that is given to them within an E. coli vector.
The Alfano laboratory is fully equipped for molecular biology research. We have 6 Dell
Precision computers all networked to make exchanging large files between computers easy.
The lab has several incubators (shaking and static) for growing bacterial cultures, including
refrigerated models that favor bacteria that grow at lower than room temperature. Either in our
lab or nearby every type of centrifuge is available. For DNA work we have several thermal
cyclers for PCR, many Bio-Rad DNA agarose gel boxes to separate DNA, and a Bio-Rad 2000
Gel documentation system to capture the data. To perform DNA gel blots we have a large area
for working with radioactively labeled DNA and two hybridization ovens. Our lab is equipped
with Hoefer protein gel vertical boxes of several different types to separate proteins and all of
the power supplies to run them and two Hoefer SemiPhor semi-dry electroblotters to transfer
proteins to membranes for immunoblots. We have a Baker EdgeGard laminar flow clean bench
for bacterial and plant cell culture. We store our bacterial culture and plasmid collection in a 80°C freezer. The culture collection is cataloged and the information is stored in a database on
one of our lab computers to make finding the cultures in the freezer easy. We have a Percival
growth chamber that has a humidifier/dehumidifier, which is ideal for reproducible plant
pathogenicity assays. Attached to the Beadle Center we have ample greenhouse space and
have access to several walk-in growth chambers. We have a Zeiss fluorescence microscope in
the lab equipped with deconvultion capabilities and DIC. A darkroom for photographic
applications as well as high quality digital photography equipment is located on our floor of the
building. The PI has an office about 100 sq. ft. Members of the PI's research group share 2
offices adjacent to the laboratory. The UNL Resource Centers have their own offices adjacent to
each facility.
Personnel on the project will visit Dr. Joe Barbieri's lab at the Wisconsin Medical College
in Milwaukee for training and advice on the experiments having to do with the identification of
HopU1's targets in mammalian cell lines.
Facilities
Page 8
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
EQUIPMENT
The Proteomic Center is split into two locations at UNL, the Beadle Center, which
performs all the front end work with protein (i.e., protein purification and 2D gels) and in
Hamilton Hall, which houses the Mass Spec equipment. This facility is well-equiped with all the
needed equipment including the following: Protein purification equipment, 2D gels rigs, a
MALDI-TOF, a Q-TOF, an autodigester, and an autopicker. The microarray equipment is
located the Genomic Core Research Facility in the Beadle Center and houses a complete
Affymetrix GeneChip Microarray System including Affymetrix Hybridization Oven 640, Affymetrix
Fluidics Station 450, a high resolution Affymetrix Genechip Scanner 3000, Affymetrix computer
workstation, Affymetrix GeneChip Operating Software (GCOS), and Affymetrix Data Mining Tool
Software (DMT). The Microscope Core Facility is located in the Beadle Center and has several
confocal and standard flurorescence microscopes (Olympus FV500 Upright and Inverted
Confocal Microscopes, and a Nikon SMZ-800 fluorescence stereo microscope) equipped with
several filters optimal for viewing GFP and YFP.
Equipment
Page 9
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
RESEARCH & RELATED Senior/Key Person Profile (Expanded)
PROFILE - Project Director/Principal Investigator
Prefix
* First Name
Middle Name
* Last Name
Dr.
James
Robert
Alfano
Position/Title: Associate Professor
Department: Plant Science Initiative
Organization Name: University of Nebraska
Division: Instit Agricultural Sciences
* Street1: 1901 Vine St.
Street2: The Beadle Center
* City: Lincoln
County: Lancaster
* Country: USA: UNITED STATES
* Zip / Postal Code: 68588-0660
Suffix
* State: NE: Nebraska Province:
*Phone Number
Fax Number
* E-Mail
402-472-0395
402-472-0395
[email protected]
Credential, e.g., agency login: XXXXXXX
* Project Role: PD/PI
Other Project Role Category:
File Name
1130-AlfanoBio.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Dr.
Anna
Middle Name
* Last Name
Suffix
Block
Position/Title: Postdoctoral Research Associate
Department: Plant Science Initiative
Organization Name: University of Nebraska
Division:
* Street1: 1901 Vine St.
Street2:
* City: Lincoln
County: Lancaster
* State: NE: Nebraska Province:
* Country: USA: UNITED STATES
* Zip / Postal Code: 68588-0660
*Phone Number
Fax Number
* E-Mail
402-472-0499
402-472-3139
[email protected]
Credential, e.g., agency login:
* Project Role: Post Doctoral Associate
Other Project Role Category:
File Name
7614-BlockBio.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Middle Name
* Last Name
Dr.
Thomas
E.
Elthon
Position/Title: Associate Professor
Department: Center for Biotechnology
Organization Name: University of Nebraska
Division:
* Street1: 1901 Vine St.
Street2: The Beadle Center
* City: Lincoln
Tracking Number:
County: Lancaster
Key Personnel
Suffix
* State: NE: Nebraska Province:
Page 10
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
* Country: USA: UNITED STATES
* Zip / Postal Code: 68588
*Phone Number
Fax Number
* E-Mail
402-472-6245
402-472-3139
[email protected]
Credential, e.g., agency login:
* Project Role: Faculty
Other Project Role Category:
File Name
4416-Elthon_biosketch_2007.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Dr.
Byeong-ryool
Middle Name
* Last Name
Suffix
Jeong
Position/Title: Research Assistant Professor
Department: Plant Science Initiative
Organization Name: University of Nebraska
Division:
* Street1: 1901 Vine St.
Street2:
* City: Lincoln
County: Lancaster
* State: NE: Nebraska Province:
* Country: USA: UNITED STATES
* Zip / Postal Code: 68588-0660
*Phone Number
Fax Number
* E-Mail
402-472-0500
402-472-3139
[email protected]
Credential, e.g., agency login:
* Project Role: Faculty
Other Project Role Category:
File Name
4645-JeongBio.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Middle Name
* Last Name
Dr.
Joseph
T.
Barbieri
Suffix
Position/Title: Professor
Department: Microbiology and Mol. Genetics
Organization Name: Medical College of Wisconsin
Division:
* Street1: 8701 Watertown Plank Rd.
Street2: BSB - 2nd floor
* City: Milwaukee
County:
* State: WI: Wisconsin Province:
* Country: USA: UNITED STATES
* Zip / Postal Code: 53226
*Phone Number
Fax Number
* E-Mail
414-456-8412
414-456-6535
[email protected]
Credential, e.g., agency login:
* Project Role: Consultant
Other Project Role Category:
File Name
4469-BarbieriBio.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
PROFILE - Senior/Key Person
Tracking Number:
Key Personnel
Page 11
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Prefix
* First Name
Dr.
Dorothee
Middle Name
* Last Name
Suffix
Staiger
Position/Title: Professor
Department: Department of Biology
Organization Name: University of Bielefeld
Division: Molecular Cell Physiology
* Street1: University Street 25
Street2:
* City: Bielefeld
County: Germany
* State: AE: APO/FPO Province:
Europe, Middle East,
and Africa
* Country: USA: UNITED STATES
* Zip / Postal Code: D-33615
*Phone Number
Fax Number
* E-Mail
++49-521-106 5609
++49-521-106 6410
[email protected]
Credential, e.g., agency login:
* Project Role: Consultant
Other Project Role Category:
File Name
5888-StaigerBio.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
PROFILE - Senior/Key Person
Prefix
* First Name
Dr.
Yuannan
Middle Name
* Last Name
Suffix
Xia
Position/Title: Research Assistant Professor
Department: Center for Biotechnology
Organization Name: University of Nebraska
Division:
* Street1: 1901 Vine St.
Street2: The Beadle Center
* City: Lincoln
County: Lancaster
* State: NE: Nebraska Province:
* Country: USA: UNITED STATES
* Zip / Postal Code: 68588-0665
*Phone Number
Fax Number
* E-Mail
402-472-0998
402-472-3139
[email protected]
Credential, e.g., agency login:
* Project Role: Consultant
Other Project Role Category:
File Name
7394-XiaBio.pdf
*Attach Biographical Sketch
Mime Type
application/pdf
Attach Current & Pending Support
Tracking Number:
Key Personnel
Page 12
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
RESEARCH & RELATED Senior/Key Person Profile (Expanded)
Additional Senior/Key Person Form Attachments
When submitting senior/key persons in excess of 8 individuals, please attach additional senior/key person forms here. Each additional form attached here, will provide you with the ability to identify another 8 individuals, up to a maximum of 4 attachments (32 people).
The means to obtain a supplementary form is provided here on this form, by the button below. In order to extract, fill, and attach each additional
form, simply follow these steps:
•
•
•
•
•
•
•
Select the "Select to Extract the R&R Additional Senior/Key Person Form" button, which appears below.
Save the file using a descriptive name, that will help you remember the content of the supplemental form that you are creating. When assigning a name to the file, please remember to give it the extension ".xfd" (for example, "My_Senior_Key.xfd"). If you do not name your file
with the ".xfd" extension you will be unable to open it later, using your PureEdge viewer software.
Using the "Open Form" tool on your PureEdge viewer, open the new form that you have just saved.
Enter your additional Senior/Key Person information in this supplemental form. It is essentially the same as the Senior/Key person form that
you see in the main body of your application.
When you have completed entering information in the supplemental form, save it and close it.
Return to this "Additional Senior/Key Person Form Attachments" page.
Attach the saved supplemental form, that you just filled in, to one of the blocks provided on this "attachments" form.
Important: Please attach additional Senior/Key Person forms, using the blocks below. Please remember that the files you attach must be Senior/
Key Person Pure Edge forms, which were previously extracted using the process outlined above. Attaching any other type of file may
result in the inability to submit your application to Grants.gov.
1) Please attach Attachment 1
2) Please attach Attachment 2
3) Please attach Attachment 3
4) Please attach Attachment 4
Filename
ADDITIONAL SENIOR/KEY
PERSON PROFILE(S)
MimeType
Filename
Additional Biographical
Sketch(es) (Senior/Key Person)
MimeType
Filename
Additional Current and
Pending Support(s)
Tracking Number:
MimeType
Key Personnel
Page 13
OMB Number: 4040-0001
Expiration Date: 04/30/2008
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
James R. Alfano
POSITION TITLE
eRA COMMONS USER NAME
Associate Professor
XXXXXXX
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Moorpark Junior College, Moorpark, CA
San Diego State University, San Diego, CA
Washington State University, Pullman, WA
Cornell University, Ithaca, NY
DEGREE
(if applicable)
B.S.
Ph.D.
Postdoc
YEAR(s)
FIELD OF STUDY
1981-1983
1986
1993
1993-1997
Biology
Microbiology
Microbiology
Plant Pathology
Please refer to the application instructions in order to complete sections A, B, and C of the Biographical
Sketch.
A. Positions and Honors
Professional Experience
1988-1993
Graduate Research Assistant, Institute of Biological Chemistry,
Washington State University, Pullman, WA
1987-1988
Teaching Assistant, Department of Microbiology,
Washington State University, Pullman, WA
1993-1997
Postdoctoral Research Associate, Department of Plant Pathology,
Cornell University, Ithaca, NY
1997-2000
Assistant Professor, Department of Biological Sciences,
UNLV, Las Vegas, NV
2000-2002
Assistant Professor, Plant Science Initiative and the Department of Plant Pathology,
University of Nebraska, Lincoln, NE
2002-Present
Associate Professor, Plant Science Initiative and the Department of Plant Pathology,
University of Nebraska, Lincoln, NE
Honors
1999
2000
2001
2002
2002
2004
2004-2006
2005
2006
2006
2007
USDA NRI, Plant Pathology Proposal Review Panel
NSF, Integrative Plant Biology Proposal Review Panel
NSF, Microbial Genetics Proposal Review Panel
USDA NRI, Biology of Plant-Microbe Association Proposal Review Panel
Senior Editor, Molecular Plant Pathology Journal
NIH Bacteriology and Mycology – 1 Study Section
Associate Editor, Microbiology Journal
Sygenta Award from Syngenta Crop Protection to an American Phytopathological
Society member for an outstanding recent contribution to teaching, research, or extension in
plant pathology.
NSF, Prokaryotic Molecular and Cellular Biology, Proposal Review Panel
NSF, Signal Transduction, Proposal Review Panel
Senior Editor, Journal of Molecular Plant Microbe Interactions
B. Selected Peer-Reviewed Publications
Alfano, J. R., and M. L. Kahn. 1993. Isolation and characterization of a gene coding for a novel aspartate
aminotransferase from Rhizobium meliloti. J. Bacteriol. 175: 4186-4196.
Biosketches
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Alfano, J. R., J. H. Ham, and A. Collmer. 1995. Use of Tn5tac1 to clone a pel gene encoding a highly alkaline,
asparagine-rich pectate lyase isozyme from Erwinia chrysanthemi EC16 mutant with deletions affecting the
major pectate lyase isozymes. J. Bacteriol. 177: 4553-4556.
Alfano, J. R., D. W. Bauer, T. M. Milos, and A. Collmer. 1996. Analysis of the role of the Pseudomonas
syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally
nonpolar hrpZ deletion mutants, truncated HrpZ fragments, and hrmA mutations. Mol. Microbiol. 19: 715728.
Alfano, J. R., and A. Collmer. 1996. Bacterial pathogens in plants: Life up against the wall. Plant Cell 8: 16831698.
Gopalan, S., D. W. Bauer, J. R. Alfano, A. O. Loniello, S. Y. He, and A. Collmer. 1996. Expression of the
Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the
hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific
hypersensitive cell death. Plant Cell 8: 1095-1105.
Alfano, J. R., H. -S. Kim, T. P. Delaney, and A. Collmer. 1997. Evidence that the Pseudomonas syringae pv.
syringae hrp-linked hrmA gene encodes an Avr-like protein that acts in an hrp-dependent manner within
tobacco cells. Mol. Plant-Microbe Interact. 10: 580-588.
Alfano, J. R., and A. Collmer. 1997. The type III (Hrp) secretion pathway of plant pathogenic bacteria:
Trafficking harpins, Avr proteins, and death. J. Bacteriol. 179: 5655-5662.
Charkowski, A. O., J. R. Alfano, G. Preston, J. Yuan, S. Y. He, and A. Collmer. 1998. The Pseudomonas
syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the
plant hypersensitive response and bind to pectate.
J. Bacteriol. 180: 5211-5217.
Kim J. F., A. O. Charkowski, J. R. Alfano, A. Collmer, and S. V. Beer. 1998. Transposable elements and
bacteriophage sequences flanking Pseudomonas syringae avirulence genes. Mol. Plant-Microbe Interact.
11: 1247-1252.
van Dijk, K., D. E. Fouts, A. H. Rehm, A. R. Hill, A. Collmer, and J. R. Alfano. 1999. The Avr (effector) proteins
HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp
(type III) protein secretion system in a temperature and pH sensitive manner. J. Bacteriol. 181: 4790-4797.
Alfano, J. R., A. O. Charkowski, W. -L. Deng, T. Petnicki, K. van Dijk, and A. Collmer. 2000. The Pseudomonas
syringae Hrp pathogenicity island has a tripartite mosaic structure comprised of a cluster of type III genes
bounded by an exchangeable effector and conserved effector loci that contribute to parasitic fitness and
pathogenicity in plants. Proc. Natl. Acad. Sci. USA. 97: 4856-4861.
Collmer, A., J. L. Badel, A. O. Charkowski, W. –L. Deng, D. E. Fouts, A. R. Ramos, A. H. Rehm, D. M.
Anderson, O. Schneewind, K. van Dijk, and J. R. Alfano. 2000. Pseudomonas syringae Hrp type III
secretion system and effector proteins. Proc. Natl. Acad. Sci. USA. 97: 8770-8777.
Alfano, J. R., and A. Collmer. 2001. Mechanisms of bacterial pathogens in plants: Familiar foes in a foreign
kingdom. pp. 179-226. In Principles of Bacterial Pathogenesis. E. Groisman, ed., Academic Press, San
Diego, California, USA.
Kim, J. F. and J. R. Alfano. 2002. Pathogenicity islands and virulence plasmids of bacterial plant pathogens. In
Current Topics in Microbiology and Immunology. J. Hacker, ed., Springer-Verlag, Berlin, Germany. Volume
264/2: 127-147.
Fouts, D. E., R. B. Abramovitch, J. R. Alfano, A. M. Baldo, C. R. Buell, S. Cartinhour, A. K. Chatterjee, M.
D’Ascenzo, M. L. Gwinn, S. G. Lazarowitz, N. –C. Lin, G. B. Martin, A. H. Rehm, D. J. Schneider, K. van
Dijk, X. Tang, A. Collmer. 2002. Genomewide identification of Pseudomonas syringae pv. tomato DC3000
promoters controlled by the HrpL-alternative sigma factor. Proc. Natl. Acad. Sci. USA. 99: 2275-2280.
Collmer, A., M. Lindeberg, T. Petnicki-Ocwieja, D.J. Schneider, and J.R. Alfano. 2002. Genome mining type III
secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors. Trends
Microbiol. 10: 462-469.
Petnicki-Ocwieja, T., D. J. Schneider, V. C. Tam, S. T. Chancey, L. Shan, Y. Jamir, L. M. Schechter, C. R.
Buell. X. Tang, A. Collmer, and J. R. Alfano. 2002. Genomewide identification of proteins secreted by the
Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000. Proc. Natl. Acad. Sci.
USA. 99: 7652-7657.
van Dijk, K., V. C. Tam, A. R. Records, T. Petnicki-Ocwieja, and J. R. Alfano. 2002. The ShcA protein is a
molecular chaperone that assists in the secretion of the HopPsyA effector from the type III (Hrp) protein
secretion system of Pseudomonas syringae. Mol. Microbiol. 44: 1469-1481.
Alfano, J. R. and M. Guo. 2002. The Pseudomonas syringae Hrp (type III) protein secretion system: Advances
in the new millennium. In Plant-Microbe Interactions. G. Stacey and N. T. Keen, eds., APS Press, St. Paul,
Minnesota, USA. Volume 6:227-258.
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Espinosa, A., M. Guo, V. C. Tam, Z. Q. Fu, and J. R. Alfano. 2003. The Pseudomonas syringae type IIIsecreted protein HopPtoD2 possesses tyrosine phosphatase activity and suppresses programmed cell
death in plants. Mol. Microbiol. 49: 377-397.
Buell, C. R., et. al. 2003. The complete sequence of the Arabidopsis and tomato pathogen Pseudomonas
syringae pv. tomato DC3000. Proc. Natl. Acad. Sci. USA 100: 10181-10186.
Chatterjee, A., Y. Cui, H. Yang, A. Collmer, J. R. Alfano, and A. K. Chatterjee. 2003. GacA, the response
regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv. syringae
DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors. Mol. PlantMicrobe Interact. 16: 1106-1117.
Schechter, L. M., K. A. Roberts, Y. Jamir, J. R. Alfano, and A. Collmer. 2004. Pseudomonas syringae type III
secretion system targeting signals and novel effectors studied with a Cya translocation reporter. J.
Bacteriol. 186: 543-555.
Ham, J. H., Y. Y. Cui, J. R. Alfano, P. Rodriguez-Palenzuela, C. M. Rojas, A. K. Chatterjee, and A. Collmer.
2004. Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants: Importance
of PelE in virulence and atypical regulation of HrpN. Mol. Plant-Microbe Interact. 17: 184-194.
Jamir, Y., M. Guo, H. -S. Oh, T. Petnicki-Ocwieja, S. Chen, X. Tang, M. B. Dickman, A. Collmer, and J. R.
Alfano. 2004. Identification of Pseudomonas syringae type III effectors that can suppress programmed cell
death in plants and yeast. Plant J. 37: 554-565.
He, P., S. Chintamanani, Z. Chen, L. Zhu, B. N. Kunkel, J. R. Alfano, X. Tang, and J. -M. Zhou. 2004.
Activation of a Coi1-dependent pathway in Arabidopsis by Pseudomonas syringae type III effectors and
coronatine. Plant J. 37: 589-602.
Jamir, Y., X. Tang, and J. R. Alfano. 2004. The genome of Pseudomonas syringae tomato DC3000 and
functional genomic studies to better understand plant pathogenesis. In Pseudomonas, Vol. I: Genomics,
Lifestyle and Molecular Architecture. pp. 113-138. Juan L. Ramos, ed., Kluwer Academic/Plenum
Publishers, Dordrecht, The Netherlands.
Shan, L., H. -S. Oh, M. Guo, J. Zhou, J. R. Alfano, A. Collmer, and X. Tang. 2004. The hopPtoF locus of
Pseudomonas syringae pv. tomato DC3000 encodes a type III chaperone and a cognate effector. Mol.
Plant-Microbe Interact. 17: 447-455.
Alfano, J. R., and A. Collmer. 2004. Type III secretion system effector proteins: Double agents in bacterial
disease and plant defense. Annu. Rev. Phytopathol. 42: 385-414.
Wehling, M. D., M. Guo, Z. Q. Fu, and J. R. Alfano. 2004. The Pseudomonas syringae HopPtoV protein is
secreted in culture and translocated into plant cells via the type III protein secretion system in a manner
dependent on the ShcV type III chaperone. J. Bacteriol. 186: 3621-3630.
Espinosa, A., and J. R. Alfano. 2004. Disabling surveillance: Bacterial type III secretion system effectors that
suppress innate immunity. Cell. Microbiol. 6: 1027-1040.
Petnicki-Ocwieja, T., K. van Dijk, and J. R. Alfano. The hrpK operon of Pseudomonas syringae pv. tomato
DC3000 encodes two proteins secreted by the type III (Hrp) protein secretion system: HopB1 and HrpK, a
putative type III translocator. 2005. J. Bacteriol. 187: 649-663.
Lindeberg, M., J. Stavrinides, J. H. Chang, J. R. Alfano, A. Collmer, J. L. Dangl, J. T. Greenberg, J. W.
Mansfield, and D. S. Guttman. 2005. Proposed guidelines for a unified nomenclature and phylogenetic
analysis of type III Hop effector proteins in the plant pathogen Pseudomonas syringae. Mol. Plant-Microbe
Interact. 18: 275-282.
Guo, M., S.T. Chancey, F. Tian, Z. Ge, Y. Jamir, and J.R. Alfano. 2005. Pseudomonas syringae type III
chaperones ShcO1, ShcS1, and ShcS2 facilitate translocation of their cognate effectors and can substitute
for each other in the secretion of HopO1-1. J. Bacteriol.187: 4257-4269.
Fu, Z.Q., M. Guo, and J.R. Alfano. 2006. The Pseudomonas syringae HrpJ is a type III-secreted protein that is
required for plant pathogenesis, injection of effectors, and for secretion of the HrpZ1 harpin. J. Bacteriol.
188: 6060-6069.
Fereirra, A.O., C.R. Myers, J.S. Gordon, G.B. Martin, M. Vencato, Alan Collmer, M.D. Wehling, J.R. Alfano, G.
Moreno-Hagelsieb, W.F. Lamboy, G. DeClerck, D.J. Schneider, and S.W. Cartinhour. 2006. Wholegenome expression profiling defines the HrpL regulon of Pseudomonas syringae pv. tomato DC3000,
allows de novo reconstruction of the Hrp cis element, and identifies novel co-regulated genes. Mol. Plant
Microbe Interact. 19: 1167-1179.
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Vencato, M., F. Tian, J. R. Alfano, C. R. Buell, S. Cartinhour, G. A. DeClerck, D. S. Guttman, J. Stavrinides, V.
Joardar, M. Lindeberg, P. A. Bronstein, J. W. Mansfield, C. R. Myers, A. Collmer, and D. J. Schneider.
2006. Bioinformatics-enabled identification of the HrpL regulon and type III secretion system effector
proteins of Pseudomonas syringae pv. phaseolicola 1448A. Mol. Plant Microbe Interact. 19: 1193-1206.
C. Research Support
Ongoing Research Support
NSF Microbial Genetics and Integrative Plant Biology
Proposal No. MCB-0544447 03/06-02/09
Secretion signals and type III chaperones in the Pseudomonas syringae type III secretion system
Role: Principle Investigator
XXXXXXX
National Institutes of Health NIAID
Proposal No. 1R56AI069146-01 05/06-04/07
Suppression of innate immunity by ADP ribosyltransferase type III effectors.
Role: Principle Investigator
Completed Research Support (within the last 3 years)
NSF Plant Genome Research
Proposal No. DBI-0077622 12/00-11/05
Functional genomics of the interactions of tomato and Pseudomonas syringae pv. tomato DC3000
Role: Principle Investigator
USDA-NRI Biology of Plant-Microbe Associations
Proposal No. 01-35319-13862 12/3-11/05
Chaperones of the type III protein secretion system of Pseudomonas syringae pv. tomato DC3000
Role: Principle Investigator
Biosketches
Page 17
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Anna Block
Postdoctoral Research Associate
eRA COMMONS USER NAME
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
John Mason School, Abingdon, England
University of Bath, Bath, England
University of Florida, Gainesville, FL
University of Florida, Gainesville, FL
Hôpital Cardiologique, Pessac, France
University of Nebraska, Lincoln, NE
DEGREE
(if applicable)
YEAR(s)
A’ levels
M Biochem
Ph.D
Post Doc
Post Doc
Post Doc
1994-1996
1996-2000
2000-2004
2004-2005
2005-2006
2006-2007
FIELD OF STUDY
Science
Biochemistry
Plant molecular biology
Plant molecular biology
Genetics
Plant Science Initiative
Please refer to the application instructions in order to complete sections A, B, and C of the Biographical
Sketch.
A. Positions and Honors
Professional Experience
2000-2004
2004-2005
2005-2006
2006-present
Graduate Research Assistant, Plant Molecular and Cellular Biology program,
University of Florida, Gainesville, FL
Postdoctoral Research Associate, Plant Molecular and Cellular Biology program,
University of Florida, Gainesville, FL
Postdoctoral Research Associate, Laboratoire d'hémobiologie
hôpital cardiologique, Pessac, France
Postdoctoral Research Associate, Plant Science Initiative,
University of Nebraska, Lincoln, NE
B. Selected Peer-Reviewed Publications
O’Donnell, P.J., E. Schmelz, A. Block, O. Miersch, C. Wasternack, J. B. Jones and H. J. Klee, 2003. Multiple
Hormones Act Sequentially to Mediate a Susceptible Tomato Pathogen Defense Response. Plant
Physiology 133: 1181-1189.
Schmelz, E., J. Engelberth, J. H. Tumlinson, A. Block and H. T. Alborn, 2004. The use of vapor phase
extraction in metabolic profiling of phytohormones and other metabolites. The Plant Journal 39: 790-808.
Block, A., E. Schmelz, P. J. O’Donnell, J. B. Jones and H.J Klee, 2005. Systemic acquired tolerance to
virulent bacterial pathogens in tomato. Plant Physiology 138: 1481-1490.
Block, A., E. Schmelz, J. B. Jones and H. J. Klee, 2005. Coronatine and Salicylic Acid: the battle between
Arabidopsis and Pseudomonas for phytohormone control. Molecular Plant Pathology 6: 79-83 (cover
article).
Auldridge, M. E., A. Block, J. Vogel, C. Dabney-Smith, I. Mila, M. Bouzayen, M. Magallanes,-Luundback, D.
DellaPenna, D. R. McCarty and H. J. Klee, 2006. Characterization of three members of the Arabidopsis
carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme
family. The Plant Journal 45: 982-993.
Biographical Sketches for each listed Senior/Key Person 2
Page 18
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Thomas E. Elthon
Associate Professor
Leader UNL Proteomic Center
UNL Center for Biotechnology
eRA COMMONS USER NAME
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Arizona State University
Iowa State University
Iowa State University
DEGREE
(if applicable)
YEAR(s)
B.S.
M.S.
Ph.D.
1977
1980
1983
FIELD OF STUDY
Biology
Botany
Botany
NOTE: The Biographical Sketch may not exceed four pages. Follow the formats and instructions on the
attached sample.
A. Positions and Honors. List in chronological order previous positions, concluding with your present position. List
any honors. Include present membership on any Federal Government public advisory committee.
Postdoctoral Fellow, August 1983 to August 1984, Biochemistry and Biophysics, University of
Pennsylvania.
Postdoctoral Fellow, August 1984 to May 1985, MSU-DOE Plant Research Lab., Michigan State
University.
NSF Postdoctoral Fellow, June 1985 to December 1987. MSU-DOE Plant Research Lab., Michigan
State University.
Visiting Assistant Professor, January 1988 to June 1989. Dept. of Biological Sciences, University of
Maryland-Baltimore County.
Assistant Professor, July 1989 to August 1995. Dept. of Biological Sciences, University of Nebraska.
Associate Professor, August 1995 to July 2004. Dept. of Biological Sciences, University of Nebraska.
Protein Core Facility Manager, August 2004 to present. Dept. of Agronomy and Horticulture, Center for
Biotechnology, Plant Science Initiative, and Dept. of Biological Sciences, Univ. of Nebraska
B. Selected peer-reviewed publications (in chronological order). Do not include publications submitted or in
preparation. For publicly available citations, URLs or PMC submission identification numbers may accompany the
full reference. Note copies of these publications are no longer accepted as appendix material.
Bhadula, S. K., T. E. Elthon, J. E. Habben, T. G. Helentjaris, S. Jiao, and Z. Ristic. 2001. Heat-stress
induced synthesis of chloroplast protein synthesis elongation factor (EF-Tu) in a heat-tolerant maize
line. Planta 212:359-366.
Corina Borghouts, Alexandra Werner, Thomas Elthon, and Heinz D. Osiewacz. 2001. Coppermodulated gene expression and senescence in the filamentous fungus Podospora anserina. Mol Cell
Biol 21:390-399.
Lund, A. A., D. M. Rhoads, A. L. Lund, R. L. Cerny, and T. E. Elthon. 2001. In vivo modifications of the
maize mitochondrial low molecular weight heat stress protein, HSP22. J. Biol. Chem. 276:2992429929.
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
Biographical Sketches for each listed Senior/Key Person 3
Biographical Sketch Format Page
Page 19
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Principal Investigator/Program Director (Last, First, Middle):
PI Name
Heckman, N. L., T. E. Elthon, G. L. Horst, and R. E. Gaussoin. 2001. Influence of Trinexapac-ethyl on
respiration of isolated wheat mitochondria. Crop Sci. 42:423-427.
Karpova, O.V., E.V. Kuzmin, T. E. Elthon, and K.J. Newton. 2002. Differential expression of alternative
oxidase genes in respiratory deficient maize mitochondrial mutants. Plant Cell 14:1-15.
Worthington, P., P. Blum, F. Perez-Pomares, and Thomas E. Elthon. 2002. Large scale cultivation of
acidophilic hyperthermophiles for recovery of secreted proteins. App Env Micro 69:252-257.
Rasmusson, A. J., K. L. Soole, and T. E. Elthon. 2004. Alternative NAD(P)H dehydrogenases of plant
mitochondria. Annual Review of Plant Physiology and Plant Molecular Biology 55:23-39.
Karpova, O.V., E.V. Kuzmin, T.E. Elthon, and K.J. Newton. 2004. Mitochondrial respiratory
deficiencies signal up-regulation of the genes for heat shock proteins. J Biol Chem 279:2067220677.
Rhoads, D. M., S. J. White, Y. J. Zhou, M. Muralidharan, and T. E. Elthon. 2005. Altered gene
expression in plants with constitutive expression of a corn mitochondrial small heat shock protein
suggests the involvement of retrograde regulation in the heat stress response. Physiologia
Plantarum 123:435-444.
Jiao, S., J. M. Thornsberry, T. E. Elthon, and K. J. Newton. 2005. Biochemical and molecular
characterization of PSI deficiency in the NCS6 mitochondrial mutant of maize. Plant Mol Biol 57:303313.
XXXXXXX
C. Research Support. List selected ongoing or completed (during the last three years) research projects (federal
and non-federal support). Begin with the projects that are most relevant to the research proposed in this
application. Briefly indicate the overall goals of the projects and your role (e.g. PI, Co-Investigator, Consultant) in
the research project. Do not list award amounts or percent effort in projects.
XXXXXXX
PHS 398/2590 (Rev. 09/04, Reissued 4/2006)
Biographical Sketches for each listed Senior/Key Person 3
Continuation Format Page
Page 20
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Byeong-ryool Jeong
Research Assistant Professor
eRA COMMONS USER NAME
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Pusan National University, Pusan, Korea
Pusan National University, Pusan Korea
Indiana University Bloomington
DEGREE
(if applicable)
B.S.
M.S.
Ph.D.
YEAR(s)
1983
1985
1998
FIELD OF STUDY
Biology
Biology
Biology (Mol. Cell. Dev.
Biology)
Please refer to the application instructions in order to complete sections A, B, and C of the Biographical
Sketch.
A. Positions and Honors.
Positions and Employment
1985-1986, Cadet (for Lieutenant) in the Korean Army
1986-1989, Research/Teaching Assistant, Pharmacology, Pusan National University Medical School, Korea
1990-1998, Associate Instructor, Biology, Indiana University Bloomington
1990-1998, Research Assistant, Biology, Indiana University Bloomington
1998-2003, Post-doctoral fellow, Biological Sciences, University of Nebraska-Lincoln
2004-2004, Senior Research Technologist, Plant Science Initiative, University of Nebraska-Lincoln
2005-present, Research Assistant Professor, Plant Pathology, Agronomy, University of Nebraska-Lincoln
Honors
1979-1982: Scholarships for excellent admittance exam and performance, Sponsored by Pusan National University,
Korea.
1998: The 1998 Outstanding Associate Instructor Award, Sponsored by Indiana Unversity Bloomington, Department of
Biology.
2000: Travel Award for Chlamydomonas Meeting, Sponsored by Genetics Society of America.
2003: Scholarship for poster presentation in Keystone Symposia, Sponsored by NIH.
2005-2006: 2005 Layman Award, Sponsored by University of Nebraska.
B. Selected peer-reviewed publications (in chronological order).
1. Hong, K. W. , Rhim, B. Y. , Lee W. S. , Jeong, B. R. , Kim, C. D. , and Shin, Y. W. 01 Nov 1989. Release of superoxidedependent relaxing factor(s) from endothelial cells. Am. J. Physiol. 257:H1340-6.
2. Wu-Scharf, D. , Jeong, B. -r. , Zhang, C. , and Cerutti, H. 10 Nov 2000. Transgene and transposon silencing in
Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Science. 290:1159-1162.
3. Byeong-ryool Jeong, Dancia Wu-Scharf, Chaomei Zhang, and Heriberto Cerutti. 22 Jan 2002. Suppressors of
transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA damaging agents and reactivate
transposable elements. Proc. Natl. Acad. Sci. USA. 99:1076-1081.
4. Zhang, C. , Wu-Scharf, D. , Jeong, B. -r. , and Cerutti, H. 01 Jul 2002. A WD40-repeat containing protein is required for
transcriptional silencing of a transgene in Chlamydomonas. Plant J. 31:25-36.
5. Rohr, J. , Sarkar, N. , Balenger, S. , Jeong, B. -r. , and Cerutti, H. 20 Aug 2004. Tandem inverted repeat system for
selection of effective transgenic RNAi strains in Chlamydomonas. Plant J. 40:611-621.
P
Biographical Sketches for each listed Senior/Key Person 4
Page 21
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
6. van Dijk, K., Marley, K.E., Jeong, B. r., Xu, J., Hesson, J., Cerny, R.L., Waterborg, J.H., and Cerutti, H. (2005).
Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas. Plant Cell
17(9):2439 2453.
C. Research Support
Completed Research Support (within the last 3 years)
XXXXXXX
Biographical Sketches for each listed Senior/Key Person 4
Page 22
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Give the following information for all new key personnel.
Copy this page for each person.
NAME
Joseph T. Barbieri
POSITION TITLE
Professor
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing.
Include postdoctoral training.)
INSTITUTION AND LOCATION
SUNY at Cortland, New York
University of Massachusetts at Amherst, Amherst,
Massachusetts
DEGREE
(if applicable)
B.S.
Ph.D.
YEAR(s)
1975
1980
FIELD OF STUDY
Biology
Microbiology
RESEARCH AND PROFESSIONAL EXPERIENCE: Concluding with present position, list, in chronological order, previous employment,
experience, and honors. Include present membership on any Federal Government public advisory committee. List, in chronological order, the titles,
all authors, and complete references to all publications during the past three years and to representative earlier publications pertinent to this
application. If the list of publications in the last three years exceeds two pages, select the most pertinent publications. DO NOT EXCEED TWO
PAGES.
PROFESSIONAL EXPERIENCE
Postdoctoral Fellow
Department of Microbiology, 1980-1984
Advisor: Dr. R. John Collier, UCLA, Los Angeles, CA
Research Associate
Department of Microbiology, 1984-1986
Advisor: Dr. R. John Collier, Harvard Medical School, Boston, MA
Asst. Professor - Professor Department of Microbiology, 1986-present
(Tenured)
Medical College of Wisconsin, Milwaukee, Wisconsin
Director
Medical Scientist Training Program, 2005-present, MCW
AWARDS AND HONORS
National Institutes of Health Individual Postdoctoral Fellowship, 1983-1984
National Institutes of Health, Research Career Development Award, 1992-1997
Visiting Professor, Department of Pharmacology, University of Freiberg, Germany, 10/1996
Session leader, Gordon Conference on Microbial Pathogenesis, July 12, 1998
Co-organizer, 6th Midwest Microbial Pathogenesis Meeting, Milwaukee, WI Sept /10-12, 1999
Division Lecturer, Division D, American Society for Microbiology, 2000
Division chairman, Division B, American Society for Microbiology, 2001
Michael Gill Lecture, Tufts Medical Center, Boston MA, 2001
Lecturer, ASM, Careers in Biomedical Research, Madison, WI, 2001
Lecturer, International Forum on Infection and Immunity, Osaka, Japan, 2001
Session leader, American Society for Microbiology, Salt Lake City, UT, 2002
Chairman Rank and Tenure, Medical College of Wisconsin, 2002-2003
Teacher of the Year; Graduate School at the Medical College of Wisconsin, 2003
Lecturer, ETOX12, Canterbury, UK, 2005
Lecturer, Japanese Society for Bacteriology, Nagazawa, Japan, 2006
Session leader, Gordon Conference on Microbial Toxins and Pathogenesis, 2006
Chair elect Gordon Conference on Microbial Toxins and Pathogenesis, 2010
STUDY SECTION MEMBERSHIP
Member-NIH-NIAID Bacteriology and Mycology-1 Study Section (10/96- 06/00): Chairman-NIH-NIAID
Bacteriology and Mycology-1 Study Section (10/98-06/00). Reviewer- Oklahoma City National Memorial
Institute for the Prevention of Terrorism (MIPT), Oklahoma City, OK 9/2000, 02/02. Reviewer- NIAIDBioterrorism. 06/02, 02/04, 01/05. Reviewer- NIH-NRSA. 05/02, 11/02, 02/03, 06/03, 06/04, 11/04. Ad-hoc
member- NIH (BM1 or RFA) 10/94, 5/95, 12/95, 3/96, 6/96, 07/00, 02/01, 04/03, 06/03, 06/04, 11/05, 03/06
ad-hoc member- Cystic Fibrosis Foundation (National) review panel 12/03.
Biographical Sketches for each listed Senior/Key Person 5
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
EDITORIAL BOARD
Member, Infection and Immunity (1990-1995)
Member, Molecular Microbiology (1997-present)
Editor, Infection and Immunity, (1997-2006).
PUBLICATIONS (2002 to present. Total-92 peer reviewed publications
63. Banwart, B., Splaingard, M., Farrell, P., Moss, J., Ehrmantraut, M.E., Frank, D.W., and Barbieri, J.T.
2002. Acute infections of infants with Cystic Fibrosis by Pseudomonas aeruginosa expressing the Type III
cytotoxin ExoS. J.I.D. 185: 269-70.
64. Krall, R, Sun, J.J., Pederson, K.J., Barbieri, J.T. 2002. In vivo modulation of Rho GTPases by
Pseudomonas aeruginosa ExoS. Infect. Immun. 70: 360-7.
65. Riese, M.J. and Barbieri, J.T. 2002. Membrane localization contributes to the in vivo ADP-ribosylation of
Ras by Pseudomonas aeruginosa ExoS. Infect. Immun. 70: 2230-2.
66. Riese, M.J., Goehring, M., Ehrmantraut, M., Moss, J., Aktories, K., Barbieri, J.T., and Schmidt, G. 2002.
Auto-ADP-ribosylation of Pseudomonas aeruginosa ExoS. J. of Biol. Chem. 277: 12082-8.
67. Pederson, K.J., Krall, R., Riese, MJ, Ning, G. and Barbieri, J.T. 2002. Intracellular localization modulates
targeting of ExoS, a type-III cytotoxins, to eukaryotic signaling proteins. Mol. Microbio. 46(5): 1381-1390.
68.Maresso, A.M. and Barbieri, J.T. 2002. Purification and characterization of two recombinant forms of the
type-III cytotoxin, ExoS. Protein Purification and Expression. 26: 432-437.
69. Fraylick JE. Riese MJ. Vincent TS. Barbieri JT. Olson JC. 2002. ADP-ribosylation and functional effects of
Pseudomonas exoenzyme S on cellular RalA. Biochemistry. 41(30):9680-02.
70. Sun, J-J and Barbieri, J.T. 2003. Pseudomonas aeruginosa ExoT ADP-ribosylates CT10-regulator of
kinase (Crk) proteins. Journal of Biological Chemistry. 278(35):32794-803
71. Maresso AW. Riese MJ. Barbieri JT. 2003. Molecular heterogeneity of a type III cytotoxin, Pseudomonas
aeruginosa exoenzyme S. Biochemistry. 42(48):14249-57.
72. Krall R. Zhang Y. Barbieri JT. Intracellular Membrane Localization of Pseudomonas ExoS and Yersinia
YopE in Mammalian Cells. 2004. Journal of Biological Chemistry. 279(4):2747-53
73. Sun, J-J, Maresso, AW, Kim, J, and Barbieri, JT. 2004. How bacterial ADP-ribosylating toxins recognize
substrates. Nature: Structural and Molecular Biology, 11(9):868-76.
74. Baldwin, M.R., Bradshaw, M, Johnson, E.A., Barbieri, J.T. 2004. C terminus of Botulinum Neurotoxin
Type A Light Chain Contributes to Solubility, Catalysis, and Stability. Prot Expr Purif. 37:187-95.
75. Maresso, AW, Baldwin, MR, and Barbieri, J.T. 2004. ERM proteins are high affinity targets for ADPribosylation by P. aeruginosa ExoS. Journal of Biological Chemistry, 279(37):38402-8.
76. Sun, J-J, Barbieri, J.T. 2004. ExoS RhoGAP activity stimulates reorganization of the actin cytoskeleton
through RhoGDI Journal of Biological Chemistry. 279(41):42936-44.
77. Rao, A.R., Splaingard, MS, Gershan, WM, Havens, PL, Thill, A, Barbieri, JT 2005. Detection of
Pseudomonas aeruginosa Type-III antibodies in children with tracheostomies Pediatr Pulmonol. 39:402-7.
78. Baldwin, MR, Tepp, WH, Pier, CL, Bradshaw, M, Ho, M, Wilson, BA, Fritz, RB, Johnson, EA, and
Barbieri, JT. 2005. Characterization of the Antibody Response to the Receptor Binding Domain of
Botulinum Neurotoxin Serotypes A and E. Infect Immun. 73(10):6998-7005.
79. Zhang Y, and Barbieri , J.T. 2005. A Leucine rich motif targets the Pseudomonas type-III cytotoxin ExoS
for intracellular transport to Golgi-ER. Infect. Immun. 73(12):7938-45.
80. Corech, R, Corech, R, Rao, A, Laxova, A, Moss, J, Rock, MJ, Li, Z, Kosorok, MR, Splaingard, ML, Farrell,
PM, and Barbieri, JT. 2005 Early Immune Response to the Type-III System of Pseudomonas aeruginosa in
Children with Cystic Fibrosis. J Clin Microbiol. 43(8):3956-62.
81. Deng,Q., Sun,JJ, and Barbieri,J.T. 2005. Uncoupling Crk-signal transduction by Pseudomonas ExoT. J
Biol Chem.; 280(43):35953-60.
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83. Chen S, Barbieri JT. 2006. Unique substrate recognition by botulinum neurotoxins serotypes A and E.
J Biol Chem. 281(16):10906-11 .
Biographical Sketches for each listed Senior/Key Person 5
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
84. Boldt GE, Kennedy JP, Hixon MS, McAllister LA, Barbieri JT, Tzipori S, Janda KD. 2006. Synthesis,
characterization and development of a high-throughput methodology for the discovery of botulinum
neurotoxin a inhibitors. J Comb Chem. 8(4):513-21.
85. Fu, Z, Chen, S., Baldwin, MR, Boldt, GE, Crawford, A, Janda, KD, Barbieri, JT, and Kim, JJ. 2006.
Structures of the Light Chain of Botulinum Neurotoxin Serotype A Bound to Small Molecule Inhibitors.
Biochemistry 45(29):8903-11.
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87. Maresso, AW, Pereckas, PS, Wakim, BT, Barbieri, JT. 2007. Pseudomonas aeruginosa ExoS ADPribosyltransferase inhibits ERM phosphorylation. Cellular Microbiology. 9(1):97-105.
XXXXXXXXX
Recent Reviews:
• Barbieri, J.T., Riese, M.J., and Aktories, K 2002. Bacterial toxins that modify the actin cytoskeleton.
Annu. Rev. Cell. Dev. Biol. 18: 315-344.
• Barbieri, J.T. and Burns, D. 2003. Bacterial ADP-ribosylating Exotoxins. In, Bacterial Protein
Toxins. Ed. Burns, D., Barbieri, J.T., Iglewski, B., and Rappuoli, R. ASM Press, Washington, DC.
• Burns, D., Barbieri, J.T. 2003. Dangerous uses of bacterial toxins. In, Bacterial Protein Toxins. Ed.
Burns, D., Barbieri, J.T., Iglewski, B., and Rappuoli, R. ASM Press, Washington, DC.
• Barbieri, J.T.Sun, JJ, and Aktories. 2004. Bacterial toxins that modulate the actin cytoskeleton. In,
Microbial toxin-A critical Review, Ed. Thomas Profit. Horizon Scientific Press.
• Barbieri, J.T. 2004. Damage by Microbial Toxins. In Schaecter’s Mechanisms of Microbial Disease.
Ed. Carrie Engleberg and Victor DiRita. Lippincott Williams and Wilkins.
• Barbieri, J.T., and Sun, J-J. 2004. Pseudomonas ExoS and ExoT., Rev Physiol Biochem Pharmacol
(2004) 152:79–92.
• Baldwin, M.J. and Barbieri, J.T. 2004. The type-III cytotoxins of Yersinia and Pseudomonas
aeruginosa that modulate the actin cytoskeleton. In, Current Topics in Microbiology and
Immunology, Edited by P. Boquet and E. Lemichezl.
• Maresso, AM, Frank, DW, Barbieri, JT. Pseudomonas aeruginosa toxins. 2004. In, The
•
•
Comprehensive Sourcebook of Bacterial Protein Toxins, Third Edition Edited by Joseph E. Alouf and
Michel R.Popoff. Academic Press (London) Aktories, K. and Barbieri, J.T. Cytotoxins, Weapons and
Tools of Bacteria to Modulate Rho and Actin Signal Pathways in Infection. Nature Reviews. 2005.
Barbieri, J.T., Bacterial Toxins That Modify the Epithelial Cell Barrier. 2006. In, Bacterial-Epithelial
Cell Cross-Talk: Molecular Mechanisms in Pathogenesis, Edited by Beth A. McCormick,
Cambridge University Press.
Baldwin MR, Kim JJ, Barbieri JT.. 2007. Botulinum neurotoxin B-host receptor recognition: it takes
two receptors to tango. Nat Struct Mol Biol. 14(1):9-10.
Biographical Sketches for each listed Senior/Key Person 5
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
RESEARCH PROJECTS ONGOING OR COMPLETED DURING THE LAST 3 YEARS
Ongoing
Acellular Vaccines against Bacterial Pathogens
Joseph T. Barbieri, PhD; P.I.
USPHS-NIH RO1, AI30162, Years 14-19, 07/01/05-06/30/10. The major goal of this grant is to characterize
the molecular and cellular properties of ExoS and ExoT of P. aeruginosa.
Vaccines and Therapies against Botulism
Joseph T. Barbieri, PhD; P.I.
USPHS-NIH U54, AI057153-0, 09/01/03 through 6/30/08.
The major goal is to develop novel strategies for the generation of a recombinant holotoxoid and subunit
vaccines against botulinum toxin and to develop strategies for therapeutic intervention to intoxication.
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High-throughput identification of BoNT inhibitors.
Joseph T. Barbieri, PhD; Co-I (Kim Janda, PI)
USPHS-NIH 1 R01 AI066507-01, 02/15/05 through 7/31/07 (no cost extension)
The major goal is to use a high throughput screen to identify small molecule inhibitors of BoNT catalysis.
Molecular pathogenesis of P. aeruginosa in CF
Joseph T. Barbieri, PhD; P.I.
USPHS-NIH R01, HL68912-01, July 1, 2002- June 30, 2007 (no cost extension).
The major goals of this grant are to examine correlations between the immune response to type-III antigens
and clinical outcome of P. aeruginosa infections in children with CF, to engineer type-III apparatus proteins,
and perform a proteomic characterization of P. aeruginosa isolates form children with CF.
Completed (Past 3 years)
Molecular Properties of Yersinia YopT.
Joseph T. Barbieri, PhD; P.I.
USPHS-NIH RO3, AI054435, 04/01/03 through 03/31/05.
The major goal of this grant is to characterize the molecular and cellular properties of YopT of Yersinia
Biographical Sketches for each listed Senior/Key Person 5
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Dorothee Staiger
eRA COMMONS USER NAME
Professor
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Eberhard-Karls-Universitaet, Tuebingen,
Germany
Ludwig-Maximilians-Universitaet, Muenchen
Max-Planck-Institut fuer Zuechtungsforschung,
Koeln, Germany
Max-Planck-Institut für Zuechtungsforschung,
Koeln, Germany
ETH Zuerich, Switzerland
DEGREE
(if applicable)
YEAR(s)
Diploma
1978-1984
1982
FIELD OF STUDY
Biochemistry
Chemistry
Ph. D.
1985-1989
Plant Molecular Biology
Postdoc
1989-1990
Plant Molecular Biology
Research
Associate
1990-2002
Plant Molecular Biology
Please refer to the application instructions in order to complete sections A, B, and C of the Biographical
Sketch.
A. Positions and Honors
Professional Experience
1984-1985
Diplomandin, Max-Planck-Institut fuer Biochemie, Muenchen-Martinsried, Dept. of Prof. Dieter
Oesterhelt
1989-1990
Postdoctoral Research Associate, Max-Planck-Institut fuer Zuechtungsforschung,
Koeln-Vogelsang, Germany, Dept. of Prof. Jeff Schell
1990-1996
Assistent, Institut fuer Pflanzenwissenschaften, ETH Zuerich
1996-2002
Oberassistent, Institut fuer Pflanzenwissenschaften, ETH Zuerich
2002-present Chair of Molecular Cell Physiology at the Faculty of Biology and Institute of Genome Research
and Systems Biology at Bielefeld University, Bielefeld, Germany
Honors
1985-1989
2002
Fellow of the Fritz Thyssen Foundation
Habilitation and Venia Legendi for Plant Biology at ETH Zurich
Ad hoc reviewer for
Chronobiology International, Cellular and Molecular Life Sciences, Molecular and General Genetics,
Nature, Physiologia Plantarum, Planta, Plant Molecular Biology, Plant Physiology, Proceedings of the National
Academy of Sciences USA, The Plant Cell, The Plant Journal, BMC Biology, BBA, Biological Rhythm
Research, Nucleic Acids Research, FEBS Letters,
National Science Foundation, US Department of Agriculture, Israel Science Foundation, United States Israel
Binational Science Foundation, German Research Council, BMBF, Council for the Earth and Life Sciences of
NOW, German Academic Exchange Service, BBSRC
Biographical Sketches for each listed Senior/Key Person 6
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
B. Selected Peer-Reviewed Publications
Neumann H, Gierl A, Tu J, Leibrock J, Staiger D, Zillig W (1983) Organization of the genes for ribosomal RNA
in Archaebacteria. Mol Gen Genet 192, 66-72.
Schreckenbach T, Werenskiold A-K, Staiger D, Allen RG, Bernier F, Lemieux G, Nations C, Palotta D
(1986) Gene expression during plasmodial differentiation. In: The molecular Biology of Physarum
polycephalum. eds.: Dove WF, Dee J, Hatano S, Haugli FB, Wohlfarth-Bottermann K-E; Plenum Publishing
Corporation. pp 131-150.
Staiger D, Kaulen H, Schell J (1989) A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is
recognized by an evolutionarily conserved nuclear protein. Proc Natl Acad Sci USA 86, 6930-6943.
Staiger D, Kaulen H, Schell J (1990) A nuclear factor recognizing a positive regulatory upstream element of the
Antirrhinum majus chalcone synthase promoter. Plant Physiol 93, 1347-1355.
Körber H, Strizhov N, Staiger D, Feldwisch J, Olsson O, Sandberg G, Palme K, Schell J, Koncz C (1991) TDNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone
antagonist in plants. The EMBO J 10, 3983-3991.
Fritze K, Staiger D, Czaja I, Walden R, Schell J, Wing D (1991) Developmental and UV light regulation of the
snapdragon chalcone synthase promoter. The Plant Cell 3, 893-905.
Staiger D, Becker F, Schell J, Koncz C, Palme K (1991) Purification of tobacco nuclear proteins binding to a
CACGTG motif of the chalcone synthase promoter by DNA affinity chromatography. Eur J Biochem 199, 519527.
Staiger D, Apel K (1993) Molecular characterization of two cDNAs from Sinapis alba L. expressed specifically at
an early stage of tapetum development. The Plant Journal 4, 697-703.
Staiger D, Kappeler S, Müller M, Apel K (1994) The proteins encoded by two tapetum-specific transcripts,
Satap35 and Satap44, from Sinapis alba L. are localized in the exine cell wall layer of developing microspores.
Planta 192, 221-23.
Heintzen C, Melzer S, Fischer R, Kappeler S, Apel K, Staiger D (1994) A light- and temperature-entrained
circadian clock controls expression of transcripts encoding nuclear proteins with homology to RNA-binding
proteins in meristematic tissue. The Plant Journal 6, 799-813.
Heintzen C, Fischer R, Melzer S, Kappeler S, Apel K, Staiger D (1994) Circadian oscillations of a transcript
encoding a germin-like protein that is associated with cell walls in young leaves of the long-day plant Sinapis alba
L.. Plant Physiol 106, 905-915.
Staiger D (1996) Clock-controlled transcripts in higher plants. In: Vistas on Biorhythmicity, eds.: H. Greppin, R.
Degli Agosti, M. Bonzon; Geneva. pp 119-133.
Heintzen C, Nater M, Apel, K, Staiger D (1997) AtGRP7, a nuclear RNA-binding protein as a component of a
circadian-regulated negative feedback loop in Arabidopsis thaliana. Proc Natl Acad Sci USA 94, 8515-8520.
Membre N, Berna A, Neuteling G, David A, David H, Staiger D, Vasquez JS, Raynal M, Delseny M, Bernier
F (1997) Sequence, genomic organization and differential expression of three Arabidopsis cDNAs for oxalateoxidase-like proteins. Plant Molec Biol 35, 459-469.
Staiger D, Heintzen C, Zecca L (1999) The RNA-binding protein AtGRP7 is a component of a clockregulated negative feedback circuit in Arabidopsis in: "News from the Plant Chronobiology Research"
Biological Rhythm Research 30, 237-248.
Staiger D, Apel K (1999) Circadian clock-regulated expression of an RNA-binding protein in Arabidopsis:
characterisation of a minimal promoter element.
Mol Gen Genet 261, 811-819.
Staiger D, Apel K, Trepp G (1999) The Atger3 promoter confers circadian clock-regulated transcription with
peak expression at the beginning of the night. Plant Mol Biol 40, 873-882.
Staiger D, Heintzen C (1999) The circadian system of Arabidopsis thaliana - forward and reverse genetic
approaches. Chronobiol Internat 16, 1-16.
Apel K, Melzer S, Staiger D (1999) Biologische Zeit - Gene und die innere Uhr der Pflanzen. ETH Bulletin
272, 26-29.
Staiger D, Heintzen C, Zecca L (1999) The RNA-binding protein AtGRP7 is a component of a clock-regulated
negative feedback circuit in Arabidopsis in: "News from the Plant Chronobiology Research". Biological Rhythm
Research 30, 237-248.
Biographical Sketches for each listed Senior/Key Person 6
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Membre N, Bernier F, Staiger D, Berna A (2000) Arabidopsis thaliana germin-like proteins: common and
specific features point to a variety of functions. Planta 211, 354-354.
Staiger D (2000) Wegbereiter der Pflanzenmolekularbiologie Biologie in unserer Zeit 30, 253.
Staiger D (2000) Biologische Zeitmessung bei Pflanzen. Biologie in unserer Zeit 30, 76-81.
Staiger D (2001) Circadian clocks: CONSTANS lends its zinc finger Trends in Plant Science 6, 293.
McWatters HG, Roden L, Staiger D (2001) Picking out parallels: plant circadian clocks in context.
Philosophical Transactions of the Royal Society 356, 1735-1743.
Staiger D (2001) RNA-binding proteins and circadian rhythms in Arabidopsis thaliana.
Philosophical Transactions of the Royal Society 356, 1755-1759.
Staiger D (2002) Biological timing in Arabidopsis: time for nuclear proteins. Planta 214, 334-344.
Staiger D, Zecca L, Wieczorek Kirk DA, Apel K, Eckstein L (2002) The circadian clock regulated RNAbinding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA.
The Plant Journal 32, 361-371.
Staiger D (2002) Chemical strategies for iron acquisition in plants. Angewandte Chemie International Edition 41,
2259-2264.
Staiger D (2002) Wie gelangt Eisen in die Pflanze? Angewandte Chemie 114, 2363-2368.
Fankhauser C, Staiger D (2002) Photoreceptors in Arabidopsis thaliana: light perception, signal transduction
and entrainment of the endogenous clock. Planta 216, 1-16.
Ziemienowicz A, Haasen D, Staiger D, Merkle T (2003) Arabidopsis transportin1 is the nuclear import
receptor for the circadian clock-regulated RNA-binding protein AtGRP7. Plant Mol Biol 53, 201-212.
Staiger D, Allenbach L, Salathia N, Fiechter V, Davis SJ, Millar AJ, Chory J, Fankhauser C (2003) The
Arabidopsis SRR1 gene mediates phyB signaling and is important for normal circadian clock function. Genes
and Development 17, 256-268.
Staiger D, Zecca L, Wieczorek Kirk DA, Apel K, Eckstein L (2003) The circadian clock regulated RNAbinding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA.
The Plant Journal 33, 361-371.
Frohnmeyer H, Staiger D (2003) Ultraviolet-B radiation (UV-B) mediated responses in plants – balancing
damage and protection. Plant Physiol 133, 1420-1428.
Rudolf F, Wehrle F, Staiger D (2004) Slave to the rhythm. The Biochemist 26, 11-13.
Schöning JC, Staiger D (2005) At the pulse of time: Protein interactions determine the pace of circadian
clocks. FEBS Letters 579, 3246-3252.
Staiger D (2005) Paradigmenwechsel im Verständnis der inneren Uhr. Biologie in unserer Zeit 35, 76-77.
Schöning JC, Streitner C, Staiger D (2005) Clockwork Green – the circadian oscillator in Arabidopsis.
Biological Rhythm Research 37, 335-352.
Staiger D (2005) Am Puls des Lebens: Biologische Zeitmessung bei Arabidopsis thaliana. BIOforum 28, 5355.
Nodop A, Suzuki I, Barsch A, Schröder AK, Niehaus K, Staiger D, Pistorius EK, Michel KP (2006)
Physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking the histidine
kinase Slr1759 and the response regulator Slr1760. J. Biosciences 61c, 865-878.
Staiger D, Streitner C, Rudolf F, Huang X (2006) Multiple and slave oscillators. In: Endogenous Plant
Rhythms, eds: Hall A, McWatters H. Blackwell Publishing, 57-83.
Schöning JC, Staiger D (2006) Being in time: the importance of posttranslational processes in circadian
clocks. BIO TECH international 18, 12-15.
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Biographical Sketches for each listed Senior/Key Person 6
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
C. Research Support
Ongoing Research Support
German Research Council STA 653/1 Research Group FOR 387 Project 7
(2003-2007)
Redox-Regulation der Wechselbeziehung zwischen Photosynthese, Respiration und N-Stoffwechsel
in Cyanobakterien
Role: Principle Investigator
German Research Council STA 653/2-1 und 2-2
(2004-2007)
Functional characterisation of a gene family encoding circadian regulated glycine-rich RNA-binding
proteins in Arabidopsis thaliana
Role: Principle Investigator
German Research Council Collaborative Research Program SFB613 Project D7
(2005-2008)
Aufklärung des molekularen Mechanismus eines circadianen “slave“ Oszillators mittels Einzelmolekülfluoreszenz- und Rasterkraftmikroskopie
Role: Principle Investigator
Bundesministerium für Verbraucherschutz, Ernährung und Landwirtschaft, Fachagentur für
nachwachsende Rohstoffe FKZ 22009402
(2005-2007)
Produktion von biologisch abbaubaren Polymeren in transgenen Kartoffelknollen Phase II
Teilvorhaben 3: Untersuchungen zum Nachweis und zur Optimierung der CyanophycinProduktion in transgenen Pflanzen
Role: Principle Investigator
Completed Research Support (within the last 3 years)
ETH Research Commission TH-34./00-3
Molecular mechanism of a clock-regulated feedback loop based on an RNA-binding protein in Arabidopsis
Role: Principle Investigator
Schweizerischer Nationalfonds 31-52475
Identification of transcripts with altered expression in transgenic plants overexpressing the RNA-binding
protein AtGRP7, a component of the circadian system in Arabidopsis
Role: Principle Investigator
Functional Genomics Center Zuerich
Identification of target transcripts of the circadian clock-regulated RNA-binding protein AtGRP7 in Arabidopsis
thaliana
Role: Principle Investigator
FIF4-Project Faculty for Biology, University of Bielefeld
Molekulare und biochemische Charakterisierung zweier neuer Proteine, die unter Kontrolle des
circadian regulierten RNA-Bindungsprotein AtGRP7 stehen und möglicherweise am Lipidstoffwechsel
bei Arabidopsis thaliana beteiligt sind
Role: Principle Investigator
Biographical Sketches for each listed Senior/Key Person 6
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
BIOGRAPHICAL SKETCH
Provide the following information for the key personnel and other significant contributors in the order listed on Form Page 2.
Follow this format for each person. DO NOT EXCEED FOUR PAGES.
NAME
POSITION TITLE
Xia, Yuannan
eRA COMMONS USER NAME
Research Assistant Professor
EDUCATION/TRAINING (Begin with baccalaureate or other initial professional education, such as nursing, and include postdoctoral training.)
INSTITUTION AND LOCATION
Peking University
China University of Science & Technology
University of Nebraska-Lincoln
DEGREE
(if applicable)
B.S.
M.S.
Ph.D.
YEAR(s)
1980
1982
1985
FIELD OF STUDY
Biochemistry
Virology
Biological Sciences
A. Positions and Employment
1982-1985 Research Assistant, Department of Plant Pathology, University of Nebraska-Lincoln
1986-1987 Postdoctoral Research Fellow, Department of Plant Pathology, University of Nebraska-Lincoln
1988-1989 Postdoctoral Research Fellow, Department of Biochemistry, University Laval
1990-1992 Research Associate, Department of Microbiology,University of Guelph.
1993-2002 Senior Research Scientist and Research Director of Molecular Biology, Restoragen Inc
2002-present Research Assistant Professor, Manager of Genomic Core Research Facility,
Center for Biotechnology,University of Nebraska-Lincoln.
B. Selected peer-reviewed publications (in chronological order)
Skrdla,M. P., D. E. Burbank, Y. Xia, R. H. Meints, and J. L. Van Etten. 1984. Structural proteins and lipids in a
virus, PBCV-1, which replicates in a Chlorella-like alga. Virology 135:308-315.
Van Etten, J. L.,Y. Xia, and R. H. Meints. 1986. Viruses of a Chlorella-like green alga. In: T. Kosuge and E.
W. Nester(ed.), Plant-Microbe Interactions, vol. II. pp.307-325, Macmillan Publishing Co. New York.
Xia, Y.,D. E. Burbank, Uher, D. Rabussay, and J. L. Van Etten. 1986. Restriction endonuclease activity
induced by PBCV-1 virus infection of a Chlorella-like green alga. Mol. Cell. Biol. 6:1430-1439.
Xia, Y. and J. L. Van Etten. 1986. DNA methyltransferase induced by PBCV-1 virus infection of a Chlorellalike green alga. Mol. Cell. Biol. 6:1440-1445.
Van Etten,J. L.,Y. Xia, K. E. Narva, and R. H. Meints. 1986. Chlorella algal viruses. In: G. Fink, R.Wickner, A.
Hinnebusch, A. Lambowitz, L. Mets, I. Gunsalus, and A. Hollaender(ed.). Extrachromosomal Elements in
Lower Eukaryotes. pp. 337-347. Plenum Press, New York.
Xia, Y., D. E. Burbank, and J. L. Van Etten. 1986. Restriction endonuclease activity induced by NC-1A virus
infection of a Chlorella-like green alga. Nucleic Acids Res. 14:6017-6030.
Xia, Y., D. E. Burbank, L. Uher, D. Rabussay,and J. L. Van Etten. 1987. Il-3A virus infection of a Chlorellalike green alga induced a DNA restriction endonuclease with novel sequence specificity. Nucleic Acids
Res. 15:6075-6090.
Xia, Y.1987. Eukaryotic algal viruses. In: Viruses and Agriculture. P. Tien and Z. Gong(ed.). Science Press,
Beijing,China. pp. 247-257.
Xia, Y.,K. E. Narva, and J. L. Van Etten. 1987. The cleavage site of the RsaI isoschizomer, CviQI, is G/TAC.
Nucleic Acids Res. 15:10063.
Xia, Y.,R. Morgan, I. Schildkraut, and J. L. Van Etten. 1988. A site-specific single strand endonuclease
activity induced by NYs-1 virus infection of a Chlorella-like green alga. Nucleic Acids Res. 16:9477-9487.
Van Etten, J. L.,Y. Xia,D. E. Burbank, and K. E. Narva. 1988. Chlorella viruses code for restriction and
modification enzymes. Gene 74:113-115.
Labbe, S., Y. Xia and P.H. Roy. 1988. BspMII and AccIII are an isoschizomer pair which differ in their
sensitivity to cytosine methylation. Nucleic Acids Res. 16:7184.
Biographical Sketches for each listed Senior/Key Person 7
Page 31
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Stefan, C., Y. Xia, and J. L. Van Etten. 1991. Molecular cloning and characterization of the gene encoding
the adenine methyltransferase, M.CviRI, from Chlorella virus XZ-6E. Nucleic Acids Res. 19:307-311.
Xia, Y., Y. Ling, P. Dobos, J. L. Van Etten, and P. J. Krell. 1993. Adenine DNA methyltransferase M.CviRI
expression accelerates apoptosis in baculovirus-infected insect cells. Virology 196:817-824.
Jin, A.,Y. Zhang, Y. Xia, E. Traylor, M. Nelson, and J. L. Van Etten. 1994. New restriction endonuclease
CviRI cleaves DNA at TG/CA sequences. Nucleic Acids Res. 22:3928-3929.
Zhang,Y., M. Nelson, J. Nietfeldt, Y. Xia, D. Burbank, S. RoRopp, and J. L. Van Etten. 1998. Chlorella virus
NY-2A encodes at least 12 DNA endonuclease/methytransferase genes. Virology 240:366-375.
Heinrich, J., Y. Xia, P. Chadha-Mohanty, F.W. Wagner, E.H.Grotjan. 1999. Bioassay for growth hormone
releasing hormone (GHRH) using a recombinant receptor and cAMP-responsive reporter system.
Molecular and Cellular Endocrinology 150:65-72.
Graziani S, Y. Xia , J.R. Gurnon, J.L.Van Etten , D. Leduc, S. Skouloubris, H. Myllykallio, U. Liebl. 2004.
Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria Chlorella
virus-1. J Biol Chem. 279(52):54340-7.
Alvarez-Venegas R., M. Sadder, A. Hlavacka, F. Balu ka, Y. Xia, G. Lu, A. Firsov, G. Sarath, H. Moriyama, J.
G. Dubrovsky, and Z. Avramova . 2006. The Arabidopsis homolog of trithorax, ATX1, binds
phosphatidylinositol 5-phosphate, and the two regulate a common set of target genes. PNAS 103: 60496054.
Alvarez-Venegas R., Y. Xia, G. Lu and Z. Avramova. 2006. Phosphoinositide 5-Phosphate and
Phosphoinositide 4-Phosphate Trigger Distinct Specific Responses of Arabidopsis Genes; Genome-Wide
Expression Analyses. Plant Signaling & Behavior 1(3): 134-139.
LaRosa P.C.,J. Miner, Y. Xia,Y. Zhou, S. Kachman and M. E. Fromm. 2006. Histological and Microarray
Analysis of the Delipidation of White Adipose Tissue of Mice Fed Conjugated Linoleic Acid Physiological
Genomics 27:282-294.
G. Lu, T. Nguyen, Y. Xia, M. Fromm. 2006. AffyMiner: mining significant genes from Affymetrix microarray
data. BMC Bioinformatics. 7 (Suppl 4): S26; http://biocore.unl.edu/affyminer/.
Alvarez-Venegas R, M.Yielmaz1, O. Le, Q. Hou, M. Sadder, A. Al-Abdallat, Y. Xia, G. Lu, and
Z.Avramova.2007. ATX1 and ATX2, Two Highly Related Homologs are Differentially Expressed and
Control Largely Non-overlapping Sets of Arabidopsis Genes. (submitted to Plant Physiology).
C. Current Research Support
NSF EPSCoR EPS-0346476 Fromm (PI)
2004-2007
Metabolite Signaling Center.
The goal of this study is to better understand the response of molecules (metabolites) in the diet and the
molecular response to these chemicals in mammals, for the purpose of improving human health.
Role: investigator
NRI Strategic Research Grant Fromm (PI)
2006-2008
Plant Chromatin Biology.
The goal of this study is to obtain genome-wide data on the interactions between chromation binding proteins
and the genome. This information is correlated with gene expression changes to create chromatin-based gene
regulation models.
Role: investigator.
Biographical Sketches for each listed Senior/Key Person 7
Page 32
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
PHS 398 Cover Page Supplement
OMB Number: 0925-0001
Expiration Date: 9/30/2007
1. Project Director / Principal Investigator (PD/PI)
Prefix:
* First Name: James
Dr.
Middle Name: Robert
* Last Name:
Alfano
Suffix:
* New Investigator?
Degrees:
● No
❍Yes
BS
PhD
2. Human Subjects
Clinical Trial?
● No
❍Yes
* Agency-Defined Phase III Clinical Trial?
❍No
❍Yes
3. Applicant Organization Contact
Person to be contacted on matters involving this application
Prefix:
* First Name: Nancy
Middle Name:
* Last Name:
Becker
Suffix:
* Phone Number: 402-472-3601
Fax Number: 402-471-9323
Email: [email protected]
* Title:
University Grants Coordinator
* Street1:
312 North 14th Street
Street2:
Alexander Bldg. West
* City:
County:
* State:
Lincoln
Lancaster
NE: Nebraska
Province:
* Country:
USA:
* Zip / Postal Code:
Clinical Trial & HESC
Tracking Number:
68588-0430
Page 33
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
PHS 398 Cover Page Supplement
OMB Number: 0925-0001
Expiration Date: 9/30/2007
4. Human Embryonic Stem Cells
* Does the proposed project involve human embryonic stem cells?
●No
❍Yes
If the proposed project involves human embryonic stem cells, list below the registration number of the
specific cell line(s) from the following list: http://stemcells.nih.gov/registry/index.asp . Or, if a specific
stem cell line cannot be referenced at this time, please check the box indicating that one from the registry will be used:
Cell Line(s):
❏
Specific stem cell line cannot be referenced at this time. One from the registry will be used.
Clinical Trial & HESC
Tracking Number:
Page 34
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
PHS 398 Modular Budget, Periods 1 and 2
OMB Number: 0925-0001
Expiration Date: 9/30/2007
Budget Period: 1
Start Date: 07/01/2007
End Date: 06/30/2008
A. Direct Costs
Funds Requested ($)
* Direct Cost less Consortium F&A
Consortium F&A
0.00
* Total Direct Costs
250,000.00
B. Indirect Costs
Indirect Cost Type
1. Modified Total Direct Costs
250,000.00
Indirect Cost
Rate (%)
47.50
Indirect Cost
Base ($)
* Funds Requested ($)
238,800.00
113,430.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) Dept. Health and Human ServicesPeter Nwaogu 214/767-3764
Indirect Cost Rate Agreement Date
02/07/2007
C. Total Direct and Indirect Costs (A + B)
Total Indirect Costs
113,430.00
Funds Requested ($)
363,430.00
Budget Period: 2
Start Date: 07/01/2008
End Date: 06/30/2009
Funds Requested ($)
A. Direct Costs
250,000.00
* Direct Cost less Consortium F&A
Consortium F&A
* Total Direct Costs
B. Indirect Costs
Indirect Cost Type
1.
Modified Total Direct Costs
Indirect Cost
Rate (%)
47.50
Indirect Cost
Base ($)
250,000.00
* Funds Requested ($)
238,464.00
113,271.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) Dept. Health Human ServicesPeter Nwaogu214/767-3764
Indirect Cost Rate Agreement Date
02/07/2007
C. Total Direct and Indirect Costs (A + B)
Modular Budget
Tracking Number:
Total Indirect Costs
Funds Requested ($)
Page 35
113,271.00
363,271.00
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
PHS 398 Modular Budget, Periods 3 and 4
OMB Number: 0925-0001
Expiration Date: 9/30/2007
Budget Period: 3
Start Date: 07/01/2009
End Date: 06/30/2010
A. Direct Costs
Funds Requested ($)
* Direct Cost less Consortium F&A
250,000.00
Consortium F&A
* Total Direct Costs
250,000.00
B. Indirect Costs
Indirect Cost Type
1. Modified Total Direct Costs
Indirect Cost
Rate (%)
47.50
Indirect Cost
Base ($)
* Funds Requested ($)
238,118.00
113,106.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) Dept. Health Human ServicesPeter Nwaogu214/767-3764
Indirect Cost Rate Agreement Date
02/07/2007
C. Total Direct and Indirect Costs (A + B)
Total Indirect Costs
113,106.00
Funds Requested ($)
363,106.00
Budget Period: 4
Start Date: 07/01/2010
End Date: 06/30/2011
Funds Requested ($)
A. Direct Costs
* Direct Cost less Consortium F&A
250,000.00
Consortium F&A
* Total Direct Costs
B. Indirect Costs
Indirect Cost Type
1.
Modified Total Direct Costs
Indirect Cost
Rate (%)
47.50
Indirect Cost
Base ($)
250,000.00
* Funds Requested ($)
237,761.00
112,937.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) Dept. Health Human ServicesPeter Nwaogu214/767-3764
Indirect Cost Rate Agreement Date
02/07/2007
C. Total Direct and Indirect Costs (A + B)
Modular Budget
Tracking Number:
Total Indirect Costs
Funds Requested ($)
Page 36
112,937.00
362,937.00
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
PHS 398 Modular Budget, Period 5 and Cumulative
OMB Number: 0925-0001
Expiration Date: 9/30/2007
Budget Period: 5
Start Date: 07/01/2011
End Date: 06/30/2012
A. Direct Costs
Funds Requested ($)
* Direct Cost less Consortium F&A
250,000.00
Consortium F&A
* Total Direct Costs
250,000.00
B. Indirect Costs
Indirect Cost
Rate (%)
Indirect Cost Type
1.
Modified Total Direct Costs
47.50
Indirect Cost
Base ($)
237,394.00
* Funds Requested ($)
112,762.00
2.
3.
4.
Cognizant Agency (Agency Name, POC Name and Phone Number) Dept. Health Human ServicesPeter Nwaogu214/767-3764
Indirect Cost Rate Agreement Date
C. Total Direct and Indirect Costs (A + B)
Total Indirect Costs
112,762.00
Funds Requested ($)
362,762.00
Cumulative Budget Information
1. Total Costs, Entire Project Period
* Section A, Total Direct Cost less Consortium F&A for Entire Project Period
$
1,250,000.00
Section A, Total Consortium F&A for Entire Project Period
$
0.00
* Section A, Total Direct Costs for Entire Project Period
$
1,250,000.00
* Section B, Total Indirect Costs for Entire Project Period
$
565,506.00
* Section C, Total Direct and Indirect Costs (A+B) for Entire Project Period
$
1,815,506.00
2. Budget Justifications
8677-Personnel_Justification.pdf
Personnel Justification
Consortium Justification
Additional Narrative Justification
Modular Budget
Tracking Number:
Page 37
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Attachments
PersonnelJustification_attDataGroup0
File Name
8677-Personnel_Justification.pdf
Mime Type
application/pdf
ConsortiumJustification_attDataGroup0
File Name
Mime Type
AdditionalNarrativeJustification_attDataGroup0
File Name
Mime Type
Modular Budget
Tracking Number:
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
PERSONNEL JUSTIFICATION
James R. Alfano, Ph.D., Principle Investigator (33% effort). Dr. Alfano will dedicate 33% effort.
He will be involved in directing all aspects of the experiments outlined in this application. Dr.
Alfano has worked on type III secretion systems since 1993. His area of expertise is bacterial
genetics, molecular microbiolgy and plant molecular biology.
Anna Block, Ph.D., Postdoctoral Researcher (100% effort). Dr. Block has much experience with
plant-microbe interaction research. She will perform many of the experiments in Aim 2.
Emerson Crabill, Ph.D. Graduate Student Researcher (100% effort). Mr. Crabill will work on
aspects of Aim 3.
Thomas Elthon, Ph.D., Associate Professor and Leader of the UNL Proteomic Facility (10%
effort). Dr. Elthon helps direct and oversees personnel working within the UNL Proteomic
Facility. He has been a very important resource for this project because of his experience with
protein chemistry. Because he handles other projects in his facility, it is difficult to predict the
amount of his effort on this project.
Byeong-ryool Jeong, Research Assistant Professor (100% effort). Dr. Jeong is exceptional with
RNA research. He will perform all of the microarray experiments and RT-PCR experiments in
Aim 3 and several experiments within Aim 1.
Andrew Karpisek, B.S., Technician. Andrew will assist in all the experiments and order needed
reagents and other lab manager duties.
Fang Tian, M.S., Ph.D. Graduate Student Researcher (100% effort). Mrs. Tian is an exceptional
student and will play a leading role on this project and will work on objectives within each Aim.
Personnel Justification
Page 39
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
OMB Number: 0925-0001
Expiration Date: 9/30/2007
PHS 398 Research Plan
1. Application Type:
From SF 424 (R&R) Cover Page and PHS398 Checklist. The responses provided on these pages, regarding the type of application being submitted, are repeated for your reference, as you attach the appropriate sections of the research plan.
*Type of Application:
❍ New
● Resubmission
❍ Renewal
❍ Continuation
❍ Revision
2. Research Plan Attachments:
Please attach applicable sections of the research plan, below.
1. Introduction to Application
3829-Introduction_to_Application.pdf
(for RESUBMISSION or REVISION only)
2. Specific Aims
2279-Specific_Aims.pdf
3. Background and Significance
0041-Background_and_Significance.pdf
4. Preliminary Studies / Progress Report
270-Preliminary_Studies.pdf
5. Research Design and Methods
3186-AlfanoResearch_Design_and_Methods.pdf
6. Inclusion Enrollment Report
7. Progress Report Publication List
Human Subjects Sections
Attachments 8-11 apply only when you have answered "yes" to the question "are human subjects involved" on the R&R Other Project Information
Form. In this case, attachments 8-11 may be required, and you are encouraged to consult the Application guide instructions and/or the specific
Funding Opportunity Announcement to determine which sections must be submitted with this application.
8. Protection of Human Subjects
9. Inclusion of Women and Minorities
10. Targeted/Planned Enrollment Table
11. Inclusion of Children
Other Research Plan Sections
12. Vertebrate Animals
5644-12._Vertebrate_Animals.pdf
13. Select Agent Research
173-13._Select_Agent_Research.pdf
14. Multiple PI Leadership
8666-14._Multiple_PD_PI_Leadership_Plan.pdf
15. Consortium/Contractual Arrangements
5371-15._Consortium_Contractual_Arrangements.pdf
16. Letters of Support
8067-CombinedSupportLetters.pdf
17. Resource Sharing Plan(s)
191-17._Resource_Sharing.pdf
18. Appendix
List of Research Plan Attachments
Tracking Number:
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Attachments
IntroductionToApplication_attDataGroup0
File Name
3829-Introduction_to_Application.pdf
Mime Type
application/pdf
SpecificAims_attDataGroup0
File Name
2279-Specific_Aims.pdf
Mime Type
application/pdf
BackgroundSignificance_attDataGroup0
File Name
0041-Background_and_Significance.pdf
Mime Type
application/pdf
ProgressReport_attDataGroup0
File Name
270-Preliminary_Studies.pdf
Mime Type
application/pdf
ResearchDesignMethods_attDataGroup0
File Name
3186-AlfanoResearch_Design_and_Methods.pdf
Mime Type
application/pdf
InclusionEnrollmentReport_attDataGroup0
File Name
Mime Type
ProgressReportPublicationList_attDataGroup0
File Name
Mime Type
ProtectionOfHumanSubjects_attDataGroup0
File Name
Mime Type
InclusionOfWomenAndMinorities_attDataGroup0
File Name
Mime Type
TargetedPlannedEnrollmentTable_attDataGroup0
File Name
Mime Type
InclusionOfChildren_attDataGroup0
File Name
Mime Type
VertebrateAnimals_attDataGroup0
File Name
5644-12._Vertebrate_Animals.pdf
Mime Type
application/pdf
SelectAgentResearch_attDataGroup0
File Name
173-13._Select_Agent_Research.pdf
Mime Type
application/pdf
MultiplePILeadershipPlan_attDataGroup0
File Name
8666-14._Multiple_PD_PI_Leadership_Plan.pdf
Mime Type
application/pdf
ConsortiumContractualArrangements_attDataGroup0
File Name
5371-15._Consortium_Contractual_Arrangements.pdf
Mime Type
application/pdf
LettersOfSupport_attDataGroup0
File Name
8067-CombinedSupportLetters.pdf
Mime Type
application/pdf
List of Research Plan Attachments
Tracking Number:
Page 41
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
ResourceSharingPlans_attDataGroup0
File Name
191-17._Resource_Sharing.pdf
Mime Type
application/pdf
Appendix
File Name
Mime Type
List of Research Plan Attachments
Tracking Number:
Page 42
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
1. Introduction to Application
This application is the second resubmission. It was reviewed by the Host Interactions with
Bacterial Pathogens Study Section on June 29, 2006. I was pleased that the reviewers noted
that the application was “highly significant” and a “strong application” and that it was much
improved over the previous submission (The original submission was viewed as “meritorious
and solid”). I thank these reviewers for providing many insightful comments that have helped us
improve and sharpen the hypotheses described in the earlier resubmission. To help reviewers
discriminate between text from the last submission and text added to this one, I have added
side-bars to the left margin to indicate where the application has been significantly changed.
Most of the Figures in the previous submission have been modified by the addition of new data
and in several instances have been combined with other Figures to save space.
We have added new data to this application that greatly strengthens the significance of
this research. The most significant new data are the following: The identification of Arabidopsis
thaliana T-DNA Atgrp7 mutant that displayed enhanced susceptibility to Pseudomonas syringae
and reduced callose deposition (Fig. 6). This indicates that the glycine-rich RNA-binding protein
AtGRP7, a HopU1 ADP-ribosyltransferase (ADP-RT) substrate, contributes to plant innate
immunity. Additionally, we demonstrate that HopU1-His ADP-ribosylates the RNA recognition
motif (RRM) domain of AtGRP7, but not the glycine-rich domain (Fig. 5C). Subsequently, we
have individually substituted lysine for each arginine residue in the RRM domain of full length
AtGRP7 (fused to GST) and have observed that two arginines (at positions 47 and 49) are
required for the ADP-ribosylation of AtGRP7 by HopU1-His. Based on comparison with
structures of RRM domains from other RNA-binding proteins, both of these residues would be
solvent-exposed and, therefore, potentially accessible to HopU1. Interestingly, arginine 49 is in
the most conserved region of the RRM domain and this residue has been implicated in RNAbinding of other RNA-binding proteins. Thus, our hypothesis is that ADP-ribosylation of AtGRP7
by HopU1 reduces or abolishes its ability to bind RNA. We believe these new results as well as
the other changes that we have made have strengthened this application.
Essentially, the main negative comment in the Summary Statement was that the study
section was not convinced that the Aim seeking to identify the in vitro and in vivo animal targets
for HopU1, localize HopU1 in animal cells, and to determine whether HopU1 suppresses animal
innate immunity would be biological interesting and questioned whether this effort would be
better spent on the more pressing issue of identifying HopU1 targets in plants. In this
resubmission I have taken this criticism to heart and have deleted Aim 3. I did retain entry level
experiments that seek to identify the in vitro substrates of HopU1 in animal cells as a sub-aim of
Aim 2. I must note that I remain excited about these experiments because even if we find that
HopU1 does not suppress animal innate immunity, HopU1 still may be useful as a
pharmacological or therapeutic tool if it disables the function of an important animal protein. We
have all of the techniques up and running in my laboratory and, therefore, this sub-aim should
not be a significant distraction to the overall project. Identifying the in vitro substrates in animal
cells lines would be a good stopping point to evaluate whether more examination of HopU1’s
affect on animal innate immunity is warranted.
Because we now have strong evidence that one of the targets of HopU1, AtGRP7, is
involved in innate immunity we have added a new specific aim (Aim 1) focused entirely on the
consequence of its ADP-ribosylation and the role this protein plays in innate immunity. At the
time of our last submission we were unsure about AtGRP7’s involvement in innate immunity and
I thought it might have been “jumping the gun” to propose detailed experiments to explore this
even though the direction of the research was clear. The other two Aims have remained largely
unchanged except for minor adjustments. Aim 2 is focused more on identifying additional
substrates of HopU1 and determining whether they are likely to be in vivo targets, while Aim 3
(formerly Aim 1 in the previous submission) is focused on the effect of HopU1 on plant-microbe
interactions and plant innate immunity.
Introduction
Page 43
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Another important update for this application is that two weeks ago most of the data in
the preliminary data section of this application was accepted in principle as an Article in the
journal Nature. Only days ago, I submitted a revised manuscript and await the editor’s final
decision, which I believe will be based on production issues (e.g., page limits, etc.) and not
scientific ones. I am grateful to NIH because the original application was awarded an R56 grant,
which allowed us to produce the additional data needed for multiple NIH submissions pertaining
to this project, which ultimately led to this manuscript. Without this award, we would most likely
have needed to publish a less complete story much sooner and in a journal less prestigious.
In summary, this new submission is more focused because of our extensive preliminary
data. The application is novel for several reasons. HopU1 is the first ADP-RT identified in a
plant pathogen, it has novel substrates for ADP-RTs, and these RNA-binding protein substrates
suggest a novel pathogenic strategy utilized by bacterial pathogens to modulate plant innate
immunity by affecting RNA metabolism and the plant defense transcriptome. This strategy is
likely not limited to plant pathogens because IpaH9.8, a type III effector from the animal
pathogen Shigella flexneri was recently shown to bind the RRM-containing mammalian splicing
factor U2AF35 resulting in the suppression of pro-inflammatory cytokines (150). While IpaH9.8’s
enzymatic activity is presently unknown, it suggests that animal pathogens may alter RNA
metabolism in a similar manner to suppress the eukaryotic immune response. Therefore, these
studies should be relevant to the important mission of the NIH
Introduction
Page 44
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Research Plan
2. Specific Aims
Eukaryotic innate immune systems act as effective barriers to infection by microorganisms.
Understanding the mechanisms that bacterial pathogens employ to circumvent innate immune
systems will improve our ability to control disease. Plants and animals use specific pattern
recognition receptors (PRRs) to recognize conserved molecules of microorganisms (known as
PAMPs). Plants have numerous PRRs that can recognize specific virulence proteins specifically
present in pathogens (known as Avr proteins). Many Gram-negative bacteria use type III protein
secretion systems to inject effector proteins into host eukaryotic cells. We have shown that a
primary role for many Pseudomonas syringae type III effectors is to suppress innate immunity.
However, the enzymatic activities and the mechanisms that type III effectors use to suppress
innate immunity are not well understood. Identifying the enzymatic activities of type III effectors
and their substrates is essential to identify important components of innate immunity and to
improve strategies to control bacterial diseases.
Our long-term goal is to elucidate the molecular basis for suppression of innate immunity
by type III effectors. The objective of this application is to identify targets of the P. syringae type
III effector HopU1, a mono-ADP-ribosyltransferases (ADP-RTs), and to determine its roles in
bacterial pathogenesis. The central hypothesis of the proposed experiments is that the targets
of the HopU1 ADP-RT type III effector will be components of innate immunity. We formulated
this hypothesis based on the literature and on our research on other type III effectors as well as
our preliminary data showing that HopU1 suppresses outputs of innate immunity. Recently, we
have shown that HopU1 can use several Arabidopsis RNA-binding proteins as high affinity
substrates in in vitro ADP-RT assays. Based on our preliminary data, one of these proteins,
AtGRP7, plays a role in innate immunity. A major goal of this application is to elucidate the
function of this protein as it relates to innate immunity. We are prepared to undertake the
proposed research because we have extensive experience in manipulating type III systems, and
we were among the first to report that certain type III effectors suppress innate immunity. In
addition, our preliminary identification of HopU1’s substrates has positioned us well to perform
the experiments described in this application. Our research team includes experts in the
following areas: type III secretion systems, proteomics and mass spectrometry, Affymetrix
microarrays, plant glycine-rich RNA-binding proteins, and animal pathogen ADP-RTs. This
qualified group of investigators will insure that our discoveries are linked to basic concepts of
pathogenesis and immunity in both plants and animals.
The Specific Aims of this application are as follows:
1. Determine the molecular consequence of ADP-ribosylation on the function of AtGRP7
and elucidate the role this protein plays in innate immunity. Our working hypothesis of this
aim is that AtGRP7 binds to immunity-related RNAs to enhance the innate immune response
and that ADP-ribosylation by HopU1 disrupts its function.
2. Identify additional substrates of HopU1 and verify their involvement in innate
immunity. Our working hypothesis is that the plant targets for the HopU1 ADP-RTs will be
important components of plant innate immunity.
3. Analyze the affect that HopU1 has on host-microbe interactions. Our working hypothesis
of this aim is that HopU1 type III effector suppresses innate immunity. This is based on our
preliminary data and in this aim we will determine to what extent this occurs with HopU1.
The proposed research is innovative because, to date, ADP-RTs have not been implicated in
the suppression of innate immune surveillance systems. Moreover, RNA-binding proteins have
not been described as substrates for ADP-RTs and, therefore, represent novel substrates for
this important group of bacterial toxins. Collectively, we expect the outcomes of these
experiments will greatly add to our understanding of the activities and roles of type III effectors,
particularly in how they suppress innate immunity in eukaryotes.
Specific Aims
Page 45
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
3. Background and Significance
3.a. Significance of the proposed research. The ability to avoid surveillance by eukaryotic
innate immune systems was likely a critical development in the evolution of bacterial
pathogenicity. We now know that one strategy bacteria use to circumvent the innate immune
system is by injecting effectors via their type III protein secretion systems (TTSSs). This
application seeks to exploit the power of the Pseudomonas syringae – Arabidopsis model
system to better understand innate immunity in eukaryotes and to understand how type III
effectors disable it. Because there are considerable similarities between the innate immune
systems in plants and mammals (see below) we expect that our findings will be relevant to the
mission of the NIH and be broadly interesting to researchers studying molecular mechanisms of
bacterial pathogenesis. One only needs to consider how the discovery of the Toll receptor in
Drosophila facilitated the discovery of Toll-like receptors in mammals (119, 135) or how
research on plant resistance proteins provided important clues that the nucleotide-binding
oligomerization domain proteins (96, 147) were involved in perception of microorganisms, to
appreciate how research in non-mammalian model systems can lead to a better understanding
of conserved biological processes. In the experiments described in this proposal we will explore
how a P. syringae type III effector suppresses innate immunity with the expectation that what we
learn about this process in plants will contribute to a better understanding of innate immunity in
animals. Our focus is on a mono-ADP-ribosyltransferase (ADP-RT) type III effector from P.
syringae and its role in pathogenesis. ADP-RTs have been well studied in animal pathogens
where they modulate several processes in eukaryotes including protein synthesis, adenylate
cyclase, and actin polymerization (16). Here we present preliminary data suggesting that the P.
syringae HopU1 ADP-RT can suppress plant innate immunity. The mechanism that HopU1 uses
to suppress innate immunity appears to be novel compared to animal pathogen ADP-RTs
because HopU1 can use RNA-binding proteins as substrates – a class of proteins not
previously identified to be ADP-RT substrates. Therefore, experiments described in this
proposal will likely increase our knowledge of this important group of bacterial toxins. Moreover,
recently non-ADP-RT type III effectors from animal and plant pathogens have been shown to
suppress innate immune surveillance systems (39, 51). While progress has been made on how
individual type III effectors accomplish this, there is a significant gap of knowledge in how
surveillance systems are breached by type III effectors. We expect that the experiments
proposed in this application will identify how this is accomplished by the HopU1 ADP-RT type III
effector.
3.b. Innate immunity surveillance in eukaryotes. Plants induce basal defense responses
upon sensing conserved molecules produced by microorganisms (22). These include chitin (55)
and ergosterol (74) from fungi and lipopolysaccharide (47), cold shock protein (54), EF-Tu
(191), and flagellin from bacteria (70). These molecules have been referred to as general
elicitors by plant pathologists, but they are conceptually equivalent to pathogen-associated
molecular patterns (PAMPs), molecules present in microbes that are recognized by the innate
immune system in animals (22, 134). PAMPs are detected in animals by specific host pattern
recognition receptors (PRRs), which are Toll-like receptors (TLRs) and nucleotide-binding
oligomerization domain (NOD) proteins (96, 133). TLRs generally sense extracellular PAMPs,
whereas NODs recognize PAMPS intracellularly (68). A similar dichotomy is seen with plant
PRRs. A plant receptor-like kinase, FLS2, recognizes bacterial flagellin from the outside of plant
cells, whereas plant resistance (R) proteins recognize bacterial avirulence (Avr) proteins
intracellularly. In addition, other similarities between plant and animal PRRs have been well
documented (44, 96, 102, 133, 146, 147).
3.c. Innate immunity signal transduction and outputs. Once animal or plant PRRs perceive
PAMPs or Avr proteins signal transduction pathways are triggered and defense-related outputs
Background & Significance
Page 46
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
are deployed. In plants, many of the signal transduction components acting downstream of Avr
protein recognition are dependent on salicylic acid (SA) (129, 143). The emerging picture is that
a complex signal network is triggered upon perception of an Avr protein. Components of signal
transduction pathways operating downstream of PAMP perception in both plants and animals
are not well understood. However, there are common features shared by both (36, 92, 146,
147). For example, mitogen-activated protein kinase (MAPK) cascades are activated in plants
and animals following PAMP perception (147). In both hosts, transcription factors are activated
following activation of MAPK – the NF-κB transcription factor in animals and WKRY transcription
factors in plants are good examples (13, 107, 118, 192). The physiological responses that occur
in plants after activation of innate immunity include generation of reactive oxygen species (ROS)
and nitric oxide (NO), changes in cytoplasmic Ca2+ levels, production of pathogen-related (PR)
gene expression, phytoalexin production, papillae formation (i.e., thickening of the cell wall), and
callose (β-1-3 glucan) deposition in the cell wall (44, 70, 85, 102). Among the defense
responses triggered is the hypersensitive response (HR), a programmed cell death response
similar to apoptosis in animals that has long been associated with defense to viral, fungal, and
bacterial pathogens (73, 84).
3.d. Animal pathogen type III protein secretion systems
Type III protein secretion systems (TTSSs) have a central role in the pathogenesis of plants and
animals (40, 62, 83). These systems are capable of injecting or translocating bacterial proteins
called effectors into eukaryotic cells. Researchers studying animal pathogen TTSSs have made
major progress in understanding type III effectors. In general, type III effectors often promote or
inhibit phagocytosis (depending on the pathogen) by co-opting the cytoskeleton of the host (10,
17, 42, 141) or they affect cellular trafficking or vesicle fusion (114, 163, 166, 183). Some animal
pathogen type III effectors can suppress innate immune surveillance systems but the
mechanisms they use to accomplish this is not well understood (39, 41).
3.e. Pseudomonas syringae is an excellent model for understanding bacterial
pathogenesis. Pseudomonas syringae is an extracellular pathogen that lives in the apoplast of
plant tissue (6, 157). P. s. pathovar tomato DC3000 is pathogenic on tomato and the genetically
amenable model plant Arabidopsis (115, 185). In susceptible hosts, DC3000 grows to high
populations and causes chlorotic and necrotic lesions. In resistant hosts, DC3000 elicits the HR
and other host immune responses. DC3000 mutants defective in type III secretion lose their
ability to elicit the HR in resistant hosts and to be pathogenic in susceptible hosts (6). The
genome sequence of P. s. tomato strain DC3000 has been published (26). The availability of
the DC3000 genome has led to the identification of over 35 type III effectors (30, 57, 77, 154,
194). The effectors that travel the P. syringae TTSS have been referred to as Avr, Hop or Vir
proteins depending on how they were initially discovered (7, 122, 123, 180). While the majority
of these effectors activities are unknown, recently there has been progress in determining their
enzymatic activities and host targets (8, 95, 100, 137). Because DC3000 infects the model plant
Arabidopsis, whose genome sequence has also been determined (http://www.arabidopsis.org/),
the genomes of both the pathogen and one of its hosts is known, which has resulted in
important community resources to study this host-microbe interaction.
3.f. Type III effectors of plant pathogens can act as suppressors of innate immunity.
There were clues in the early 1990s that the TTSSs of pathogens were capable of suppressing
plant innate immunity. For example, Jakobek et al. (98) showed that a virulent P. syringae
pathovar, P. s. pv. phaseolicola, suppressed induction of defense-related mRNA and
phytoalexins in bean that were separately induced by an avirulent (i.e., nonpathogenic due to
triggering innate immunity) P. syringae pathovar and nonpathogenic E. coli. These early studies
suggested the TTSS of bacterial pathogens was involved in defense suppression. More recent
Background & Significance
Page 47
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
studies suggested that specific type III effectors altered innate immunity (97, 176). Recently,
several P. syringae effectors have been identified as suppressors of the HR and other
responses associated with defense (1, 14, 24, 52, 99, 100, 108, 124, 127, 145). A subset of
these also has been shown to suppress other hallmarks of innate immunity (24, 33, 99).
Moreover, P. syringae type III effectors suppressed Bax-induced programmed cell death (PCD)
in yeast and plants (99). While the mechanism of suppression is unknown, suppression of PCD
pathways in yeast suggests that these effectors may act on conserved pathways in eukaryotes.
Some type III effectors are able to suppress basal defenses triggered by PAMPs. For example,
Hauck et al. (82) found that transgenically expressed AvrPto in Arabidopsis suppressed the
expression of a set of genes predicted to encode proteins that are secreted cell wall and
defense proteins, which are normally expressed in a manner independent of SA. Other effectors
suppress SA-dependent responses (46). Moreover, when P. syringae TTSS defective mutants
were infected into transgenic Arabidopsis plants expressing specific type III effectors these
mutants grew to significantly higher levels than control strains suggesting that plant defenses
induced by these mutants were suppressed by these type III effectors (33, 82).
3.g. Mono-ADP-ribosyltransferases. Mono-ADP-ribosyltransferases (ADP-RTs) represent
some of the best described bacterial toxins from animal pathogens (16). These enzymes
catalyze the transfer of ADP-ribose of β-NAD onto a host target protein usually inactivating it.
The substrates for these enzymes include elongation factor 2, heterotrimeric G proteins, small G
proteins, actin, and actin-binding proteins (3, 4, 35, 37, 43, 93, 94, 120, 136, 172, 174, 184).
The effect of ADP-RT toxin activity on the host cell varies from inhibition of protein synthesis,
stimulation or inhibition of adenylate cyclase, or suppression of actin polymerization. Many of
these toxins are classic A-B toxins that have the enzyme as part of the A domain and a B
domain that directs the toxin to a specific cell type. The P. aeruginosa ADP-RTs ExoS and ExoT
are unusual ADP-RTs in that they are bi-functional toxins that lack a B domain and are injected
into host cells via its TTSS (17). The C-terminal ADP-RT domain of ExoS ADP-ribosylates a
diverse group of proteins including Ras GTPases family members and inhibit phagocytosis (15,
58, 65, 132, 160, 179). The ADP-RT domain of ExoT, also C-terminal, ADP-ribosylates a more
restricted set of substrates including CT10 regulator of kinase (Crk) proteins (172), which are
adapter proteins that are involved in signaling complexes leading to phagocytosis. These
enzymes also both have N-terminal Rho GTPase activating protein (GAP) domains that are
active on Rho, Rac, and Cdc42 resulting in disruption of the host cytoskeleton and inhibition of
phagocytosis (69, 105, 112, 113). Therefore, ExoS and ExoT suppress innate immunity by
inhibiting phagocytosis. The subject of this application is the P. syringae HopU1 ADP-RT, which
is type III-injected into host cells and also can suppress innate immunity. However, the nature of
this suppression must be fundamentally different because plant cells are enclosed within a cell
wall and do not possess phagocytic cells as components of their innate immunity.
3.h. Chloroplast RNA-binding proteins, glycine-rich RNA-binding proteins, and their
possible involvement in innate immunity. Post-transcriptional metabolism of RNA includes a
variety of processes including the processing, transport, translation, stabilization, and
degradation of RNA (34, 53). Several classes of RNA-binding proteins (RBPs) play important
roles in these processes and they are emerging as important, often multifunctional, cellular
regulatory proteins (27, 29, 53, 56, 138). RBPs are quite diverse and grouped based on their
conserved RNA-binding motifs. These include RBPs that contain one or more of the following
motifs: RNA-recognition motif (RRM), the pentatricopeptide repeat, the K-homology motif, the
arginine-rich motif, RGG boxes, and double-stranded RNA-binding domains (5, 64).
The largest group of RBPs are those that contain RRMs (27, 158). The functional
relatedness between RRM-containing RBPs has been shown to be reflected in the sequence
similarity of their RRMs (5, 61, 109). Analysis of RRM-containing RBPs on this basis separates
Background & Significance
Page 48
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
these proteins into classes that include poly(A)-binding proteins, splicing factors, nucleolins,
prokaryotic RBPs, and a final class that includes glycine-rich RNA-binding proteins (GR-RBPs)
and chloroplast RNA-binding proteins (CP-RBPs)(5, 61, 109, 130). GR-RBPs and CP-RBPs are
further associated in that they both show an affinity for poly(G) and poly(U) sequences (50, 91,
121, 126). As shown in the Preliminary Studies section, the P. syringae type III effector HopU1
ADP-ribosylates in vitro a subset of Arabidopsis CP-RBPs and GR-RBPs. The evolutionary
studies on RBPs support that these two classes of proteins are more related to each other than
they are to the other RRM-containing RBPs, which may explain the specificity of HopU1 for
these two RBP classes. GR-RBPs are not well understood and they likely have multiple
functions. Most have one RRM and a C-terminal glycine-rich domain (5). However, they have
long been known to be induced by abiotic and biotic stresses in several different plant species.
These stresses include drought (20, 72), salinity (11), cold temperatures (50, 110, 130),
circadium rhythm (28, 86), wounding (171), and pathogenesis (139). GR-RBPs have generally
been found to localize to the nucleus and, in certain cases, the nucleolus (5, 45, 66, 87). Coldinduced GR-RBPs (named CIRPs) also have been identified in humans, and other mammals,
as well as amphibians (79, 144, 155, 161). In mice and humans CIRPs suppress cell growth in
response to cold temperatures (79, 144). The human CIRP homolog Rbm3 was shown to
reduce microRNA levels and enhance global protein synthesis (48). Several GR-RBPs are
phosphorylated in vitro and in vivo (50, 59, 168). Relevant to this application is that a soybean
GR-RBP has been shown to be phosphorylated in response to a bacterial Avr protein (168),
which suggests that phosphorylation may functionally activate this protein to participate in an
Avr-triggered innate immune response. Taken together, these reports suggest that GR-RBPs
are involved in RNA metabolism and that they could affect these processes during abiotic and
biotic stresses. Relatively recently, MA16, a GR-RBP from corn, was shown to interact with a
novel DEAD box RNA helicase (66), suggesting that GR-RBPs may function as part of
ribonucleoprotein complexes. We found that the type III effector HopU1 modifies the
Arabidopsis GR-RBPs AtGRP7 and AtGRP8 (177) and recently we determined that an
Arabidopsis T-DNA insertion mutant lacking AtGRP7 is more susceptible to P. syringae. This
suggests that AtGRP7 functions in plant innate immunity and experiments described in this
application will address this hypothesis.
Like GR-RBPs, CP-RBPs are not well understood. However, CP-RBPs have been shown to
stabilize and process mRNA and tRNA in the plant chloroplast (53, 138, 142). These proteins
contain two RRMs and an acidic N-terminal domain. While there is not extensive evidence that
abiotic and biotic stresses induce these proteins, CP-RBP mRNAs have been found to be
elevated by salt stress (23). As shown in the Preliminary Data section of this application, several
Arabidopsis CP-RBPs are ADP-ribosylated by HopU1. It is truly remarkable that HopU1 can
ADP-ribosylate GR-RBPs and CP-RBPs. We are unaware of any demonstration in the literature
that ADP-RTs act on these classes of proteins. However, an animal RBP has been shown to be
ADP-ribosylated by an unknown animal protein (156). Interestingly, IpaH9.8, a type III effector
from the animal pathogen Shigella flexneri that contains leucine-rich repeats, was recently
shown to bind to a mammalian splicing factor resulting in the suppression of pro-inflammatory
cytokines (150). This splicing factor, U2AF35, is an RBP with an RRM (186). The substrates of
the HopU1 ADP-RT suggest a novel strategy utilized by bacterial pathogens to modulate plant
innate immunity by indirectly affecting host RNA status. That is, GR-RBPs may act as key posttranscriptional regulators through either the trafficking, stabilization, or processing of specific
mRNAs in response to pathogen stress and the ADP-ribosylation of the GR-RBPs by HopU1
may disrupt their activity. By disabling the function of GR-RBPs the pathogen may reduce the
amount of immunity-related mRNAs available in the plant and tip the balance of the interaction
in favor of the pathogen.
Background & Significance
Page 49
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
A
HopU1
HopO1-1
HopO1-2
LT toxin
CT toxin
Chicken ART
C3 toxin
SpvB
VIP2
ExoS
ExoT
*
GTPLYREVNNYLR
TSCLYRPINHHLR
TSCLYRPINHHLR
GDRLYRADSRPPD
DDKLYRADSRPPD
CYYVYRGVRGIRF
DMKVYRGTDLNPL
HRVVYRGLKLDPK
NITVYRWCGMPEF
VVKTFRGTRGGDA
VVKTFRGTQGRDA
97
125
135
20
20
159
275
465
344
314
317
183
201
215
69
69
176
306
491
376
333
336
*
GGRYVEPAFMSTTRIKDSA
GNTYRDDAFMSTSTRMDVT
GKTYRDEAFMSTSTHMQVS
GFVRYDDGYVSTSLSLRSA
GFVRHDDGYVSTSISLRSA
GKSVYFGQFTSTSLRKEAT
GKTFKDDGFMSTALVKESS
GNIIIDKAFMSTSPDKAWI
NTIKEDKGYMSTSLSSERL
GKVGHDDGYLSTSLNPGVA
GQVGHDAGYLSTSRDPGVA
224
238
253
119
119
212
345
526
417
370
374
* *
ISGSSQAPSEEEIM
IGPFSKNPYEDEAL
IGPFSKNPYEDEAL
LGVYSPHPYEQEVS
LGAYSPHPDEQEVS
IKQFSFFPSEDEVL
VSKISYFPDEAELL
LGDVAHFKGEAEML
LSAIGGFASEKEIL
VSGISNYKNEKEIL
VSEISIEGDEQEIL
T
T
W
W
Number of bacteria
log [cfu/cm2]
W
T
Δh
op
U1
W
T
Δh
op
U
DN 1
A
4. Preliminary Studies
4.a. Pseudomonas syringae pv.
tomato DC3000 has at least 3 genes
that may encode mono-ADPribosyltransferases. Genome-wide
Region 1
Region 2
Region 3
searches for type III effector genes
within the genome of P. s. tomato
B
C
DC3000 identified 3 ORFs, hopPtoS1,
00505 00504 shcF hopF2 hopU1 00500
hopU1
hopPtoS2, and hopPtoS3, which
1
2
C
1
2
appeared to encode mono-ADPRT
No RT
500 bp
ribosyltransferases (ADP-RTs) based
D
E
8
DC3000
on the presence of putative ADP-RT
ΔhrcC
in planta CyaA activity
ΔhopU1
7
catalytic sites (Fig. 1A) (38, 77, 152,
Strains
pmol/μg of cAMP
6
DC3000(pavrPto1-cyaA)
140.0 +/- 24.0
154). These genes have been recently
ΔhrcC(pavrPto1-cyaA)
1.1 +/- 0.1
5
renamed as hopO1-1, hopU1, and
DC3000(phopU1-cyaA)
104.3 +/- 10.2
4
ΔhrcC(phopU1-cyaA)
2.2 +/- 1.3
hopO1-2, respectively (123). HopO1-1
3
is about 72% identical to HopO1-2,
DC3000(phopU1 -cyaA)
115.0 +/- 7.9
2
ΔhrcC(phopU1 -cyaA)
1.1 +/- 0.1
2
4
0
whereas HopU1 is far less similar to
Day
these ADP-RTs (about 21% identical).
Also, included in the protein
Fig. 1. HopU1 is a putative mono-ADP-ribosyltransferase
comparisons in Figure 1A is P.
that contributes to virulence. (A) Alignment of the conserved
aeruginosa’s ExoS ADP-RT, which
regions of known mono-ADP-ribosyltransferases (ADP-RTs) with
putative DC3000 ADP-RTs. Conserved residues are shown in
like these proteins is associated with
red and the invariant amino acids of the CT group of ADPthe TTSS. Interestingly, DC3000
RTs(80) are marked with asterisks. (B) hopU1 (white box) is
putative ADP-RTs and ExoS have
downstream of a type III-related promoter, the shcF type III
biochemically similar N-termini that
chaperone gene, and the hopF2 effector gene (hatched boxes).
likely represent type III secretion
(C) RNA was isolated from DC3000 (WT) or the ΔhopU1 mutant
grown in either rich media (1) or a minimal medium that induces
signals (154). However, ExoS is
type III-related genes (2) and used in RT-PCR reactions. A DNA
significantly larger than the DC3000
control (C) and no RT controls (No RT) were included. (D)
ADP-RTs and contains a GTPAdenylate cyclase (CyaA) assays were carried out by infiltrating
activating protein (GAP) domain not
DC3000 and a ΔhrcC mutant (defective in TTSS) carrying
apparent in the DC3000 ADP-RTs
constructs that produced HopU1-CyaA, HopU1DD-CyaA (HopU1
catalytic mutant), or AvrPto1-Cya (a type III effector known to be
(69). A DC3000 mutant defective in
injected), respectively, into Nicotiana benthamiana. cAMP levels
HopO1-1 was reduced in its ability to
were determined 10 h after infiltration. (E) Bacterial strains were
multiply in plant tissue and disease
inoculated into A. thaliana Col-0 leaves by dipping plants into
symptom production indicating that it
bacterial suspensions.
contributes to virulence (76). We have
recently reported that hopO1-1 and hopO1-2 are both transcribed and that HopO1-1 and
HopO1-2 are secreted and translocated into plant cells via the DC3000 TTSS (76).
DD
DD
4.b. The DC3000 hopU1 gene is transcribed and encodes a type III effector that is injected
into plant cells. To begin to characterize the DC3000 effector genes that potentially encode
ADP-RTs we focused on hopU1, which is downstream of an apparent type III promoter and the
shcF type III chaperone gene and the hopF2 effector gene in the DC3000 chromosome (Fig.
1B). Semi-quantitative RT-PCR experiments indicated the hopU1 gene is transcribed and its
expression was elevated when DC3000 was grown in a medium that induces the expression of
the T3SS (Fig. 1C). HopU1 was type III-injected into plant cells based on adenylate cyclase
translocation assays (Fig. 1D). A DC3000 ΔhopU1 mutant was reduced 6 fold in its ability to
Preliminary Studies/Progress
Page 50
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
multiply in plant tissue and cause disease symptoms in A. thaliana Col-0 (Fig. 1E). Thus, HopU1
is expressed in DC3000 and translocated into plant cells by the TTSS.
A
4.c. HopU1 suppresses innate immune responses in a
manner dependent on its ADP-RT active site. We earlier
reported that DC3000 mutants defective in type III effectors that
can suppress the hypersensitive response (HR), a programmed
DC3000
ΔhopU1 ΔhopU1
cell death of plant cells associated with innate immunity, often
(phopU1)
B
display an enhanced ability to elicit an HR (99). To investigate
HR
No HR
HR
whether the ΔhopU1 mutant shared this phenotype we
108
infiltrated wild type DC3000 and the ΔhopU1 mutant at different
cell densities into Nicotiana tabacum cv. Xanthi (tobacco).We
ΔhopU1 ΔhopU1 ΔhopU1
consistently found that the ΔhopU1 mutant elicited an HR in
(pvector) (phopU1) (phopU1DD)
C
tobacco at cell densities below the threshold needed for wild
Col-0
HopU1-HA HopU1DD-HA
type DC3000 (Fig. 2A). When hopU1 was expressed in trans in
the ΔhopU1 mutant it complemented this phenotype (Fig. 2A).
To determine whether the predicted ADP-RT activity of HopU1
was required for suppression of the HR, we ectopically
D
1600
Col-0
expressed in the ΔhopU1 mutant a HopU1 derivative
HopU1-HA
1400
HopU1 -HA
1200
(HopU1DD) that had its glutamic acid residues in its putative
1000
ADP-RT active site (Fig. 1A) substituted with aspartic acids.
800
600
This strain elicited an enhanced HR similar to the ΔhopU1
400
mutant control suggesting that the suppression of the HR
200
required a functional ADP-RT active site (Fig. 2B). Thus, these
0
1
2
3
Experiment Number
results provide genetic evidence that HopU1 acts as a
suppressor of the nonhost HR and that HR suppression activity
Fig. 2. HopU1 suppresses
of HopU1 requires a functional ADP-RT catalytic site.
outputs of plant innate immunity.
We reasoned that HopU1 may be capable of
(A) DC3000, ΔhopU1 mutant, and
suppressing other innate immune responses. To test this
the ΔhopU1 mutant expressing
hopU1 were infiltrated into tobacco
Arabidopsis ecotype Col-0 plants were transformed using an
leaves at threshold cell densities (1
Agrobacterium-mediated floral dip method (19). Homozygous
6
X 10 cells/ml). After 24 h the tissue
lines were established that expressed HopU1 or the ADP-RT
was assessed for HR production.
catalytic mutant derivative (HopU1DD) tagged with a
(B) The ΔhopU1 mutant carrying a
vector control (pvector), a hopU1
hemagglutinin (HA) epitope constitutively expressed from the
construct (phopU1), or a hopU1
cauliflower mosaic virus 35S promoter. Bacterial flagellin often
ADP-RT catalytic mutant construct
acts as a pathogen-associated molecular pattern (PAMP), a
(phopU1DD) were infiltrated into
7
conserved molecule from a microorganism recognized by
tobacco at 1 X 10 cells/ml and
animal and plant innate immune systems (2, 103, 147). A
assessed for HR production after
24 h. (C) Callose deposition was
conserved peptide from bacterial flagellin, flg22, has been
visualized in A. thaliana plants
shown to be effective at triggering callose (β-1-3 glucan)
expressing HopU1-HA or the
deposition (71). We infiltrated wild type Arabidopsis ecotype
HopU1DD-HA mutant 16 h after
Col-0 and HopU1-HA-expressing Col-0 leaves with 1 μM of
treatment with flg22. (E) Callose
deposition was quantified by
flg22. After 12 h the chlorophyll was cleared by bathing the
counting the number of callose foci
leaves in an alcoholic lactophenol solution. The leaves were
per field of view. Twenty leaf
then stained with 0.01% aniline blue in 150 mM K2HPO4
regions (4 fields of view from 5
solution and viewed with fluorescence microscopy.The HopU1different leaves) were averaged
and error bars (s.e.m.) are
HA-expressing transgenic plants treated with flg22 produced
indicated.
significantly reduced amounts of callose compared to wild type
plants (Fig. 2C and D). Importantly, plants expressing the HopU1DD-HA no longer suppressed
callose production (Fig. 2C and D). HopU1-HA-expressing plants also elicited a delayed atypical
Number of callose foci
No HR
HR
No HR
DD
Preliminary Studies/Progress
Page 51
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
HR in response to the type III effector AvrRpt2, which is recognized by the RPS2 resistance
protein present in A. thaliana Col-0 (185) (data not shown). Together these data indicate that
HopU1 can suppress innate immune responses that are triggered by both a type III effector
protein or a PAMP.
32.5
B
2500
Specific Activity
Ho
pU
1Hi
Ho
s
pU
1
DD
-H
is
A
Coomassie blue
2000
1500
1000
1
500
Ho
pU
1Hi
Ho
s+
pU
Ar
1ab
Hi
.
s+
Ho
To
pU
b.
1
Ho DD -H
is
pU
+
1
Ar
DD ab
Hi
.
s+
To
b.
HopU1 HopU1DD BSA
C
32.5
25
6.5
Autoradiograms
Fig. 3. HopU1-His ADP-ribosylates polyL-arginine and proteins in Arabidopsis
and tobacco. (A) SDS PAGE of partially
purified HopU1-His and HopU1-His
catalytic mutant (HopU1DD-His) used in
ADP-RT assays. (B) HopU1-His (stippled
bar), HopU1DD-His (white bar), and a BSA
control (black bar) were incubated with
poly-L-arginine in the presence of [32P]NAD. 32P-labeled products were quantified
with liquid scintillation. The specific activity
32
unit is μmole P transferred/min/μmole of
HopU1 times 10-10. The experiment was
performed twice and the standard errors
are indicated. (C) Autoradiograms of ADPRT assays with either Arabidopsis (At) or
tobacco (Nt) extracts with HopU1-His or
HopU1DD-His.
4.d. Recombinant HopU1-His ADP-ribosylates polyL-arginine demonstrating that HopU1 is an ADP-RT.
hopU1 was cloned into pQE30, which fused 6 histidine
codons to the 5’ end of hopU1. Site-directed
mutagenesis was performed on this construct using the
QuikChange mutagenesis kit (Stratagene) to change
the codons corresponding to amino acids 233 and 235
in region 3 of HopU1 from glutamic acids to aspartic
acids (See Fig. 1A). These changes were confirmed by
sequencing the hopU1 carried on the resulting
construct. The constructs that encoded HopU1 and
HopU1DD were expressed separately in E. coli and each
protein was purified to ~ 90% purity with immobilized
metal affinity chromatography (Fig. 3A). Most ADP-RTs
that have two glutamic acids in Region 3 modify
arginine residues and the N-terminal glutamate is
required for this modification (81, 153). To determine if
HopU1 could ADP-ribosylate poly-L-arginine we
incubated HopU1 or HopU1DD with poly-L-arginine in
the presence of [32P]-NAD using a standard protocol
(167). As shown in the bar graph in Fig. 3B, HopU1-His
was capable of ADP-ribosylating poly-arginine.
HopU1DD-His was greatly reduced in its ability to ADPribosylate poly-arginine producing 32P material similar in
amount to the BSA negative control. Therefore, HopU1
is an active ADP-RT that can modify arginine residues.
4.e. HopU1 ADP-ribosylates proteins in Arabidopsis
and other plant extracts. To begin to determine the
substrates for the HopU1 ART we made crude extracts
from the leaves of A. thaliana ecotype Col-0 and N.
tabacum Cv. Xanthi (tobacco) and assayed them using
a standard ADP-RT assay (172). Briefly, we added 20
μg of plant extract, 200 ng HopU1-His or HopU1DD-His,
32
and 0.2 μM [ P]-NAD to each reaction and incubated the reactions for 45 m. The samples were
subjected to SDS-PAGE, stained with Coomassie blue, and exposed to X-ray film. As shown in
Fig. 3C, we identified at least 2 major ADP-ribosylated products in Arabidopsis extracts and 3
major products in tobacco extracts. No labeled products were detected from reactions using the
inactive HopU1DD-His (Fig. 3A). Moreover, novobiocin has been used as an inhibitor of ADPRTs (174) and when this inhibitor was included in the reactions no labeled products were
detected (data not shown).
4.f. HopU1-His ADP-ribosylates several Arabidopsis RNA-binding proteins. To begin to
characterize the plant proteins that are ADP-ribosylated by HopU1 an Arabidopsis extract was
used in ADP-RT assays and the reaction was separated with two-dimensional PAGE followed
by autoradiography (Fig. 4A). Several protein spots are radiolabeled suggesting that HopU1-His
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
A
pH 4
IEF
B
pH 7
Fraction 22 23 24
33 34 35
41 42 43
can use multiple Arabidopsis proteins as
substrates. One protein spot that co32.5
75
migrated with a spot that was radiolabeled
25
37
16.5
was well isolated on a Coomassie blue6.5
25
stained polyacrylamide gel was cut out,
C
IEF
pH 4
pH 7
15
digested with trypsin and glutamyl
endopeptidase, and analyzed with tandem
mass spectrometry (Fig. 4A and Table 1).
37
This protein spot corresponded to
25
20
Arabidopsis protein At4g24770.1, which is
37
predicted to encode a chloroplast RNA25
binding protein (CP-RBP) similar to cp31.
15
The other ADP- RT activity spots did not co37
migrate with Coomassie-stained protein
pH 4
pH 7
IEF
25
spots suggesting that these proteins were in
20
lower abundance in Arabidopsis extracts. To
pH 4
pH 7
enrich for the less abundant ADP-RT
IEF
substrates more concentrated Arabidopsis
Fig. 4. Separation of HopU1-His ADP-RT reactions
with two-dimensional (2D) SDS-PAGE. (A) A. thaliana
extracts were made, ultracentrifuged, and
soluble protein extract was incubated with HopU1-His in
separated further using ion exchange
the presence of [32P]-NAD. The reaction mix was
chromatography. Fifty 1 ml fractions were
subjected to preparative 2D SDS-PAGE and stained with
collected from an ion exchange column and
Coomassie blue (top panel). The dried polyacrylamide
ADP-RT assays were performed on each
was exposed to X-ray film producing the shown
autoradiogram (bottom panel). The black arrowhead in
fraction by adding HopU1-His and the other
the top panel identifies a protein that possessed the
components of the reaction mix to an aliquot
same migration as an ADP-riboslyated activity spot in the
of each fraction followed by SDS-PAGE and
bottom panel, also marked with a black arrowhead. (B) A
autoradiography. The major ADP-RT activity
crude A. thaliana Col-0 extract was separated with an ion
exchange column and fractions were tested for proteins
was found in 3 different groups of fractions
that could be ADP-ribosylated by HopU1-His. ADP-RT
as shown in Fig. 4B. Aliquots from these
assays were carried-out on all fractions and
fractions were used in additional ADP-RT
autoradiograms from the fractions that contained the
assays and subsequently subjected to twomajority of substrates of HopU1-His based on a high
32
dimensional PAGE and autoradiography. A
level of P incorporation are shown. (C) A. thaliana
extract fractions after ion exchange chromatography
representative two-dimensional
were used in ADP-RT assays and subjected to 2D SDSpolyacrylamide gel and autoradiogram of an
PAGE. A representative gel (top panel) stained with
ADP-RT reaction using fraction 42 from Fig.
Coomassie blue and an autoradiogram (lower panel)
4B is shown in Fig. 4C. Two Coomassiecontaining aliquots from fraction 42 from B are shown.
The black arrowheads present in the top panel identify
stained protein spots co-migrated with the
protein spots that co-migrated with protein spots that
radioactive ADP-RT activity spots that were
were modified by HopU1-His in the lower panel.
detected on the autoradiogram (Fig. 4C).
Molecular weight markers in kilodaltons are indicated on
These protein spots were cut out of the gel
the left (A-C).
and analyzed with tandem mass
spectrometry and found to contain predominately two proteins At2g37220.1 and At5g50250.1,
which are homologous to the CP-RBPs cp29 and cp31, respectively (Fig. 4C and Table 1)(149).
ADP-RT reactions using an aliquot from fraction 22 was treated similarly and 2 protein spots
that co-migrated with ADP-RT activity spots were analyzed with mass spectrometry and found
to be At2g21660.1 and At4g39260.1, which encode glycine-rich RNA-binding proteins (GRRBPs) AtGRP7 and AtGRP8, respectively (177). Table 1 summarizes the different RBPs
identified with mass spectrometry.
62
150
SDS-PAGE
SDS-PAGE
SDS-PAGE
SDS-PAGE
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
A
GS
T
At con
2g
tr
At 372 ol
5g
20
At 502 .1-G
4g 5 0
ST
At 2477 .1-G
GR
0.1 ST
At P7- -GS
GR GS
T
P8
T
-G
ST
Ho
+ A pU1
tG DD -F
Ho RP7- LAG
+ A pU1 HA
tG -FL
Ho RP7 AG
+ A pU1 -HA
tG -FL
RP AG
7R
47
K -H
A
Table 1. RNA-binding proteins (RBPs) identified with mass spectrometry to be ADP-ribosylated
by HopU1-His
pI and mass
% of protein coverage
a
(kD) of
pI and mass (kD) of
by
ADP-RT Substrate
b
c
protein
ADP-RT spot
MS peptides
(Accession No.)
AtRBP31 chloroplast RBP
4.3, 35.7
4.3, 32
15
(At4g24770.1)
cp29-like chloroplast RBP
4.8, 30.7
4.5, 25
49
(At2g37220.1)
cp31-like chloroplast RBP
4.6, 31.8
4.5, 27
37
(At5g50250.1)
AtGRP7 glycine-rich RBP
5.9, 16.9
5.2, 20
27
(At2g21660.1)
5.3, 16.6
5.2, 17
19
AtGRP8 glycine-rich RBP
(At4g39260.1)
a
Substrates were isolated based on their co-migration with protein spots on 2D
polyacrylamide gels that possessed ADP-RT activity and identified with MS.
b
Estimated isoelectric points and molecular masses based on the actual migration
on 2D polyacrylamide gels of protein spots with ADP-RT activity.
c
The percent of coverage of the identified protein from the databases with peptides identified.
D
83
62
47.5
32.5
HA
FLAG
R141
R152
R153
R124
R125
Immunoblots
R77
R87
R96
R104
R43
R47
R49
R9
B
R22
Autoradiogram
AtGRP7
C
Glycine-rich domain
GS
T
At co
GR ntr
o
RR P7 l
M
Gl
y-r
ich
R9
K
R2
2
R4 K
3K
R4
7K
R4
9
R7 K
7K
R8
7K
RRM
47.5
32.5
47.5
32.5
Autoradiogram
AtGRP7
immunoblot
Fig. 5. HopU1-His ADP-ribosylates recombinant RNAbinding proteins in vitro and in planta. (A) Recombinant
CP-RBP and GR-RBP glutathione S-transferase (GST)
fusions and a GST control were used in ADP-RT assays with
HopU1-His. ADP-RT reactions were subjected to SDS-PAGE
followed by autoradiography. The substrate fusions are listed
with their A. thaliana locus or protein name. (B) Schematic
representation of the RRM and glycine-rich domains within
AtGRP7 and the locations of the arginine residues (R)
throughout the protein. Black and grey sections within the
RRM domain depict RNP1 and RNP2, respectively, two
conserved regions within RRM domain (27). The locations of
the arginine residues (R) are indicated. (C) In vitro ADP-RT
assays were done with HopU1-His and various AtGRP7-GST
fusions. These included an RRM-GST fusion, a glycine-rich
domain-GST fusion, and AtGRP7-GST mutants that have
each arginine within the RRM domain separately substituted
with lysine. Autoradiograms (top panel) indicate the AtGRP7GST derivatives that maintained the ability to be ADPribosylated and immunoblots (bottom panel) indicate the
relative stability of each AtGRP7-GST derivative. (D) AtGRP7
fused to an HA epitope (AtGRP7-HA) or an AtGRP7-HA
derivative that is unable to be ADP-ribosylated (AtGRP7R47K –
HA) were transiently expressed in N. benthamiana with
HopU1-FLAG or the ADP-RT catalytic mutant HopU1DDFLAG. After 40 h leaf samples were subjected immunoblot
analysis using anti-HA and anti-FLAG antibodies. (A, C)
Molecular mass markers (kDa) are indicated.
4.g. Confirmation that E. coli-produced recombinant RBPs are ADP-ribosylated by
HopU1-His. cDNAs of each of the Arabidopsis RBP genes identified to encode proteins ADPribosylated by HopU1-His using mass spectrometry (see Table 1) were amplified from
Arabidopsis RNA and were separately ligated into a GST fusion vector. E. coli strains carrying
each construct were grown and each RBP-GST fusion was partially-purified with glutathione
Sepharose and used in ADP-RT reactions containing HopU1- His, [32P]-NAD, and one RBPGST product. SDS-PAGE and autoradiography confirmed that each of the RBP-GST fusions
could act as substrates for HopU1-His (Fig. 5A). Thus, we confirmed that CP- and GR-RBPs
can act as in vitro substrates for the HopU1 ADP-RT and because we were unable to identify
any HopU1 substrates other than these in our extensive mass spectrometry analyses it
suggests that the in vivo substrates of HopU1 belong to these protein classes.
Preliminary Studies/Progress
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
4.h. Two arginines within the RRM of AtGRP7 are required for ADP-ribosylation
CP-RBPs and GR-RBPs belong to a group of RNA-binding proteins that all have in common an
RNA-recognition motif (RRM), a protein domain demonstrated to be necessary and sufficient for
RNA binding (27, 101). We performed localization experiments and found that HopU1-GFP (and
HopU1-GUS) and the GR-RBPs AtGRP7 and AtGRP8 GFP fusion proteins were similarly
localized to the cytoplasm and possibly to the nucleus, while the CP-RBP-GFP fusions were
discretely localized to the chloroplast (data not shown). Because of our localization experiments
and the association of GR-RBPs with abiotic and biotic stress, we focused on the GR-RBPs as
putative physiological targets of HopU1. The GR-RBP AtGRP7 and AtGRP8 are homologous to
each other sharing 76.9% identity and likely perform related functions. AtGRP7 has been shown
to bind RNA and influence mRNA oscillations in response to circadian rhythms at the posttranscriptional level (87, 170). AtGRP7 contains 14 arginine residues that represent putative
sites of ADP-ribosylation (Fig. 5B). We found that the RRM domain-GST fusion was ADPribosylated by HopU1-His, while the glycine-rich domain-GST fusion was not (Fig. 5C). To
determine the arginine residues required for ADP-ribosylation, each arginine of RRM was
individually mutated to lysine in full length AtGRP7-GST fusions. When arginines in positions 47
or 49 of AtGRP7 were substituted with lysine these AtGRP7-GST derivatives were no longer
ADP-ribosylated suggesting that one of these residues is the site of the ADP-ribose modification
while the other may be required for substrate recognition (Fig. 5C). Interestingly, based on the
RRM domain structure in other RNA-binding proteins both of these residues would likely be
solvent-exposed (128). Moreover, the arginine in position 49 is within RNP1, the most
conserved region of the RRM domain and one that has been implicated in RNA binding (27).
4.i. The AtGRP7 substrate can be ADP-ribosylated by HopU1 in planta
To determine if AtGRP7 can be ADP-ribosylated by HopU1 in planta, we co-expressed HopU1FLAG or HopU1DD-FLAG and AtGRP7-HA or AtGRP7R47K-HA (an AtGRP7 derivative that cannot
be ADP-ribosylated) in N. benthamiana using Agrobacterium transient assays. After 40 h, plant
extracts isolated from leaf tissue were separated on SDS-PAGE gels and analyzed with
immunoblots using anti-FLAG or anti-HA antibodies. We consistently observed an increase in
the molecular mass of AtGRP7-HA when it was expressed in planta with HopU1-FLAG, but not
when expressed with HopU1DD-FLAG or when HopU1-FLAG was expressed with AtGRP7R47KHA (Fig. 5D) suggesting that the increased molecular mass was due to ADP-ribosylation.
Therefore, HopU1-FLAG can ADP-ribosylate AtGRP7-HA inside the plant cell.
4.j. An Arabidopsis mutant lacking AtGRP7 showed enhanced susceptibility to P.
syringae. To determine the extent that RBPs affect bacterial pathogenicity and innate immunity
we are in the process of acquiring or making transgenic Arabidopsis plants that are defective (TDNA insertional mutants or reduced (RNAi knocked-down plants) in RBPs that were modified by
HopU1. We identified an A. thaliana SALK homozygous T-DNA insertion line in the AtGRP7
locus, and confirmed that it did not produce AtGRP7 mRNA and protein (Fig. 6A and data not
shown). To determine if this mutant, designated Atgrp7-1, was altered in its responses to P.
syringae, we infected wild type A. thaliana Col-0 and the Atgrp7-1 mutant plants with wild type
DC3000 and a ΔhrcC mutant defective in the TTSS. Each strain grew to higher levels on
Atgrp7-1 plants compared to wild type Col-0 plants (Fig. 6B), indicating that Atgrp7-1 plants
were more susceptible to P. syringae. The growth difference was even more pronounced for the
ΔhrcC mutant, which is likely due to the fact that this strain cannot inject any type III effectors,
many of which suppress innate immunity. Importantly, DC3000 caused enhanced disease
symptoms on Atgrp7-1 mutant plants compared to wild type Col-0 (Fig. 6C). Additionally, we
found that flg22-induced callose deposition was reduced in Atgrp7-1 plants compared to wild
type A. thaliana Col-0 (Fig. 6D), further supporting that Atgrp7-1 mutant plants were impaired in
their innate immune responses. Similar phenotypes were observed for an independent T-DNA
Preliminary Studies/Progress
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Ho
p
+ C U1-H
ol0 is
Ho
p
+ A U1-H
tgr
i
p7 s
-1
Co
l-0
A
Atg
rp7
-1
mutant designated Atgrp7-2 (data not shown). Thus, we now have strong evidence that AtGRP7
plays a role in innate immunity and one major goal of this proposal is to determine how ADPribosylation by HopU1 disables this protein.
Fig. 6. Analyses of A. thaliana Atgrp7-1 mutant plants
suggest AtGRP7 plays a role in innate immunity. (a)
Immunoblot analysis (left panel) of soluble proteins from
leaves of A. thaliana Col-0 and Atgrp7-1 mutant plants
using anti-GRP antibodies, which recognizes AtGRP7.
HopU1-His ADP-RT assays were performed with the
same samples. The autoradiogram (right panel) shows
the absence of an ADP-RT high-affinity band in the
Atgrp7-1 lane (indicated with an arrow). (b) Bacterial
growth assays of wild type DC3000 and the ΔhrcC
mutant spray-inoculated at a cell density of 2 X 108
cells/ml onto Atgrp7-1 and wild type A. thaliana Col-0
plants. (c) Disease symptoms in Atgrp7-1 plants and A.
thaliana Col-0 plants after spray-inoculation with at a cell
density of 2 X 108 cells/ml. Pictures were taken after 5
days. The experiments in ‘b’ and ‘c’ were repeated five
times with similar results. (d) Callose deposition was
determined in A. thaliana Col-0 and Atgrp7-1 mutant
plants 16 h after treatment with flg22. The number of
callose foci per field of view for 20 leaf regions (4 fields of
view from 5 different leaves) were averaged ± s.e.m. The
experiment was repeated three times with similar results.
C
Col-0
25
17
Atgrp7-1
Anti-GRP Autoradiogram
Immunoblot ADP-RT assay
8
7
Col-0/DC3000
Atgrp7-1/DC3000
Col-0/hrcC
Atgrp7-1/hrcC
6
5
4
3
2000
1500
1000
500
0
0
Day
4
-0
At
gr
p7
-1
9
2500
Co
l
Number of Bacteria
log [cfu/cm2]
B
D
Number of callose foci
7
Fig. 7. Proposed model of
suppression of plant innate
immunity by the HopU1 ADP-RT
type III effector. P. syringae pv.
tomato DC3000 injects HopU1 and
other type III effectors into plant
cells via its type III secretion
system. Innate immunity can be
triggered by a type III effector
(depicted above as the ‘A’-labelled
diamond) if it is recognized directly
or indirectly by a resistance (R)
protein or by a PAMP perceived by
PAMP receptors (e.g., FLS2).
Activated type III effector- and
PAMP-triggered signal
transduction pathways result in
transcriptional changes in the plant
U1
A
cw
pm
Callose, HR, &
other responses
FLS2
R
A
Reduced
immune response
RBP
U1
ADPRBP
2.
RBP
nm
Immunity-induced
mRNA
1.
resistance to the pathogen. Our model predicts that in the absence of HopU1 (U1) (as in 1), innate immunityinduced mRNAs (i.e., the plant defense transcriptome) are stabilized, transported, or processed by RNA-binding
proteins (RBPs) in the nucleus or the cytoplasm (as shown) resulting in the production of immunity-related
products that facilitate the outputs of innate immunity, including the deposition of callose and the HR. In the
presence of HopU1 (as in 2), one or several RBPs are ADP-ribosylated by HopU1 altering their normal function
and, subsequently, affecting RNA metabolism. For example, the ADP-ribose modification may prevent an RBP
from binding mRNA as depicted. This results in a suppressed innate immune response favoring the survival of
the pathogen. cw, cell wall; pm, plasma membrane; nm, nuclear membrane.
The substrates of the HopU1 ADP-RT suggest a novel strategy utilized by bacterial
pathogens to modulate plant innate immunity by indirectly affecting host RNA status. That is,
GR-RBPs may act as key post-transcriptional regulators through either the trafficking,
stabilization, or processing of specific mRNAs in response to pathogen stress and the ADP-
Preliminary Studies/Progress
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
ribosylation of the GR-RBPs by HopU1 may disrupt their activity (Fig. 7). By disabling the
function of GR-RBPs the pathogen may reduce the amount of immunity-related mRNAs
available in the plant and tip the balance of the interaction in favor of the pathogen. Our central
hypothesis is that HopU1 substrates such as AtGRP7 are important components of the innate
immune system and that ADP-ribose modification of these by HopU1 disables their function. In
the case of AtGRP7, we will determine whether the plant defense transcriptome is altered when
AtGRP7 is absent or ADP-ribosylated. Collectively, our preliminary results position us well to
undertake the experiments described below.
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
5. Research Design and Methods
The experimental plan makes use of methodologies that are well established in our
laboratory or the laboratories of collaborators. Thus, in the interest of space, only unusual or
new manipulations are described in detail and references and detailed procedures for standard
molecular biological procedures have been omitted. The goals of these experiments are to
exploit the P. syringae – Arabidopsis thaliana model system to learn about strategies bacterial
pathogens employ to circumvent eukaryotic innate immunity.
5.a. AIM 1: Determine the molecular consequence of ADP-ribosylation on the function of
AtGRP7 and elucidate the role this protein plays in innate immunity.
a. Rationale. Our preliminary data supports the involvement of in innate immunity
because Arabidopsis plants lacking AtGRP7 are more susceptible to P. syringae and produce
less callose deposition upon flagellin treatment (Fig. 6). The implication is that AtGRP7
enhances the plant defense transcriptome and the pathogen targets it to diminish the innate
immune response. In this Aim, we will determine how ADP-ribosylation affects AtGRP7 function
and whether AtGRP7 binds immunity-related RNAs.
b. Identification of the arginine residue within AtGRP7 that is ADP-ribosylated by
HopU1. From our preliminary data we suspect that HopU1 ADP-ribosylates an arginine residue
within RRM of AtGRP7, either arginine 47 (R47) or arginine 49 (R49), since site-directed
AtGRP7-GST mutants of these residues were no longer ADP-ribosylated (Fig. 5C). R49 is the
residue likely to be ADP-ribosylated by HopU1 as it is located in the RNP1, the most conserved
part of the RRM, and has been implicated in RNA binding in other RRM-containing proteins
(27). Partially-purified AtGRP7-GST will be ADP-ribosylated by HopU1 in vitro and subjected to
SDS-PAGE. Importantly, one ADP-RT reaction will use the HopU1DD-His catalytic mutant and,
therefore, in this reaction AtGRP7 will not be ADP-ribosylated. Coomassie blue-stained
AtGRP7-GST bands (from reactions with HopU1-His and HopU1DD-His ) will be cut from
polyacrylamide gels and analyzed with LC-MS-MS to determine the mass difference between
the modified and the unmodified AtGRP7. These analyses should confirm that AtGRP7 is only
ADP-ribosylated at one residue. These proteins (modified and unmodified AtGRP7-GST) will be
digested with trypsin and their peptides will be analyzed with ESI-MS-MS to identify the ADPribosylated residue.
Anticipated problems and alternate strategies. It is possible that ADP-RT reactions
containing active HopU1-His may retain a significant fraction of unmodified AtGRP7-GST. This
would make the mass spectrometry analyses more difficult to interpret because there would be
a mixture of modified and unmodified peptide fragments. If this occurs we will perform ADP-RT
assays using biotinylated NAD (6-Biotin-17-NAD, Trevigen). This will result in biotinylation of the
ADP-riboyslated AtGRP7 fraction allowing purification of ADP-ribosylated AtGRP7-GST using
avidin affinity chromatorgraphy. We have used biotinylated NAD in ADP-RT reactions with
HopU1-His and AtGRP7-GST and know that HopU1 can use it as a substrate.
c. Determine the extent that ADP-ribosylated AtGRP7 is altered in its ability to
bind RNA. This sub-aim is critical to our understanding of HopU1 because it has the potential of
identifying a consequence of ADP-ribosylation of AtGRP7. The mostly likely result of ADPribosylation of AtGRP7 is that it reduces or abolishes the ability of AtGRP7 to bind RNA
because HopU1 modifies an arginine residue within the RRM of AtGRP7 (Fig. 5c). We have a
well established collaboration with Dr. Dorothee Staiger at the University of Bielefeld, Germany
(See enclosed letter). Dr. Staiger studies the involvement of AtGRP7 and AtGRP8 in circadian
rhythms in Arabidopsis (86, 87, 169, 190). She has agreed to act as a consultant in these
assays (and in other experiments involving AtGRP7 described below). AtGRP7 has been shown
to bind to the 3’ untranslated region (UTR) of its own transcript (87, 170). The RNA
Research Design & Methods
Page 58
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
electrophoretic mobility shift assays to determine whether a protein binds RNA are well
established and have been used successfully with AtGRP7 (170).
For our experiments, we will use partially-purified AtGRP7-GST preparations from ADPRT assays with either HopU1-His or HopU1DD-His (the catalytic mutant). Importantly, in these
experiments AtGRP7-GST will be modified with unlabelled ADP-ribose. The following synthetic
oligoribonucleotides will be labeled with T4 polynucleotide kinase and γ [32P]-ATP (the
ribonucleotides in bold indicate where the oligomers differ): 5’-auu uug uuc ugg uuc ugc uuu
aga uuu gau cu-3’ (wild type UTR) and 5’-auu uua uuc uaa uuc ugc uuu aga uuu aau cu-3’
(mutated UTR). The wild type oligoribonucleotide from the Atgrp7 transcript has been shown to
bind AtGRP7-GST, while the mutated oligoribonucleotide is unable to bind AtGRP7-GST (170).
The binding assays will contain 50 fmoles of labeled oligoribonucleotides, 0.5 μg of recombinant
AtGRP7-GST, 10 U RNasin (a ribonuclease inhibitor), and the rest of the components of the
reaction mix (170). Unlabelled competitor wild type UTR oligoribonucleotide and tRNA will be
used at increasing concentrations (2-500 pmole range) to determine whether any interactions
detected are specific to the Atgrp7 transcript. The binding reactions will be resolved on 6%
polyacrylamide gels in 40 mM Tris-acetate and analysed with a phosphorimager located in a
neighboring laboratory. It may be obvious from the polyacrylamide gels whether ADPribosylated AtGRP7-GST fails to bind RNA in a side-by-side comparison with unmodified
AtGRP7-GST. This would be visualized by the absence of a higher molecular mass RNAprotein complex band. However, the assay is quantitative and we should be able to determine
the dissociation constants (Kd) for unmodified AtGRP7-GST and the ADP-ribosylated form by
plotting the log (complex/free probe) against the log (protein concentration), which will yield the
log (Kd) as the x-intercept. Since RNA-binding assays have already been successfully employed
using this protein we are confident these assays will be straightforward. Thus, we should be
able to determine whether ADP-ribosylated AtGRP7-GST is reduced in its ability to bind RNA. If
it is reduced in RNA binding, we will also include AtGRP7R47K-GST and AtGRP7R49K-GST
proteins in our binding assays. R49 is present in RNP1 region of the RRM domain and has been
shown to be required for RNA binding in other RRM-containing proteins (27). Thus,
AtGRP7R49K-GST will likely be unable to bind RNA. However, if we find that AtGRP7R47K-GST
retains its ability to bind RNA, this would provide further evidence that R49 is the arginine
residue that is ADP-ribosylated by HopU1.
Arabidopsis plants that over-express AtGRP7 possess an alternatively spliced Atgrp7
transcript not detectable in wild type plants (170). This is the main evidence that AtGRP7 is
involved in processing Atgrp7 RNA. If the in vitro experiments described above indicate that
ADP-ribosylated AtGRP7-GST is less able to bind Atgrp7 RNA, we will utilize the HopU1-HAexpressing Arabidopsis plants (Fig. 2) to determine if HopU1 alters the Atgrp7 transcript in vivo.
In these experiments we will grow HopU1-expressing plants and wild type A. thaliana Col-0 on
MS plates. After 2 weeks plant tissue will be harvested, RNA will be isolated using standard
procedures, and RNA blots will be performed using Atgrp7 cDNA as a probe. If the pattern of
unspliced pre-mRNA, alternatively spliced RNA, and spliced mRNA differ between wild type and
HopU1-HA-expressing plants this would suggest that HopU1 affects the Atgrp7 transcript in
vivo.
d. Microarray analysis of the Arabidopsis Atgrp7 mutant. To provide clues to
AtGRP7’s RNA targets we propose to assess the gene expression profile differences between
wild type Arabidopsis and Atgrp7 mutant plants using Affymetrix microarrays. RNA targets of an
RNA-binding protein can be reduced in organisms lacking the corresponding RNA-binding
protein (164). Our experiments will be carried out in the Genomic Core Research Facility at the
University of Nebraska, which has an Affymetrix GeneChip Microarray System (See enclosed
letter from Dr. Yuannan Xia, the manager of the facility). We will isolate RNA from untreated
Arabidopsis plants and Atgrp7 mutant plants. Because AtGRP7 may be interacting with
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immunity-induced RNAs, we will also isolate RNA from both of these plants after infection with
both virulent DC3000 and DC3000(pavrRpt2), an avirulent strain that triggers Avr-dependent
immunity. Total RNA will be isolated from 4 week old Arabidopsis plants using Trizol, and further
purified using phenol/chloroform extractions and the RNeasy kit. High quality RNA is essential
to reduce variation between experiments and, therefore, the RNA quality will be monitored by
measuring 260/280 absorbance, with agarose gel electrophoresis, RT-PCR, and an Agilent
2100 Bioanalyzer.
Purified RNA will be used for cDNA synthesis, which will then be used to make
biotinylated cRNA using the GeneChip IVT labeling kit. Biotinylated cRNA will be purified and
hybridized to Arabidopsis ATH1 genome arrays and stained with Streptavidin-Phycoerythrin
conjugate on an Affymetrix Fluidics Station 450. The Arabidopsis ATH genome array contains
more than 22,500 probe sets representing about 24,000 genes. Affymetrix GeneChip Operating
Software (GCOS) will be used for washing, staining, scanning, and basic data analysis.
Scanning of microarrays will be done on a recently updated Affymetrix GeneChip Scanner 3000
7G. A potential pitfall with these experiments is not effectively discriminating between the useful
data and background noise, especially if the gene expression is not that different between wild
type and mutant. To reduce this problem, each experiment will be replicated three times. We will
group genes whose expression is increased or decreased 2-fold relative to control plants using
GCOS. The generated data will be further analyzed with compatible software such as
GeneSpring (Silicon Genetics) or ArrayAssist (Stratagene). The UNL Genomic Core Facility has
recently purchased the Rosetta Resolver System, which is software that helps in the storage,
retrieval and analysis of gene expression data and we will use this software in our data analysis.
Biostatisticians in the UNL Center for Bioinformatics will assist in data analysis.
e. RNA immunoprecipitation with AtGRP7 followed by microarray analysis. To
identify globally the interactions that AtGRP7 has with RNA we propose to perform RNA
immunoprecipitation followed by microarray chip analysis (RIP-chip). This technique is a
modification of chromatin immunoprecipitation microarrays (ChIP-chip) techniques recently
developed (25). In RIP-chip, an RNA-binding protein is immunoprecipitated and the interacting
RNA that is co-immunoprecipitated is used in microarray experiments to identify the precipitated
transcripts. This technique has been used successfully in yeast, humans, and plants and it has
also been used successfully with an RRM-containing RNA-binding protein (49, 63, 67, 90, 164,
165, 175). In our experiments we will use transgenic Atgrp7 mutant plants that express Atgrp7
fused to HA and express AtGRP7-HA under the control of either its native promoter or the
constitutive CaMV 35S. The reason we propose to at least initially use both promoters is that I
see differing accounts in the literature whether native expression is better (164, 175). Indeed, a
recent paper has used this technique successfully with recombinant RNA-binding protein added
to isolated RNA in vitro (175).
Arabidopsis protein extracts will be made from uninfected plants, DC3000-infected, or
plants infected with an avirulent DC3000 strain. Immunoprecipitations will be carried out with
anti-HA beads (Roche) following standard procedures (151). Precipitation of AtGRP7-HA will be
confirmed with immunoblot analysis using anti-HA antibodies (Roche). RNA extractions will be
done using Trizol. Purified RNA will be used for cDNA synthesis and biotinylated using the
GeneChip IVT labeling kit. For our reference samples, we will use RNA from the
immunoprecipitation supernatants. The resulting cDNA preparations will by hybridized to
Affymetrix Arabidopsis gene chip tilling arrays. Several labs at the University of Nebraska are
performing ChIP-chip experiments in the Nebraska Genomic Core Research Facility. Thus, the
Center has experience in data analysis of similar experiments. The bioinformaticists in the
Center are developing programs to statistically analyze microarray data and they will help us in
our data analysis. One could argue that RIP-chip experiments may result in a higher signal to
noise ratio than ChIP-chip experiments since RNA is at a higher copy number than genomic
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DNA. Data will be analysed and filtered to remove elements to clarify the signal to noise ratio.
Experiments will be done in triplicate and any signal that is not consistent between experiments
will be discarded. From our results we will rank transcripts that have high signals and these will
be confirmed with co-immunoprecipitation experiments followed by real-time RT-PCR.
.
f. Identification of proteins that interact with AtGRP7. Identifying proteins that
interact with AtGRP7 may provide important information on how this protein is involved in innate
immunity. There has been surprising little analyses of this protein in terms of its protein-protein
interactions. It has been shown to interact with the Arabidopsis nuclear import receptor TRN1
(190). To identify Arabidopsis proteins that interact with AtGRP7 we will use a BD Matchmaker
yeast two hybrid system (Clontech). My research group has had much experience with yeast
two hybrid screens. We have ligated Atgrp7 cDNA into BD Matchmaker yeast two hybrid vector
pGBKT7, which will express AtGRP7 fused to GAL4 DNA-binding domain (DBD). As part of
another project we have made Arabidopsis cDNA libraries in pGADT7, a vector that will express
GAL4 activation domain (AD) fusions. The cDNA libraries were made to untreated wild type
plants, P. syringae infected plants, and plants infected with an avirulent P. syringae strain. We
will perform yeast two hybrid assays by mating yeast strains that express AtGRP7-DBD or
Arabidopsis AD fusions screening for rescue of leucine and adenine auxotropy (growth on
plates lacking Leu and Ade) and α-galactosidase activity (blue pigmentation on X-α-Gal plates).
Any positive interactors will be retested with each insert in the other vector. Sequencing the
inserts will allow us to make a ranked list of putative interactors, which will then be tested in
GST pull-down assays. We will perform GST-pull-down assays by first labeling the plant protein
with 35S by cloning the cDNA into pSP64(polyA) vector (Promega, Madison, WI). We will use the
TnT Coupled Wheat Germ Extract in vitro Translation System (Promega) to transcribe and
translate the putative interactor. The resulting [35S]-labeled product will be incubated with
Glutathione Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, NJ) that were
previously incubated with semi-purified AtGRP7-GST. Any interaction between AtGRP7-GST
and a plant protein would be detected by SDS-PAGE analysis followed by autoradiography. We
will be especially interested in interactors that are known to be associated with innate immunity.
However, since little is known about AtGRP7, this sub-aim will likely shed additional light on how
AtGRP7 functions in plants.
g. Determine the innate immunity-related phenotypes in plants that over-express
AtGRP7 and in complementation experiments with a site-directed Atgrp7 mutant. Atgrp7
mutant plants are more susceptible to P. syringae based on symptom production and bacterial
multiplication in planta (Fig. 6B and C). To determine if plants over-expressing AtGRP7 display
increased resistance we will perform similar pathogenicity assays on these plants. To overexpress AtGRP7-HA we will also transform wild type A. thaliana Col-0 with Atgrp7 downstream
of the CaMV 35S promoter or dexamethasome (Dex)-inducible promoter (12). If these plants
display increased resistance to P. syringae it would suggest that the plant defense
transcriptome can be more efficiently translated into components of innate immunity with higher
amounts of AtGRP7 present. This would provide additional evidence that AtGRP7 plays a role
in innate immunity. These plants would be tested against other pathogens as described in Aim
3c below. Additionally, These over-expressing AtGRP7 plants may be useful in experiments that
seek to identify RNA targets of AtGRP7 described above.
An AtGRP7 site-directed mutant (AtGRP7R47K-HA, see Fig. 5C) substituted with lysine,
which can no longer ADP-ribosylated by HopU1 will be introduced into Arabidopsis using the
Agrobacterium-mediated floral dip method (18). We will also transform the Atgrp7 mutant with
wild type Atgrp7 fused to HA. Importantly, these transgenes will be expressed from their native
promoter, which we have functionally defined using transient expression in tobacco (data not
shown). Transgenic plants that express AtGRP7R47K-HA will be used to determine whether the
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identified phenotypes shown in Fig. 6 are observed when the Atgrp7 mutant plant is
complemented with an AtGRP7 derivative that cannot be ADP-ribosylated. For example, is
callose deposition restored in the complemented Atgrp7 mutant in response to flg22 (Fig. 6D)?
We expect that wild type AtGRP7 will complement reduced callose deposition as well as the
enhanced susceptibility to P. syringae. If callose deposition is no longer reduced when Atgrp7
plants are complemented with the AtGRP7 mutant it would suggest that HopU1 suppresses
callose deposition by modifying R47 residue within AtGRP7. Importantly AtGRP7 mutant that
we use in complementation experiments will first be tested to determine that it retains its ability
to bind RNA. In these experiments, we are most interested in AtGRP7 derivatives that cannot
be ADP-ribosylated by HopU1, but retain their RNA binding activities. It is possible that plants
expressing such a protein may have increased resistance to a bacterial pathogen delivering
HopU1.
Anticipated problems and alternate strategies. We have all of resources in hand to carry
out these experiments and foresee no major problems with the techniques. However, if we fail
to identify an AtGRP7 derivative that cannot be ADP-ribosylated but retains normal RNA binding
this would prevent us from addressing whether a plant expressing such a derivative would be
more resistant to a pathogen injecting HopU1. However, this result would strengthen the idea
that ADP-ribosylation of AtGRP7 prevents it from binding to RNA. These derivatives would be
included in the RNA binding experiments described above.
5.b. AIM 2: Identify additional substrates of HopU1 and verify their involvement in innate
immunity.
a. Rationale. The goal of the first objective in this Aim is to determine if the other in vitro
substrates of HopU1 identified in the Preliminary Studies section and any others identified
represent in vivo substrates. Additionally, another approach is proposed to identify in vivo
HopU1 substrates independent of our in vitro studies. Other goals of this Aim are to determine
the role these substrates play in the innate immune system. Once HopU1 in vivo substrates are
confirmed we will elucidate their function in host cells by various strategies including the use of
available Arabidopsis T-DNA mutants or RNA interference (RNAi) approaches to knock-out or
knock-down substrate expression.
b. Determining the role that HopU substrates have on plant innate immunity. This
sub-aim will primarily be focused on functional analyses of confirmed HopU1 substrates.
Confirmed ADP-RT substrates will be investigated further to determine their contribution to
innate immunity. The general strategy that we will use is to test plants that are reduced or no
longer express individual substrates for differences in how they respond to bacterial infections
and defense responses. We have acquired available T-DNA mutants from the Salk Institute
Genomic Analysis Laboratory (SIGnAL, http://signal.salk.edu/)(9) or, if there is none available
for a specific substrate gene, we will use RNAi technologies to knock-down expression of
specific substrates and then test these knocked-out or knocked-down plants in several of the
assays described in Aim 3. Any T-DNA mutants that we receive will be confirmed to have the
correct T-DNA insert with PCR amplification using T-DNA border primers and primers that
anneal to the substrate gene. Plants homozygous for the T-DNA insertions will be identified by
screening self-fertilized progeny from the mutant using PCR amplification and confirmed not to
make the corresponding mRNA with RT-PCR. Such plants will be infected with P. syringae
strains (pathogenic, nonpathogenic, and avirulent strains) to determine how resistant they are to
wild type DC3000, DC3000 hrcC mutant, and DC3000(pavrRpt2) based on bacterial
multiplication in planta and disease symptom production. These plants will also be tested in
several of the other assays described in Aim 3 including callose deposition, elicitation of the HR,
oxidative burst, and phenolic accumulation. T-DNA substrate gene mutants showing innate
immunity-related phenotypes will be complemented by making transgenics with the wild type
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
substrate gene using Agrobacterium-mediated transformation with a binary vector with a
hygromycin selectable marker. These transgenics will be used in the above assays to insure
that the phenotypes observed were due to the T-DNA insertion and not due to an unrelated
mutation or polymorphism. If we identify T-DNA substrate mutants that display a reduction in
innate immunity outputs, we will determine the effect that over-expression of the specific
substrate has on innate immunity.
If T-DNA mutants are not available or the ADP-RT substrate gene is a member of a
large gene family, we will use RNAi techniques to knock-down the expression of a substrate
gene to determine its contribution to innate immunity (117, 182). RNAi approaches have
advantages over T-DNA mutants because they potentially could allow for the design of knockdown constructs that reduce the expression of several members of a gene family. Additionally,
inducible RNAi approaches would also allow investigation of essential gene products, which
would be absent from the Arabidopsis T-DNA mutant library. The disadvantage of this approach
is that it generally requires stably transformed Arabidopsis and the targeted gene usually
remains expressed to a certain extent, albeit at a much reduced level (i.e., knocked-down). To
make RNAi constructs we will recombine small fragments (about 300 bp) of the substrate gene,
amplified using PCR, into the Gateway destination vector p*7GWIWG(II) (104). This vector is
very similar in design to the popular pHELLSGATE vectors (88) and contains two recombination
sites in opposite orientations on either side of an intron. When this construct is transformed into
plants, the dual copies of the inserted fragment are expressed from a 35S promoter producing
an RNA hairpin (hpRNA), which initiates post-transcriptional silencing of RNA (182). The choice
of the fragment expressed will determine the specificity of the RNA silencing. In most cases, we
will likely choose a fragment from within the 3’ UTR of the substrate gene. We will make
transgenic Arabidopsis plants expressing hpRNA of substrate genes and confirm that the
HopU1 substrate mRNA is reduced with Northern analysis or RT-PCR. These plants will be
used in assays described above to determine if they are altered in innate immunity. Certain
substrate genes may be required for plant development and may not tolerated being
constitutively silenced. In these cases, we will use inducible expression systems by modifying
binary vectors that we routinely use or by acquiring binary vectors with inducible promoters
made for these applications (31, 75).
c. Identification of additional in vitro substrates of the HopU1 ADP-RT. As shown in
the preliminary data, we have identified several in vitro substrates of HopU1. To accomplish this
we scaled-up the Arabidopsis extracts and used ion exchange chromatography to fractionate
the soluble proteins for use in ADP-RT activity assays. Each identified HopU1 substrate was
confirmed to be a substrate by expressing each substrate-GST fusion in E. coli, partiallypurifying each one, and using each substrate-GST fusion in ADP-RT assays to confirm that it
was ADP-ribosylated. As described in Table 1 and Figure 5A, all of the identified and confirmed
substrates, thus far, are either chloroplast RNA-binding proteins (CP-RBPs) or glycine-rich
RNA-binding proteins (GR-RBPs). We are at a point in these experiments where we suspect
that the high-affinity in vitro substrates of HopU1 belong solely to these two classes of proteins.
However, there are several additional experiments described below that we will do before we
formally arrive at this conclusion.
There are several ADP-RT activity spots that do not align with a Coomassie-stained spot
suggesting that the substrate’s concentration may be too low for identification. To attempt to
identify these proteins we will modify the plant extract preparation to further concentrate and
fractionate the ADP-RT substrates. For example, we can use either acetone or ammonium
sulfate precipitation to concentrate plant extracts because these do not interfere with the ADPRT activity of HopU1 (Fu and Alfano, unpublished). Additionally, we have separated plant
extracts with preparative isoelectric focusing using the Rotofor system (Bio-Rad) to fractionate
the proteins based on their pI. We will also determine if we can achieve better purification and
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
separation using hydrophobic interaction and/or gel filtration chromatography. Finally, as an
alternative to the [32P]-NAD approach, we have recently found that HopU1 can use biotinylated
NAD to ADP-ribosylate proteins in plant extracts. This may be useful in the identification of
substrates that are in low abundance because it allows HopU1 substrates to be tagged with
biotin facilitating their isolation and concentration with avidin affinity chromatography. This
approach has been used to identify the substrates for other ADP-RTs (188, 189).
The remaining HopU1 in vitro substrates may be other CP-RBPs and GR-RBPs. The
Arabidopsis genome has 8 known CP-RBP and at least 18 GR-RBP potential genes ((125) and
database searches). With the exception of alternatively spliced versions, we have cloned all of
the known Arabidopsis CP-RBP cDNAs and we are currently determining which can act as
HopU1 substrates. To determine whether HopU1-His could ADP-ribosylate other CP-RBPs and
GR-RBPs we will clone additional RBP cDNAs into GST fusion vectors to determine if their
protein products are ADP-ribosylated by HopU1. This is important to insure that we do not
overlook an important substrate for HopU1, which may be less abundant in our protein extracts.
We are also cognizant that there may be other unidentified CP-RBP and GR-RBP genes
present in the Arabidopsis genome.
As part of Aim 2 we are identifying T-DNA mutants and RNAi knock-down Arabidopsis
lines that are defective in different RBP substrates of HopU1 to determine the extent of their
involvement in innate immunity. We will also use these plants as a source for extracts for in vitro
ADP-RT assays, which will have two purposes: First, they will confirm that the identified activity
spots on earlier ADP-RT two dimensional gels corresponded to specific RBPs because a
specific activity spot should be absent from ADP-RT assays using Arabidopsis defective in the
corresponding RBP (e.g., see Fig. 6A); secondly, this line of experimentation should be useful
because it may allow us to identify lower affinity substrates. That is, by using extracts that lack
high affinity substrates, it may allow substrates that are less abundant or have a lower affinity for
HopU1 to be ADP-ribosylated and, therefore, become visible on ADP-RT autoradiograms.
d. in vivo ADP-RT activity in Arabidopsis. We would like to study bacterial-delivered
HopU1 ADP-ribosylation. Therefore, we will initiate in vivo ADP-ribosylation experiments using
two different approaches. Dorothee Staiger has supplied us with Sinapis alba GRP (SaGRP)
antibodies (86) that recognize AtGRP7 and AtGRP8 for use in immunoblot analyses to
determine if the mobility of these GR-RBPs are altered on 1D and 2D SDS-PAGE gels in a
HopU1-dependent manner after HopU1 is injected by the TTSS. In these experiments we will
deliver HopU1 into plant cells using wild type DC3000 and P. fluorescens(pLN18)(99). pLN18
encodes a functional P. syringae TTSS and no effector proteins. Expressing HopU1 in this
strain allows for the type III delivery of near native levels of HopU1 into plant cells with no other
type III effector traffic (in contrast to DC3000, which injects >30 effectors). After different time
points, ranging from several hours to several days (determined empirically), we will harvest leaf
tissue infiltrated with bacteria. These samples will be subjected to 1D and 2D PAGE and
immunoblot analysis using SaGRP antibodies (170). We will include several bacterial strains as
controls in these experiments including the DC3000 hopU1 mutant, a DC3000 TTSS- mutant, P.
fluorescens(pLN18) without hopU1, P. fluorescens(pLN18) with hopU1DD (which encodes the
HopU1 active site mutant), and P. fluorescens carrying a pLN18 derivative encoding a
nonfunctional TTSS. As shown in Fig. 5D, we can detect ADP-ribose modifications on 1D SDSPAGE gels with immunoblot analyses and these differences should be more pronounced using
2D PAGE. Thus, if we are able to see a shift in mobility with AtGRP7 in a HopU1-dependent
manner, we will conclude that this substrate is modified in vivo by HopU1.
We will perform similar experiments as those described above for other substrates
where antibodies that recognize them are not available by making transgenic Arabidopsis plants
that produce each substrate fused to an epitope that can be detected with commercially
available antibodies. To accomplish this, we are currently cloning the cDNAs of the other
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substrates downstream of their native promoters in a cloning vector such that they produce the
RBP fused to an HA epitope. Where possible we have relied on information on their promoters
from the literature, but in the absence of such information, we will clone 1.5 kb upstream of the
start codon of each substrate gene. Ultimately, gene fusions will be re-cloned into a pPZP212
derivative (78), an Agrobacterium binary vector that lacks a promoter. We will perform
Agrobacterium transient assays to determine whether the substrate can be detected with
immunoblots using an anti-HA antibody (Roche). We have performed Northern blot analysis on
HopU1 substrate genes and mRNA can be detected in Arabidopsis leaf tissue for each gene
indicating that they are expressed in adult Arabidopsis leaves (Jeong and Alfano, data not
shown). Arabidopsis plants will be transformed with each construct and T1 plants that express
epitope-tagged HopU1 substrates will be identified by immunoblot analysis. These plants will be
infiltrated with P. fluorescens(pLN18)(phopU1) and P. fluorescens(pLN18)(phopU1DD) and leaf
tissue will be harvested and analyzed with 1D and 2D SDS-PAGE and immunoblots to
determine if the mobility of the substrate is altered in the presence of HopU1. Because it is not
critical at this juncture to be working with homozygous lines, these plants should be relatively
easy to produce. This approach will allow us to determine if the in vitro HopU1 substrates
represent in vivo substrates.
The second approach to identify in vivo substrates of HopU1 is independent of the
identified HopU1 in vitro substrates. In this approach, we will directly identify ADP-ribosylated
proteins from plant suspension-cultured cells by introducing [32P]-NAD into cells with and
without HopU1 and then analyzing plant extracts from these cells with SDS-PAGE and
autoradiography. Commassie-stained protein spots that co-migrate with [32P]-labeled proteins
will be identified using mass spectrometry as described in our Preliminary Studies section. For
these experiments, we will use Arabidopsis Col-0 and tobacco cultivar Bright Yellow 2 (BY2)
suspension-cultured cells. We maintain both types of cells at Nebraska in the Plant
Transformation Center. We will deliver hopU1 DNA into cultured cells with Agrobacterium.
Based on pilot experiments delivering GUS DNA into these cells, as many as 30% of the cells
are transiently transformed. The cells will be electroporated in a solution containing 1 μm [32P]NAD, incubated for 2 h, and sonicated. The cell lysates will be subjected to SDS-PAGE and
autoradiography to detect [32P]-labeled proteins. We have several parameters to determine
empirically. These include the introduction of [32P]-NAD and HopU1, and the amount of total
cells needed to detect in vivo ADP-ribosylated substrates. If our initial attempts do not succeed,
we will use transformed suspension-cultured cells that express HopU1 or deliver HopU1 into
plant cells via a type III secretion system. The main obstacle in these experiments is to
introduce enough [32P]-NAD into the plant cells to allow in vivo labeling of the ADP-RT
substrates. If not enough NAD is introduced into cells by electroporation, we will use tetanolysin
(List Biologicals), a protein that makes pores in plasma membranes, which has been used
successfully to introduce impermeable molecules into animal cells (21) including NAD for in vivo
ADP-ribosylation experiments (159). It is possible that the plant cell wall may prevent tetanolysin
from reaching the plasma membrane and if this occurs, we will treat the cells with the
macerating enzymes cellulase and pectolyase to partially remove the cell wall to enhance
tetanolysin treatments. This may require the presence of an osmoticum such as mannitol to
prevent the damaged cells from lysing. If tetanolysin proves unhelpful, we will try other agents
that form pores in membranes such as lysolecithin or digitonin (89). Personnel from the Alfano
laboratory will visit the Barbieri laboratory (See enclosed letter) to learn how to perform similar
experiments using mammalian cells lines, which should also help in adapting these experiments
for use with plant suspension-cultured cells.
e. Identify the mammalian proteins ADP-ribosylated by HopU1 in vitro. This
objective is essentially the same as sub-aim 2c except here we will be using animal cell extracts
instead of plant extracts. These experiments will be done in collaboration with Dr. Joe Barbieri
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at the Medical College of Wisconsin (please see enclosed letter). The Barbieri laboratory has
done similar studies identifying substrates for ADP-RTs from P. aeruginosa and they will provide
reagents and expertise to facilitate the identification and the characterization of the animal
HopU1 targets. We will use HeLa cells for these experiments because this cell line was used to
identify many of the substrates for P. aeruginosa ExoS and ExoT these cells contain HopU1
substrates (data not shown). We will follow the Barbieri protocol for preparing the cell line
extracts, which includes an acetone precipitation step. [32P]-NAD ART assays will be perform as
describe above and subjected to 2D SDS-PAGE and autoradiography. Protein spots that align
with ADP-RT activity spots will be identified using MALDI-TOF and Q-TOF MS. Importantly,
partially-purified ExoS-His will be used as a positive control in these experiments, which also
necessitates the inclusion of its co-factor FAS (60). This is important because it will allow a
comparison between the ADP-RT activity of HopU1 and ExoS. We do not foresee problems with
these experiments. Based on the intensity of the labeling on the autoradiogram (data not
shown), HopU1 has a strong affinity for this mammalian substrate. It will be of great interest to
determine if it is similar to the substrates identified from Arabidopsis.
f. Expected Outcomes. We have tried to articulate an experimental plan that is
systematic, well prioritized, and focused. At the conclusion of these experiments we expect to
know the identity of the major plant substrates of the HopU1 ADP-RT. Additionally, we expect to
know the contribution of the major substrates of HopU1 to innate immunity. We also will attempt
to establish an in vivo ADP-RT assay. And finally, we will know the identity of HopU1 substrates
in animal cells.
5.c. AIM 3: Analyze the affect that HopU1 has on host-microbe interactions.
a. Rationale. In this aim we will perform a number of experiments to determine the
status of the innate immune system in Arabidopsis expressing HopU1. As shown in Fig.2, our
preliminary data demonstrates that HopU1 suppresses specific innate immune responses.
Therefore, in this Aim we will determine to what extent the innate immune system is impaired in
these plants. Importantly, this Aim also seeks to establish additional assays to monitor innate
immunity, which will be useful in several of the experiments elsewhere in this application.
b. Determine the extent that virulent and nonvirulent pathogens grow and cause
disease in Arabidopsis transgenic plants expressing HopU1. In these experiments we will
infect wild type Arabidopsis Col-0 plants and Col-0 transgenic plants expressing HopU1 or
HopU1DD plants with P. syringae or the Oomycete pathogen Peronospora parasitica and
determine whether the HopU1-expressing plants enable these microorganisms to grow better
and cause enhanced disease symptoms. As shown in the preliminary data, we already have
made transgenic Arabidopsis plants constitutively expressing HopU1. These plants express
HopU1 from the 35S promoter and appear identical to wild type plants in terms of their growth
and propagation. We are in the process of making transgenic plants using a chemicallyinducible system that expresses HopU1 after induction with estradiol (193).
The Arabidopsis lines expressing HopU1 will be infected with 3 different strains of P. s.
tomato DC3000: Wild type DC3000, which is pathogenic on Arabidopsis Col-0; DC3000 hrcC, a
TTSS mutant that is severely reduced in its ability to multiply in plants and is essentially
nonpathogenic, and DC3000(pavrRpt2), a strain that expresses AvrRpt2, which is recognized
by the RPS2 R protein triggering innate immunity. We chose these strains because they differ in
the innate immune responses that they induce. Because DC3000 is pathogenic on Arabidopsis
it successfully suppresses innate immunity long enough to cause disease; the DC3000 hrcC
mutant does not inject type III effectors, but it does induce PAMP-triggered immune responses
(Jamir and Alfano, unpublished); and DC3000(pavrRpt2), a strain that expresses the AvrRpt2
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Avr protein, which is a well described Avr protein (116) that induces innate immune responses
on Col-0 plants, including the HR.
To determine whether the HopU1 ADP-RT plants exhibit enhanced susceptibility we will
also test the transgenic plants with another Arabidopsis pathogen, the Oomycete Peronospora
parasitica, which causes Downy mildew (162). In a similar strategy as described above, we
chose 3 different P. parasitica isolates because they differ in virulence on Arabidopsis Col-0.
Emco5 is fully pathogenic on Col-0, Cala2 is weakly virulent, and Hiks1 induces a strong innate
immune response due to the presence of an Avr protein (33, 131). P. parasitica
sporangiophores at a concentration of approximately 5 X 104 spores/ml will be sprayed onto 7
day old Arabidopsis seedlings. The plants will be grown in a growth chamber set to a 10 h light
cycle and 18°C. The production of sporangiophores will be determined 8 days after inoculation
by counting spores on both sides of the cotyledons as described (131). We will also determine
whether any of the isolates can elicit an HR in our plants by staining with lactophenol-trypan
blue as described (111). If we have difficulties with P. parasitica assays we will consult Dr. John
McDowell at the Virginia Polytechnic Institute, he provided the P. parasitica isolates to us and
has agreed to help us establish these assays in our laboratory.
c. Determine the innate immune responses that are altered in HopU1-expressing
Arabidopsis plants. In this sub-objective we will determine to what extent the physical outputs
of innate immunity are suppressed in Arabidopsis plants expressing different HopU1. One assay
that we will continue to rely on is the callose deposition assay, which stains callose in the plant
cell wall after challenging the plant with either a PAMP or an Avr. Our experiments shown in Fig.
2 indicate that HopU1-expressing plants suppress this response when challenged with flg22 (an
active peptide from flagellin). For these experiments and others using flg22 it is important to
note that we will use two different negative controls, flg22 from Agrobacterium, which is not
recognized by Arabidopsis, and an Arabidopsis FLS2 mutant, which no longer recognizes flg22
(both kindly provided by Dr. Thomas Boller, Basel, Switzerland). We will also determine whether
Avr-triggered callose deposition is suppressed in HopU1 plants. In these experiments we will
deliver a type III effector that is recognized by A. thaliana Col-0 (e.g., AvrRpm1 or AvrRpt2)
using P. fluorescens carrying a construct that encodes an Avr protein and cosmid pLN18, which
encodes a functional P. syringae TTSS. This system has been used successfully to identify
several DC3000 type III effectors that suppress the HR (99). Additional assays will be included
to assess the defense response in similarly challenged plants. For example, we will determine
whether the production of defense-related phenolic compounds are induced in HopU1
transgenic plants by using a toluidine blue based assay (106, 148). In this assay, leaf tissue is
cleared of chlorophyll by treating with methanol and stained with toluidine blue. The tissue is
then viewed with light microscopy (100-400X magnification) for blue/green regions in the plant
cell wall indicative of phenol accumulation. We will also view these plants with fluorescence
microscopy because tissue regions that have substantial amounts of phenolics autofluoresce.
d. Microarray analyses of HopU1-expressing plants challenged with different
activators of innate immunity. We will determine the mRNA expression profile of wild type
and HopU1 transgenic Arabidopsis challenged with flg22, DC3000, DC3000 hrcC, and
DC3000(pavrRpt2) with Affymetrix microarrays. It is important to note these experiments will
use HopU1 transgenic plants that we are currently making that expresses HopU1 upon
exposure to estradiol (193). There have been recent studies profiling Arabidopsis gene
expression in response to flg22 (140, 192), DC3000, and DC3000(pavrRpt2) using Affymetrix
chips (173). Therefore, we have a significant data set to help establish these experiments at
Nebraska. We are interested in the differences in gene expression between ADP-RT expressing
plants and control plants because this may indicate signal transduction pathways that are
affected by the ADP-RT transgenes. One potential problem with these experiments is that any
Research Design & Methods
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
difference in gene profiling between control and experimental plants may not be due to the
ADP-RT activity of these proteins, but instead to a nonspecific effect. To distinguish between
these two possibilities, in addition to a standard negative control, we will make transgenic plants
expressing a catalytically inactive ADP-RT (e.g., HopU1DD). Patterns should emerge that
provide clues to the role of HopU1 in host cells. Also, It will be important to compare the sets of
differentially expressed genes with related studies (140, 173, 192) and other Arabidopsis
profiles exhibited in response to plant hormones or abiotic and biotic stresses (32, 178, 181,
187).
e. Expected outcomes. Many of the objectives of this Aim will be useful for experiments
described in the other Aims. The biotic stress experiments described in Aim 3b will determine
whether HopU1 plants better support the growth of pathogens in an ADP-RT dependent
manner. The remaining experiments of this Aim test innate immunity by quantifying the outputs
(Aim 3c), or the differences in gene expression (Aim 3d) in plant expressing HopU1. We expect
these to be extremely useful because they will allow us to better categorize the innate immunity
suppression activity of HopU1. And, finally, in terms of the central hypothesis of this application,
we expect these experiments to shed light on which innate immunity pathways are targeted by
HopU1.
TIMELINE. The timetable for the experiments proposed in this proposal are indicated in the
table below.
TIMETABLE
AIMS/TASKS
Year 1
Year 2
Year 3
Year 4
Year 5
Aim 1
sub-aim b
sub-aim c
sub-aims d-e
sub-aims f-g
Aim 2
sub-aims b-c
sub-aim d
sub-aim e
Aim 3
sub-aims b-c
sub-aim d
Future Directions. At the conclusion of these experiments we expect to know the consequence
of the ADP-ribosylation of AtGRP7 by the HopU1 ADP-RT. Additionally, we should know other
RNA targets of AtGRP7 and whether the plant defense transcriptome is enhanced by AtGRP7.
Moreover, we will identify other in vivo ADP-RT substrates of HopU1and we will have laid the
ground work for future experiments. These will dissect the molecular details that explain how
ADP-ribosylation can interfere with innate immune systems.
Research Design & Methods
Page 68
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
12. Vertebrate Animals
Not applicable.
Vertebrate Animals
Page 69
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
13. Select Agent Research
Not applicable.
Select Agent Research
Page 70
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
14. Multiple PD/PI Leadership Plan
Not applicable.
Multiple PI Leadership Plan
Page 71
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
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15. Consortium/Contractual Arrangements
No consortium/contractual arrangements have been made for the research described in
this application.
Consortium/Contractual
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Letters of Support
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Letters of Support
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Letters of Support
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Letters of Support
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
17. Resource Sharing
1. The Data Sharing Plan is not applicable to this application.
2. Sharing Model Organisms. We will make available all resources that result from
experiments described in this proposal at the time of publication. P. syringae bacterial
mutants will be made available to other researchers through the Pseudomonas syringae
website at http://pseudomonas-syringae.org/. Arabidopsis transgenic plants made for
these experiments will be available through the Arabidopsis Biological Resource Center
at The Ohio State University (url: http://www.biosci.ohiostate.edu/~plantbio/Facilities/abrc/abrchome.htm). Our microarray results as well as any
protocol that we develop or refine using Arabidopsis will be made available through the
Arabidopsis Information Resource webpage (http://www.arabidopsis.org/) maintained at
Stanford University.
Sharing—Data and Model Organism
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Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
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source(s). Otherwise, leave this section blank.
*Budget Period *Anticipated Amount ($)
*Source(s)
5. Assurances/Certifications (see instructions)
In agreeing to the assurances/certification section 18 on the SF424 (R&R) form, the authorized organizational representative agrees to
comply with the policies, assurances and/or certifications listed in the agency's application guide, when applicable. Descriptions of
individual assurances/certifications are provided at: http://grants.nih.gov/grants/funding/424
If unable to certify compliance , where applicable, provide an explanation and attach below.
Explanation:
Checklist
Tracking Number:
Page 90
Principal Investigator/Program Director (Last, first, middle): Alfano, James, Robert
Attachments
CertificationExplanation_attDataGroup0
File Name
Mime Type
Checklist
Tracking Number:
Page 91