Progress 12/15/06 to 12/14/07
Outputs
OUTPUTS: Transgenic AGP17-Expressing BL-1 Arabidopsis Lines In collaboration with Dr. Stanton Gelvin at Purdue University, we have generated eight independent transgenic Arabidopsis BL-1 lines expressing the AGP17 arabinogalactan protein. We hypothesize that expressing the putative receptor in a transformation and attachment deficient ecotype of Arabidopsis will result in improved transformation efficiency and increased attachment to roots. These experiments will allow us to demonstrate more directly the role of AGP17 in the infection of plants by the tumorigenic A. tumefaciens bacteria. We have analyzed several AGP17-expressing BL-1 transgenic Arabidopsis lines using a modified MUG fluorometric readout assay. Our initial results suggest that several of these lines exhibit enhanced transformation efficiency, consistent with what would be expected if AGP17 were to promote bacterial attachment. Expression of AGP17 in BY-2 Tobacco Cells Arabinogalactan proteins make up a family of
extensively glycosylated plant cell wall associated components. The arabinogalactan sugar additions are unique to plants and not commonly found elsewhere. The protein backbone of arabinogalactan proteins is thought to be largely inaccessible because of the sugar additions and is therefore unlikely to interact directly with other factors. Unfortunately the methods for purifying arabinogalactan proteins from plants isolates many different arabinogalactans, and it is not possible to isolate an individual arabinogalactan protein species. Antibody-based isolation methods are also unlikely to succeed as it is difficult to generate specific antibodies against the protein backbone of these extensively glycosylated proteins. Isolating a specific arabinogalactan protein by overexpression in bacteria or yeast is unlikely to yield a physiologically functional arabinogalactan as the essential sugar additions will not be made in these cell systems. Our modified AGP17 construct contains a Strep-tag
at the C-terminus and we have eliminated the site predicted to be modified by GPI addition. As a result of not having the GPI addition, the modified construct will be secreted from the BY-2 plant cells as opposed to bound to the cell membrane or cell wall. The presence of the epitope tag will allow us to purify the AGP17 protein from BY-2 culture supernatants. This approach we hope will allow us to isolate correctly glycosylated, highly purified AGP17 that we can use directly in binding assays with A. tumefaciens bacteria. We have now begun work with a synthetic AGP17 construct that has been modified to incorporate the best predicted tobacco codon usage. The synthetic construct was synthesized commercially by DNA2.0. We are now in the process of cloning the synthetic AGP17 construct into a vector that can be used to express the gene in BY-2 cells. This project is currently being carried out by undergraduate Stacie Ann Newell. Ms. Newell was awarded a prestigious American Society of
Microbiology summer research fellowship to work on this project. She presented her work in a poster on May 23, 2007, at the international ASM General Meeting in Toronto, Canada.
PARTICIPANTS: During the reporting period 12/15/2006 to 12/14/2007 the PI, one graduate student (Anna Petrovicheva) and four undergraduates (Leslie Aguirre*, Anna Cheban, Stacie Ann Newell*, and Lourdianne Pierre-Charles*; * indicates minority student)developed techniques and materials that have advanced our work on the study of the role of the arabinogalactan (AGP), AtAGP17, in attachment and infection by Agrobacterium tumefaciens. Outcome/impact - Change in Knowledge: The participants listed above all benefited and learned from the work conducted on this project. Anna Cheban's research experience in the lab helped in earning her a position in medical school during this period. Stacie Ann Newell was awarded and ASM-MURF fellowship for her work on this project and presented her results at the annual ASM meeting in Toronto (2007). Her research experience has also helped in acceptance into a nursing program. Lourdianne Pierre-Charles has used her research experience to enter a training
program that will help prepare her for future entry in medical school. Leslie is still an undergraduate at Brooklyn College and intends to continue with her research on this project in the lab. Anna Petrovicheva continues to work on this project as a graduate student. She presented her most recent findings at the annual Crown Gall meeting in December 2007. The PI used the advances made on this project and several of the assays developed in this work to develop an NSF-CCLI (Course Curriculum and Laboratory Innovation) proposal. The proposal was funded in the spring of 2007, and the PI is now working on further developing the assays and adapting aspects of the project for inclusion in an authentic research based microbiology lab for undergraduate students (funded by the NSF-CCLI proposal and additional leveraged support from Brooklyn College). During this period the PI was also promoted to Associate Professor.
TARGET AUDIENCES: The PI used the advances made on this project and several of the assays developed in this work to develop an NSF-CCLI (Course Curriculum and Laboratory Innovation) proposal. The proposal was funded in the spring of 2007, and the PI is now working on further developing the assays and adapting aspects of the project for inclusion in an authentic research based microbiology lab for undergraduate students (funded by the NSF-CCLI proposal ). Several assays developed as a part of the USDA funded AGP project during this period were adapted for introduction as pilot exercises in the undergraduate microbiology lab sections. These undergraduate training efforts reached 32 students during this time period.
PROJECT MODIFICATIONS: One objective of this proposal, expressing the AtAGP17 modified protein in BY-2, cells is currently under modification. For reasons that are not clear the construct could not be maintained in bacterial cells, possibly because of toxicity to these cells. The AtAGP17 construct will be moved into other inducible vectors that may avoid this problem.
Impacts
During the reporting period 12/15/2006 to 12/14/2007 the PI, one graduate student (Anna Petrovicheva) and four undergraduates (Leslie Aguirre*, Anna Cheban, Stacie Ann Newell*, and Lourdianne Pierre-Charles*; * indicates minority student)developed techniques and materials that have advanced our work on the study of the role of the arabinogalactan (AGP), AtAGP17, in attachment and infection by Agrobacterium tumefaciens. Outcome/impact - Change in Knowledge: The participants listed above all benefited and learned from the work conducted on this project. Anna Cheban's research experience in the lab helped in earning her a position in medical school during this period. Stacie Ann Newell was awarded and ASM-MURF fellowship for her work on this project and presented her results at the annual ASM meeting in Toronto (2007). Her research experience has also helped in acceptance into a nursing program. Lourdianne Pierre-Charles has used her research experience to enter a
training program that will help prepare her for future entry in medical school. Leslie is still an undergraduate at Brooklyn College and intends to continue with her research on this project in the lab. Anna Petrovicheva continues to work on this project as a graduate student. She presented her most recent findings at the annual Crown Gall meeting in December 2007. The PI used the advances made on this project and several of the assays developed in this work to develop an NSF-CCLI (Course Curriculum and Laboratory Innovation) proposal. The proposal was funded in the spring of 2007, and the PI is now working on further developing the assays and adapting aspects of the project for inclusion in an authentic research based microbiology lab for undergraduate students (funded by the NSF-CCLI proposal and additional leveraged support from Brooklyn College).
Publications
- No publications reported this period
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Progress 12/15/04 to 12/14/07
Outputs
OUTPUTS: Student training: Over the course of this grant 17 undergraduate students (Leslie Aguirre*, Helen Basyuk, Katherine Bernal, Anna Cheban, Darnelle Delva*, Dahima Elvin*, Avi Harari, Khudeja Mir, Stacie Anne Newell*, Hamit Ozcan, Lourdianne Pierre-Charles*, Mamunur Rahman, Nicole Romeo*, Riza Seit-Khalil, Nel Trasybule*, Michelle Watson*, Esan Wilkinson*), 2 masters students (Kalpana Soni, Kwame McCartney*), one graduate student (Anna Petrovicheva), and one high school student (Danielle Baum) have participated for at least one semester, or one summer, on research projects associated with the role of AtAGP17 in A. tumefaciens infection of host cells. Ten of these students are minority students (indicated by "*"), and more than half of the students are women. In addition to these students who worked extensively in the lab on research projects, two of the root transformation assays developed as a part of this grant were introduced into the undergraduate microbiology lab course
(course number 52.2) taught by the PI. Through this route four sections of 16 undergraduate students in the microbiology lab had an opportunity to learn these assays. Funding: Results generated during the course of this grant period were incorporated into a number of successful grant applications submitted by the PI, including an NSF-Major Research Instrumentation grant and an NSF-Course Curriculum and Laboratory Improvement grant. In 2005 the PI and a colleague, Dr. Dan Eshel, were awarded an NSF-MRI ($210,582) to purchase a BD Biosciences FACSCanto benchtop flow cytometer. We anticipate using the flow cytometer to measure the binding of purified AGP17 to A. tumefaciens. The flow cytometer will also be used in the undergraduate microbiology labs as part of a lab designed to compare a number of cell quantification methods. In 2007 the PI was awarded an NSF-CCLI grant ($197,000) titled Authentic Research Experience in Microbiology for Undergraduate Students at Brooklyn College. The
primary aim of the NSF-CCLI grant is to introduce exciting, authentic, hands-on, research to the undergraduate microbiology labs. Several of the assays and experimental modules that will be used in the modified undergraduate labs were developed as a part of this USDA funded grant. Dissemination (Meetings and conferences): Results generated during the course of the grant were presented at a number of local, national and international meetings. The most significant of these include the annual Crown Gall meetings (a total of four posters over three years), the American Society of Microbiology Annual meetings in Toronto (2007) and the XIII International Congress on Molecular Plant-Microbe Interactions in Sorrento, Italy (2007). Ms. Stacie Anne Newell, an undergraduate student working in the PI's lab, was awarded a prestigious American Society of Microbiology summer research fellowship to conduct research important to this USDA proposal. She presented her work in a poster on May 23, 2007,
at the international ASM General Meeting in Toronto, Canada.
PARTICIPANTS: Individuals: The PI is the only individual who has worked on this proposal. Partner organizations: none. Collaborators and contacts: During the course of this project the PI has continued collaborations with Dr. Stanton Gelvin and his lab at Purdue University. Two undergraduate minority students who have worked on this project, Esan Wilkinson and Darnelle Delva, also spent time at Purdue working in Dr. Gelvin's lab (two summers for Esan Wilkinson and one summer for Darnelle Delva). The PI has also initiated collaborations with Dr. Tom Burr at Cornell University involving a closely related Agrobacterium species, A. vitis. Training or professional development: Over the course of this grant 17 undergraduate students (Leslie Aguirre*, Helen Basyuk, Katherine Bernal, Anna Cheban, Darnelle Delva*, Dahima Elvin*, Avi Harari, Khudeja Mir, Stacie Anne Newell*, Hamit Ozcan, Lourdianne Pierre-Charles*, Mamunur Rahman, Nicole Romeo*, Riza Seit-Khalil, Nel Trasybule*, Michelle
Watson*, Esan Wilkinson*), 2 masters students (Kalpana Soni, Kwame McCartney*), one graduate student (Anna Petrovicheva), and one high school student (Danielle Baum) have participated for at least one semester, or one summer, on research projects associated with the role of AtAGP17 in A. tumefaciens infection of host cells. Ten of these students are minority students (indicated by "*"), and more than half of the students are women. Two laboratory technicians and two graduate student teaching assistants had an opportunity to learn the root infection assay that was introduced in the undergraduate microbiology lab sections.
TARGET AUDIENCES: Target audiences: This project served undergraduate, masters and PhD students at Brooklyn College. The Borough of Brooklyn itself has a population of ~2.5 million people, ~70% of whom are minorities (nearly one million African-Americans and five hundred thousand Hispanics), making it the largest and most diverse of New York City's five boroughs. The undergraduate population of Brooklyn College itself is 31% Black (non-Hispanic), 12% Hispanic, 12.9% Asian/Pacific Islander, and 1.5% Native American. This diversity is represented in the students who conducted independent research in the PI's lab on this project, as well as in the undergraduate students in the microbiology lab sections who worked on modules developed as a result of this project. Efforts: In 2007 the PI was awarded an NSF-CCLI grant ($197,000) titled Authentic Research Experience in Microbiology for Undergraduate Students at Brooklyn College. The primary aim of the NSF-CCLI grant is to introduce
exciting, authentic, hands-on, research to the undergraduate microbiology labs. Several of the assays and experimental modules that will be used in the modified undergraduate labs were developed as a part of this USDA funded grant.
Impacts
Changes in knowledge: Findings and results: Transgenic AGP17-Expressing BL-1 Arabidopsis Lines In collaboration with Dr. Stanton Gelvin at Purdue University, we have generated eight independent transgenic Arabidopsis BL-1 lines expressing the AGP17 arabinogalactan protein. We have analyzed several AGP17-expressing BL-1 transgenic Arabidopsis lines using a MUG fluorometric readout assay. Our results suggest that several of these lines exhibit enhanced transformation efficiency, consistent with what would be expected if AGP17 were to promote bacterial attachment. Expression of AGP17 in BY-2 Tobacco Cells Arabinogalactan proteins make up a family of extensively glycosylated plant cell wall associated components. The arabinogalactan sugar additions are unique to plants and not commonly found elsewhere. Our modified AGP17 construct contains a Strep-tag at the C-terminus and we have eliminated the site predicted to be modified by GPI addition. As a result of not having the
GPI addition, the modified construct will be secreted from the BY-2 plant cells as opposed to bound to the cell membrane or cell wall. The presence of the epitope tag will allow us to purify the AGP17 protein from BY-2 culture supernatants. This approach we hope will allow us to isolate correctly glycosylated, highly purified AGP17 that we can use directly in binding assays with A. tumefaciens bacteria. We have begun work with a synthetic AGP17 construct that has been modified to incorporate the best predicted tobacco codon usage. The synthetic construct was synthesized commercially by DNA2.0. We are now in the process of cloning the synthetic AGP17 construct into a vector that can be used to express the gene in BY-2 cells. Examination of Arabidopsis Plants Containing Mutations in Arabinogalactan Genes: We have searched a collection of Arabidopsis mutants from the Arabidopsis Information Resource (TAIR) and identified at least ten mutant lines harboring T-DNA disruptions within AGP
loci. We now have several mutant lines growing in lab and have begun to examine them for phenotypes. Currently we are looking at the root development of these AGP mutants, and their ability to bind and be transformed by A. tumefaciens using the modified MUG fluorometric assay. So far none of the mutants we have examined have exhibited any significant differences from wild type Arabidopsis. Techniques: Quantitative Measurement of the Attachment of A. tumefaciens to Vitronectin Coated Microspheres Using the BD FACSCanto Flow Cytometer. There is very little known about what host factors A. tumefaciens bacteria recognize on the surface of plant cells. We have designed a protocol for covalently attaching vitronectin to fluorescent polystyrene microspheres, and we are in the process of measuring the attachment of A. tumefaciens to these beads relative to control beads. A quantitative MUG assay has also been developed for assaying the infection of Arabidopsis root segments by A.
tumefaciens.
Publications
- No publications reported this period
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Progress 12/15/04 to 12/14/05
Outputs
Progress Report: The Role of Arabinogalactan, AtAGP17, in A. tumefaciens Infection of Host Cells 1. Expression of AtAGP17 in Arabidopsis ecotype known to be poor hosts for A. tumefaciens and measuring bacterial attachment relative to the parental ecotypes. In collaboration with Dr. Stanton Gelvin we have successfully engineered transgenic Arabidopsis lines expressing the putative attachment factor, AtAGP17. AtAGP17, driven by the CMV promoter, was introduced into the poorly transformed, and poorly bound, Bl-1 Arabidopsis ecotype. We have analyzed 15 independent transgenic lines by the Arabidopsis root tumor assay and by using a newly developed MUG expression assay (described below). Several of the transgenic lines exhibit transformation levels that are significantly increased relative the parental Bl-1 transformation levels. These results are consistent with the possibility that AtAGP17 serves as a receptor for A. tumefaciens at the surface of plant cells infected by
A. tumefaciens. 2. We have recently received $210,00 in funding from the NSF to purchase a BD FACSCanto flow cytometer (NSF-Major Research Instrumentation awarded to D. Eshel, PI and T. Muth, Co-PI). As a result of this acquisition we now have unrestricted access to a flow cytometer and have been able to dramatically increase the amount of time we spend on the flow cytometer developing and improving our flow cytometry assay to measure bacterial attachment to Arabidopsis cells. We are use the SYTO BC dye to fluorescently label A. tumefaciens and then incubate the labeled bacteria with Arabidopsis root segments. Following co-incubation for various time periods, we wash bacteria from the root segments and use the flow cytometer to determine the number of bacteria released from the root segments. Using this assay we will be able to compare the number of bacteria attaching to our parental Bl-1 root segments to the transgenic AtAGP17-expressing root segments. 3. We have recently developed
an improved Arabidopsis root segment transformation protocol. In order to shorten the time of the assay we have switched to using a modified A. tumefaciens strain that transfers a beta-glucuronidase (uidA) marker gene into the cells of successfully transformed root segments. The infection process is the same as the standard root tumor assay, but the expression of the beta-glucuronidase gene can be detected within 5 days by using a fluorometric MUG (4-methylumbelliferyl-beta-D-glucuronide) detection assay. This assay allows us to quickly (less than a week) determine the percentage of transformed root segments as well as giving us quantitative information on the transformation level of each individual root segment. This is a major improvement over the root tumor assay, which takes 4-5 weeks for tumors to develop, and can only give very rough semi-quantitative data on the degree of transformation of each individual root segment.
Impacts
A more detailed understanding of the molecular-level interactions that are involved in the attachment of the bacterial pathogen A. tumefaciens to host plant cells will aid future studies aimed at preventing infections in susceptible crops. This information will also help agricultural biotechnologists improve their protocols for using A. tumefaciens to insert desirable genes into plant species that are currently recalcitrant to infection. Improvements along both these lines may help to reduce economic losses from A. tumefaciens infections, as well as speeding the development of enhanced crops that may better serve farmers and consumers.
Publications
- No publications reported this period
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