Stanton B Gelvin ; Purdue Research Foundation
Identification of Plant Genes Involved in Agrobacterium-Mediated Transformation

Participant Individuals:
CoPrincipal Investigator(s) : Vitaly Citovsky
Post-doc(s) : Veena Veena; Vipin Hallan; HoChul Yi; Ranjith K Tharachaparamba
Technician, programmer(s) : April Clikeman-Johnson
Graduate student(s) : Hau-Hsuan Hwang; Praveen K Rao
Undergraduate student(s) : Anthony Kaiser; Andrea Kopecky; Aneeza Salim; Richard Carl
Post-doc(s) : Luis C Zapata; Esther Mayda; Jaime Mendenez-Humara; Anna Swiderska; Michal Swiderski; Lisa Valentine; Yanmin Zhu
Technician, programmer(s) : Kui Lin
Graduate student(s) : Sang-Ic Kim
Undergraduate student(s) : Joe Lee; Rooz Golshami; Seth Kreger; Carla Reese; Emerald Screws; Amber Lafoon; Angela Valadez
Technical school student(s) : Joerg Spantzel Senior personnel(s) : Alicja Ziemienowitz
Technician, programmer(s) : Pawel Gawalski Post-doc(s) : Tzvi Tzfira
Technician, programmer(s) : Manjusha Vaidya
Senior personnel(s) : Barbara Hohn
Undergraduate student(s) : Adam Stoklosa
Technician, programmer(s) : Toshinori Fujinuma
Post-doc(s) : Kerstin Glans
Technician, programmer(s) : Cynthia Ramos
Post-doc(s) : Benoit Lacroix
Graduate student(s) : Wojciech Strzalka
Technician, programmer(s) : Aftab Kahn
Undergraduate student(s) : Karen Oskins
Technician, programmer(s) : Maxim Radionenko; Badam Yadav
Senior personnel(s) : Lan-Ying Lee
Technician, programmer(s) : Bo Li; Vijay Nadella

Partner Organizations:
Corporation for Plant Biotechnology Rese: Financial Support; Personnel Exchanges

CPBR is headed by Dr. Dorin Schumacher. It is funded by the DOE and the EPA. Some postdocs supported by the CPBR in the Gelvin lab are working on characterizing rat mutants and have been working with postdocs on this grant. For several years, Dr. Gelvin (PI) has received funding from the Corporation of Plant Biotechnology Research, and this funding has been renewed for two more years (until mid-2005). Projects funded by the CPBR complement, but do not overlap with, projects funded by this NSF grant. The major goals of the NSF grant include identification of plant proteins and genes involved in Agrobacterium-mediated transformation using forward genetic, yeast two-hybrid, and microarray/macroarray approaches. Thus, the NSF grant is more of a 'survey' to identify and perform only a superficial characterization of the major players in the transformation process. Dr. Gelvin�s work funded by the CPBR takes these initial observations and extends them into specific plant genes. For example, Dr. Gelvin identified the Arabidopsis histone H2A-1 gene HTA1 as being involved in T-DNA integration. The CPBR grant involves a further investigation of this gene, including patterns of expression, and whether other histone genes are also involved in T-DNA integration. In addition, the CPBR grant includes a screen of T-DNA activation-tagged Arabidopsis lines for hat (hyper-susceptible to Agrobacterium transformation) mutants, whereas the NSF grant funded us to identify rat (resistant to Agrobacterium transformation) mutants. So far, both approaches have yielded interesting mutants and plant genes involved in Agrobacterium-mediated transformation.

Biotechnology Research and Development Corporation: Financial Support

BRDC has provided matching funds for Gelvin's CPBR grant. These funds are being used to indentify and characterize Arabidopsis hat (hyper-susceptible to Agrobacterium transformation) mutants, and to characterize further the use of the histone H2A-1 (HTA1) and other histone genes to increase the efficiency of Agrobacterium-mediated transformation. BRDC is also providing funds to Tzvi Tzfira (Citovsky laboratory-affiliate) to investigate further the use of the VIP1 gene to increase the efficiency of Agrobacterium-mediated transformation.

Other collaborators:

We have initiated interactions with Dr. C.S. Prakash (Tuskegee University) to screen for rat mutants with undergraduates who will visit Purdue University for a summer, then continue working on the screening in his laboratory. During the summer of 2001, two students (Carla Reese and Emerald Screws) visited Purdue University where they learned to screen for rat mutants. They returned to Tuskegee University and continued this work for the 2001-2002 academic year.

There were several purposes of this program: To train minority students in plant genetic and molecular biology research, to encourage minority students to seek a career in science, to establish a close link between laboratories at Purdue University and Tuskegee University, and to serve as a recruitment tool for minority students to the graduate program at Purdue University. The initial program was highly successful. Two excellent students (Carla Reese and Emerald Screws) visited Dr. Gelvin�s laboratory, learned the Agrobacterium/rat mutant screening system, and returned to Tuskegee University where they continued their research throughout their senior year. Dr. Gelvin is very pleased that both of these students are still involved in science, and are seeking advanced degrees; he still maintains contact with these students and is involved in writing recommendation letters for them. Unfortunately, after the first year of this program, Dr. Prakash told Dr. Gelvin that he no longer had the time to help train the students, and that he personally would have to drop out of this program.

For the past two years, we have had a collaboration with Dr. Ted Muth (Brooklyn College, NY). During the summers of 2004 and 2005, Ted sent to the Gelvin lab a minority undergraduate student, Esan Wilkinson. Esan worked in Dr. Gelvin's lab, then returned to Ted Muth's lab and continued his work during the academic year. This relationship helped firmly establish a research collabortion between the two labs. We hope to recruit Esan to Dr. Gelvin's laboratory as a graduate student at Purdue.

Activities and findings:

Research and Education Activities:

The following personnel worked on projects sponsored by this grant during the entire grant period:

1. Identification and characterization of Arabidopsis rat mutants and molecular cloning of RAT genes (Gelvin, Citovsky, Hohn, and Ziemienowicz laboratories, with help from undergraduate students at Tuskegee University).

When we initiated this grant, we had already screened approximately 3000-4000 T-DNA mutagenized lines (Feldmann collection in ecotype Ws) for mutants that were resistant to Agrobacterium transformation (rat mutants). During the course of this grant, we screened approximately 10,000 additional lines from the Feldmann collection, resulting in approximately 14,000 total lines screened from this library. Of the 64 pools of 100 individually mutagenized plants in this collection, we screened representatives from all pools. It is therefore likely that we have approached saturation in screening of this T-DNA insertion library.

Of the lines from the Feldmann collection screened during the course of this grant, approximately 700 appeared to have the rat phenotype upon first screening. After several additional rounds of screening, we have confirmed the following number of rat mutants from this collection:

From efforts previous to this grant: 21
From efforts during the course of this grant: 63
Total rat mutants from forward genetic screening of all libraries: 84
rat mutants from reverse genetic screening (see below): 42
Total rat mutants from all screenings: 126

Of the new rat mutants identified recently, most seem to be blocked at an early stage of the Agrobacterium transformation process; i.e., both transient and stable transformation is blocked. However, a few mutants may be blocked at a later stage (i.e., they can be transiently but not stably transformed) and therefore may be deficient in T-DNA integration. Mutant ratL3 (identified by Lisa Valentine in the Hohn laboratory) is of particular interest as it has a putative DNA binding domain and could be involved in T-DNA integration into the plant genome. A role for the RATL3 gene in T-DNA transformation was confirmed by analyzing tumor assays of allelic mutants obtained from an independent mutant collection (SALK). Both independent allelic mutants had a strong rat phenotype, arid1 mutant having 18-38% and arid2 having 18-36% in the tumor assay. We are currently pursuing further analysis of these mutants.

We have obtained part of the INRA/Versailles collection of T-DNA insertion mutants (in ecotype Ws); of the approximately 4500 lines generated, only approximately half are available from the stock centers. We initiated screening of the available mutant lines from this collection. Much of the screening of this library was undertaken by the Citovsky laboratory. We screened 2,200 plants corresponding to 50 pools of the INRA T-DNA mutant collection. Seven putative mutants were identified in the first round of screening and three of them were confirmed in the second round.

We originally had planned to screen the Amasino/Sussman collection of T-DNA insertion mutants in ecotype Ws. This collection contains approximately 64,000 members. However, we subsequently learned that this disruption library contains within the T-DNA an AP3 gene and, as a consequence, approximately 15% of the lines show lethality. We decided not to use these lines because of our inability to determine whether a rat phenotype would be caused by the T-DNA disruption or a combination of the disruption and the effects of the AP3 gene. Therefore, in collaboration with Dr. Ray Bressan of Purdue University, we generated a new T-DNA disruption library (approximately 70,000 members) in ecotype Ws. We screened this library for rat mutants. To date, approximately 3,000 plants from this line were screened in the Gelvin, Hohn, and Ziemienowitz laboratories. Of the 600 plants screened in the Hohn laboratory, 53 putative mutants were identified in the first round. Seven mutants were confirmed in the second round of screening.

In all, the various laboratories associated with this project screened approximately 20,000 lines from the three T-DNA insertion libraries. We have not identified the same mutant twice (although one mutant was identified both in a forward and a reverse genetic screen). Thus, we do not think that we have approached saturation of the mutant collections.

Vitaly Citovsky sent a postdoctoral research fellow to Stan Gelvin�s laboratory in June, 2000 for a period of one month to receive training in rat mutant screening. He continued screening in the Citovsky laboratory. In February, 2001 three scientists from Barbara Hohn�s and Alicja Ziemienowitz�s laboratories came to Purdue University for one month to receive training in rat mutant screening. In the summer of 2003, another student from Alicja Ziemienowitz�s was trained in the Gelvin laboratory. These scientists will continue screening in their home laboratories, using the Bressan library.

In May, 2001 two undergraduates from Dr. C.S. Prakash�s laboratory at Tuskegee University joined the Gelvin laboratory at Purdue University for the summer. They received training in rat mutant screening, and continued this work in Dr. Prakash�s laboratory during the subsequent academic year. Salaries for these two students were funded by a special supplement to this grant. In the summers of 2004 and 2005, Esan Wilkinson, an undergraduate from Dr. Ted Muth�s laboratory at Brooklyn College, NY received training in rat mutant screening in Dr. Gelvin�s lab. After each summer, he returned to Dr. Muth�s lab and continued working on the project.

We used both plasmid rescue and TAIL PCR to identify T-DNA/plant DNA junctions from the rat mutants. To date, we have sequenced junctions from 36 rat mutants. Many of these junctions lie in or near 'unknown' or 'hypothetical' proteins. We have attempted TAIL-PCR to obtain T-DNA/plant DNA junctions from a large subset of rat mutants. We have obtained junctions from many, but not all, mutants. In many instances, the junctions did not reveal much about the mutagenized gene, either because they were �intergenic�, were in retroelements, or were in �unknown� or �hypothetical� genes. In all instances except one investigated, the wild-type homologue of the mutant gene restored transformation proficiency when complementation was attempted. In one instance (rat17), the T-DNA-encoded marker (kanamycin-resistance) did not co-segregate with the rat phenotype. It is certainly possible (likely) that these lines contain multiple T-DNA insertions, and perhaps the junction that we identified was not responsible the mutant phenotype. We just have not had the personnel to attempt genetic complementation of all 126 rat mutants.

For these rat mutants, we have successfully complemented rat1, rat3, rat4, rat5, ratA2, ratJI, ratT5, ratT16, ratT17 and uta1. In addition, we have complemented the following rat mutants identified in a reverse genetics screen: act4, act7, and importin a-4.

We have also used a PCR-based 'reverse genetics' approach to test 'candidate' genes of suspected importance for the rat phenotype. Some of this screening has been conducted in concert with a NSF Plant Genomics grant on Chromatin Structure and Function (SBG is a subcontractor from the University of Arizona). Among the T-DNA insertional or RNAi mutants that we have identified as rat mutants are insertions in or near the following genes:

Other genes that we have identified using a 'reverse genetics'
approach and funds from this grant, include:

Genes encoding three 'unknown' proteins (These proteins interact with the processed VirB2 pilin protein; these mutations were made using anti-sense RNA and RNAi technologies)

Finally, we have begun a massive rat phenotype screening of T-DNA insertions in more than 100 different chromatin genes (these mutants have been identified in the Ecker Salk lines).

A complete description of the rat mutants identified to date can be found at: https://www.bio.purdue.edu/People/faculty/gelvin/gelvinweb/RatMutantCollTable.html

2. Identification of Arabidopsis proteins that directly interact with Agrobacterium Vir proteins (Citovsky and Gelvin laboratories).

a. Efforts of the Gelvin laboratory:

We used the Clontech system and an Arabidopsis cDNA library furnished by Dr. Vitaly Citovsky. We are currently using two Vir proteins as baits. VirB2 is the putative pilin protein. As a bait, we have used the processed (but not cyclized) form of the protein. We have screened more than three million colonies containing the VirB2 bait and the prey cDNA library and have identified two classes of strongly interacting proteins that do not also interact with controls (empty prey vector and lamin C as a prey). One of these classes of proteins is a membrane rab GTPase. The other class is a three-gene family of 'hypothetical' proteins. These latter proteins have predicted membrane-spanning regions. In addition, many Arabidopsis plants harboring anti-sense or RNAi constructions to these proteins show a rat phenotype. We have noted abnormalities in the root hairs of many RNAi plants: the hairs are �bulged�, �branched� and otherwise distorted in appearance. Some of these plants are also deficient in binding Agrobacterium cells. We are further characterizing these genes and proteins.

b. Efforts of the Citovsky laboratory:

We used the yeast two-hybrid screen with an Arabidopsis cDNA library and the nopaline-type Agrobacterium VirE2 protein as a bait. Screening of ca. 3x106 transformants resulted in identification and isolation of several independent cDNA clones producing VirE2 interactors. Two of these clones encoded the same cDNA, designated VIP1 (VirE2-interacting protein 1). The largest clone, representing the full-length cDNA of VIP1, was characterized in detail. Amino acid sequence analysis of the predicted protein encoded by the VIP1 cDNA revealed a homology to plant but not animal or yeast proteins containing a bZIP motif. VIP1 allowed nopaline VirE2 to be imported into the nuclei of yeast and mammalian cells and was required for VirE2 nuclear import and Agrobacterium-induced tumor formation in tobacco plants.

Another cDNA clone coded for a VirE2-interacting protein designated VIP2 (VirE2 interacting protein 2). Amino acid sequence analysis of VIP2 identified homology to the Rga protein of Drosophila, proposed to mediate interaction between chromatin proteins and the transcriptional complex. Unlike VIP1, VIP2 was unable to direct VirE2 into the yeast cell nucleus. However, VIP2 and VIP1 interacted with each other in the two-hybrid system. In uninfected cells, VIP1 and VIP2 may be involved in transcription, associating with the chromosomal DNA either directly or through other components of transcription complexes. Thus, it is tempting to speculate that VIP1, VIP2 and VirE2 may function in a multiprotein complex which performs a dual function: it may first facilitate nuclear targeting of VirE2 and then mediate intranuclear transport of VirE2 and its cognate T-strand to chromosomal regions where the host DNA is more exposed and, thus, better suitable for T-DNA integration.

We have further characterized VirE2 interactors, VIP1 and VIP2, identified in the previous years. Both VIP1 and VIP2 sequences were deposited to GenBank under Accession numbers AF225983 and AF295433, respectively. We are screening for VIP1 and VIP2 insertional mutants using the T-DNA tagged lines. One VIP1 mutant has already been isolated (insertion ca. 100 bp upstream of the ATG) and it is being crossed to homozygocity. Several pools of mutagenized plants with potential VIP1 disruptions have also been identified; we are continuing to screen these pools to isolate the mutated line. We also showed that, in tobacco plants, overexpression of VIP1 from the 35S promoter results in a significant increase of susceptibility to Agrobacterium infection. Conversely, suppression of VIP1 expression in anti-sense tobacco plants resulted in dramatically reduced tumorigenicity, transient T-DNA expression, and VirE2 nuclear import. Together, these results support the feasibility of our proposed approach to increase or decrease plant susceptibility to Agrobacterium transformation by overexpression or repression of cellular Vir-interacting proteins.

We are continuing to characterize VirE2 interactors, VIP1 and VIP2, identified in the previous funding years. Our reverse genetics experiments isolated one VIP1 mutant; however, the T-DNA insert, which occurred upstream of the translation initiation codon, did not appear to significantly alter the production of the VIP1 protein. We are continuing the screen for VIP1 mutants. We also identified one VIP2 mutant and are subcloning the T-DNA integration junction to determine the insertion site.

We completed our studies to enhance tobacco transformability by over-expression of VIP1. Our results show that elevated intracellular levels of VIP1 in VIP1 transgenic plants render these plants significantly more susceptible to transient and stable genetic transformation by Agrobacterium. Currently, we are testing whether or not overexpression of VIP1 also increases Agrobacterium tumorigenicity in other plant species (e.g., soybean).

We have additionally constructed an Arabidopsis cDNA library in the CytoTrap two-hybrid 'prey' vector allowing screening for protein-protein interactions which take place outside of the cell nucleus (unlike the conventional two-hybrid assay in which the interactions are always nuclear). VirB2, VirB5, and VirF open reading frames were subcloned into the CytoTrap 'bait' vector and we are presently conducting screening experiments. We used the yeast Arabidopsis cDNA library in the CytoTrap two-hybrid 'prey' vector and the octopine-type Agrobacterium VirF protein as bait. Screening of ca. 4x106 transformants resulted in identification and isolation of a VirF interactor, designated FIP1 (VirE2-interacting protein 1). The largest clone, representing the full-length cDNA of FIP1 has now been characterized. The FIP1 sequence was deposited to GenBank under Accession number AF332565. We initiated reverse genetics experiments to search for FIP1 insertional mutants. One such mutant was found following PCR-based screening of 64,000 T-DNA-tagged lines. Presently, we are sequencing the T-DNA insertion junction to determine where the FIP1 gene was disrupted. Concurrently with these experiments, the mutant line is being crossed to homozygocity in preparation to its biological characterization (i.e., susceptibility to specific stages of Agrobacterium infection).

VIP1 may be involved in uncoating of the T-complex within the host cell nucleus. VirE2 is thought to coat the transported T-DNA molecule, forming the T-complex, and, through its interaction with VIP1, assist the T-complex nuclear import. Once in the host cell nucleus, however, the T-DNA must become exposed for transcription and integration. The mechanism by which such uncoating could occur is completely unknown. Recently, an Agrobacterium host range factor, the VirF protein, has been shown to contain an F-box sequence that binds ASK1, the Arabidopsis homolog of the yeast Skp1 protein, suggesting that VirF may be involved in targeted proteolysis by participating in the E3 ubiquitin ligase or SCF (named after its original yeast protein components Skp1/Cdc53-Cul1/F-box) complex. We hypothesized that VirF may be involved in uncoating of the T-complex by targeting its protein components for proteolysis. To test this idea, we first used the yeast two-hybrid assay to examine whether VirF interacts with any of the proteins known to associate with the T-complex, e.g., VirD2, VirE2, and VIP1. In these experiments, protein-protein interaction was assessed from activation of the HIS3 reporter gene, which allows yeast cells to grow on a histidine-deficient medium.

VirF indeed interacted with ASK1, promoting histidine prototrophy. Co-expression of VirF with VirD2 or VirE2 did not result in cell growth, indicating the lack of interaction between these proteins. In contrast, yeast cells expressing VirF and VIP1 exhibited strong growth in the absence of histidine. VIP1, which is known to bind VirE2, did not interact with VirD2. Finally, no interaction between ASK1 and VIP1 or VirE2 was detected. In control experiments, under the non-selective conditions, all combinations of the tested proteins resulted in the efficient cell growth, indicating that neither of the tested proteins was toxic to yeast cells. These observations suggest that VirF specifically recognizes and interacts with VIP1 in the yeast two-hybrid system. This interaction was then confirmed using in vitro co-immunoprecipitation. Furthermore, we produced an extensive series of deletion mutants of VirF and delineated its amino acid sequence responsible for interaction with VIP1.

Next, we analyzed the effects of VirF-VIP1 interaction on VIP1 and VirE2 stability. Previously, protein stability and its changes due to the SCF-dependent degradation have been examined using fusions to a reporter protein; decrease in the reporter activity indicated decreased stability. We employed a similar approach to examine the effect of VirF on the stability of three proteins, VIP1, VirE2, and VirD2; initially, these experiments were performed in yeast cells, which are known to be infected by Agrobacterium. First, VIP1, VirE2, and VirD2 were fused to the GFP reporter and each of them was co-expressed in yeast cells with VirF driven by a methionine-repressible promoter. VirF produced in the absence of methionine significantly depleted the amount of GFP-VIP1, reducing it to about 10% of that observed under VirF-repressing conditions, i.e., in the presence of methionine. Furthermore, VirF expression also caused destabilization of up to 90% of GFP-VirE2 when coexpressed with VIP1. This destabilization was VIP1-dependent because GFP-VirE2 coexpressed with VirF in the absence of VIP1 remained stable. The VirF-mediated destabilization of VIP1 and VirE2 was specific as it did not occur when GFP-VirD2 was co-expressed with VirF and VIP1. VirF-induced destabilization of both VIP1 and VirE2 was not complete, explaining why interactions between these proteins could be detected in the yeast two-hybrid system; most likely, the residual levels of these proteins were sufficient to induce expression of the reporter genes in the two-hybrid assay.

Further support for the involvement of targeted proteolysis in the VirF-mediated destabilization of GFP-VIP1 and GFP-VirE2 was obtained using a yeast temperature-sensitive mutant in the Skp1 component of the SCF complex, skp1-4. VirF expressed in the skp1-4 strains destabilized GFP-VIP1 at the permissive temperature (25�C) but not at the restrictive temperature (37�C) at which GFP-VIP1 levels remained high. GFP-VirE2 co-expressed with VIP1 and VirF in skp1-4 also became destabilized at 25�C whereas, at 37�C, no significant changes in the levels of GFP-VirE2 fluorescence were detected.

Collectively, these results suggest that VirF specifically destabilizes VIP1 and VirE2 via the ubiquitin-mediated targeted proteolysis by the VirF-containing SCF complex (SCF^VirF). Also, because VirF binds VIP1 but not VirE2, its effect on VIP1 stability was most likely direct whereas the stability of VirE2 was affected indirectly, presumably through the VIP1-VirE2 interaction.

We have identified T-DNA insertions in the VIP1, VIP2, and FIP1 genes. In the previous years of this award we identified Arabidopsis proteins that interact with Agrobacterium VirE2 (VIP1 and VIP2) or VirF (FIP1). To understand better the biological function of these interactors we isolated homozygous T-DNA insertion mutants in their genes. Initially, plants heterozygous for T-DNA insertions into each gene were identified in the T-DNA mutant collection of SIGnAL (Salk Institute Genomic Analysis Laboratory). Having confirmed the insertion site, we derived homozygous lines, which were verified by PCR. Currently these mutants are being tested for their susceptibility to different stages of Agrobacterium infection. FIP1 may negatively affect Agrobacterium infection. Our very recent results indicate that the insertional mutant in the FIP1 gene is hyper-susceptible to Agrobacterium infection. We are examining a hypothesis that interaction between FIP1 and VirF targets FIP1 for proteolysis, enhancing the ability of Agrobacterium to infect its host plant. We are in the process of developing a system to detect proteins that are exported from Agrobacterium into the host cell cytoplasm. To date, several Agrobacterium proteins are known to be exported into the plant cell: VirE2, VirD2, VirF, and VirE3. Potentially, Agrobacterium exports numerous additional proteins to optimize the infection process. We are designing a genetic assay system to easily detect such proteins. Briefly, the tested protein is fused to a short gene activation sequence (GV) and expressed in Agrobacterium cells. These cells are used to infect plants transgenic for a reporter protein (GUS or GFP) under the control of a GV-inducible promoter. If a GV-fused tested protein is exported into the plant cell, it will activate expression of the reporter at the site of infection.

One of the central events in the Agrobacterium-mediated genetic transformation of plant cells is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein, VirE2. VirE2 is distinguished by its nuclear targeting which occurs only in plant but not in animal cells, and which is facilitated by the cellular VIP1 protein. The molecular mechanism of the VIP1 function is still unclear. We utilized in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin _. Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin _, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. We demonstrated that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin _, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an �adapter� molecule between VirE2 and karyopherin _lpha and �piggy-backing� VirE2 into the host cell nucleus. Because VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.

3. Identification of Arabidopsis genes induced or repressed during the initial stages of Agrobacterium transformation (Gelvin laboratory).

In order to identify differentially expressed genes during Agrobacterium-mediated transformation, we have used tobacco BY-2 cell suspension cultures and a super-virulent agropine-type strain of Agrobacterium (At804). Strain At804, in addition to a 'disarmed' Ti-plasmid, contains the binary vector pBISN1 (containing a nos-nptII gene and a super-promoter-gusA-intron gene). As a control, we have used strain At793 that lacks a Ti-plasmid but contains pBISN1. Strain At793 was included as a control to identify plant genes that are differentially expressed as a result of infection by Agrobacterium but not as a result of T-DNA transfer.

In the present study we used BY-2 cell suspension cultures showing an efficiency of transformation in the range of 90-100% after 2 days of Agrobacterium infection. Total RNA was isolated from BY-2 cells without infection and after 0, 3, 6, 12, 24, 30, and 36 hr of infection with At793 and At804. cDNA from these samples taken at 12 hours was prepared and used for suppressive subtractive hybridization experiments using the ClonTech 'gene select' kit. From approximately 8,000 'forward subtracted' and 10,000 'reverse subtracted' clones, we identified 33 clones representing genes induced upon Agrobacterium At804 infection, and 83 clones representing repressed genes. The genes were sequenced, and the clones identified as shown in the tables below:

Sequence analysis of clones identified from Forward Subtracted Library

Sequence analysis of clones identified from Reverse Subtracted Library

Based on sequence similarities with other known genes, most of the genes which showed induced level of expression (identified from forward subtracted library) belong to core histone gene families (histone H2A, H2B, H3 and H4) or genes families encoding proteins involved in cell division/cell proliferation such as ribosomal proteins. However the genes that showed down regulation (reverse subtracted library) predominantly contained the genes that are induced in response to pathogen attack or stress.

Northern blot analysis indicated that members of all core histone gene families, and several selected ribosomal protein genes, were induced late in infection upon co-cultivation of tobacco cells only with A. tumefaciens At804 (the transformation-proficient strain) but not with A. tumefaciens At793 (the transformation-deficient strain). Thus, induction of histone genes is dependent upon T-DNA and/or Vir protein transfer. Similarly, the repression of stress/defense response genes occurs late in infection (30-36 hr), and only by the transfer-competent strain A. tumefaciens At804. We have selected a number of genes identified as �down-regulated� by reverse suppressive subtracted hybridization, and used these genes as hybridization probes on Northern blots. Most of these genes are involved in plant defense responses (PR genes, GST genes, etc.). Northern blot analyses confirmed that these genes were down-regulated in response to transfer-competent (but not transfer-deficient) Agrobacterium strains late (30-36 hours) in the transformation process. A complete analysis of these genes can be found in: Veena, Jiang, H., Doerge, R.W., and Gelvin, S. B. 2003. Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression. Plant J. In press (July, 2003 issue).

We conducted additional expression profiling analyses following infection of tobacco BY-2 suspension culture cells with Agrobacterium strains that can transfer only Vir proteins but not T-DNA, or T-DNA but not VirE2. Results of these experiments indicate that both Vir protein and T-DNA transfer may trigger induction or repression of the genes mentioned above.

These data suggest that infection of tobacco BY-2 cells with a transfer-competent, but not a transfer-deficient Agrobacterium strain results in induction of plant genes necessary for T-DNA integration (such as histone genes) while actively suppressing the host defense response.

We repeated the expression profiling experiments using Affymetrix microarrays, using Arabidopsis suspension cells that were either uninfected, or infected with transfer-competent or transfer-deficient Agrobacterium strains (6 and 24 hr time points, with biological replicates). The results of these assays were similar to those of the tobacco BY-2 cell assays: numerous genes were up- or down-regulated. In particular, the expression of defense- and stress-related genes was affected by Agrobacterium transformation, with many genes up-regulated more by transfer-deficient strains than by transfer-competent strains.

Because our macroarray and microarray results suggested that plant defense response genes are down-regulated following infection by a transformation-competent (but not by a transformation-deficient) Agrobacterium strain, we tested several Arabidopsis mutants for susceptibility to Agrobacterium-mediated transformation. We found that the cep1 mutant (constitutive expression of PR genes) was resistant to transformation. On the other hand, Arabidopsis mutants that were compromised for defense responses (eds5, jar1, pad4, npr1, ssi1) were hyper-susceptible to Agrobacterium transformation, as was an Arabidopsis plant expressing the nahG gene. In addition, chemical elicitation of defense responses using salicylic acid, methyjasmonate, ethephon, and BTH resulted in a marked decrease in transformation of treated Arabidopsis roots. This is the first demonstration that plant defense genes play a role in Agrobacterium-mediated transformation.

4. T-DNA ligation assays (Hohn and Ziemienowicz laboratories).

An assay [Ziemienowicz et al.,(2000) Mol. Cell Biol. 20; 6317-6322] has been established to look specifically at ligation of the 5' terminus of the T-complex [single stranded DNA (8mer) with VirD2 protein covalently attached at the 5' end]. As a template for the ligation a 19-mer and a radio-labeled 13-mer were annealed, the sequence for these oligonucleotides was based on a known T-DNA integration site. Ligation was catalyzed by extracts from etiolated pea and the efficiency visualized on a denaturing polyacrylamide gel as the ratio of 13-mer to 21-mer (8 + 13) present. This assay was originally carried out with pea and was to be transferred to Arabidopsis with a view to characterizing the rat mutants which seemed to be impaired at the level of integration. In order to ensure that only nuclear proteins are assayed it was necessary to introduce a nuclei isolation step to the protocol:

Nuclear extracts from Arabidopsis were capable of catalyzing the ligation assay. The use of an anti-body against Arabidopsis ligase I and a peptide antibody against all ligases indicated that there are active ligase enzymes in the extract that could be inhibited.

In vitro experiments designed to analyze T-DNA integration using isolated nuclei from Arabidopsis root extracts have proven difficult to reproduce. As a result it was decided to try a different approach to analyze the DNA repair pathways thought to be involved in T-DNA integration. These experiments are based on a plasmid repair assay which was developed in the Hohn lab by Pawel Pelczar (submitted J. Mol. Biol.). In this paper, pBluescript plasmid was linearized in the coding region of the lacZ gene. The fidelity of repair was analyzed in different systems by comparing the ratio of blue to white colonies. An intact plasmid (kan^R) was always co-transfected as a control.

5. Involvement of the actin cytoskeleton in Agrobacterium-mediated transformation (Gelvin laboratory).

Using three different approaches, we have shown that actin microfilaments, but not microtubules, are involved in Agrobacterium-mediated transformation:

a. GST fusions of VirD2 and VirE2, but not GST alone, co-sediment with pre-polymerized f-actin. The KD�s for these interactions are in the range of 4-6 mM. GST fusions of these Vir proteins do not co-sediment with pre-polymerized microtubules. We have mapped domains within VirD2 and VirE2 responsible for these interactions. Mutation of these domains results in either loss or decrease in interaction. A VirE2 mutant that decreases interaction with f-actin also results in a lowered transformation efficiency. The actin-interacting domain of VirD2, when fused to GST, confers microfilament interaction upon the GST fusion protein.

b. Arabidopsis mutants containing T-DNA insertions into actin genes that are expressed in the root (act2 and act7) show the rat phenotype. Introduction of the corresponding wild-type gene into these rat mutants restores transformation-competence. However, a mutant containing a T-DNA insertion into an actin gene that is expressed in pollen (act12) is not a rat mutant. Similarly, a mutant containing a T-DNA insertion into a myosin gene expressed in the root (myo2) is a rat mutant. A T-DNA insertion into a tubulin gene expressed in the root (bot1) is not a rat mutant. The rat phenotype was assayed using both transient and stable transformation assays.

c. Using tobacco BY-2 suspension culture cells and a GUS assay for transient transformation, we have shown that pharmacological inhibitors of the actin cytoskeleton and the myosin motor, but not inhibitors of the microtubule cytoskeleton, reversibly inhibit transformation in a dose-dependent manner. The doses of inhibitors that decrease transformation are not toxic to either the plant or bacterial cells.

6. Biological characterization of Arabidopsis genes involved in Agrobacterium-mediated transformation.

These genes are described in Section 1 above

7. Improvement of agronomically important crops using Arabidopsis genes essential for Agrobacterium infection (Gelvin and Citovsky laboratories).

We (with the help of Dr. Kan Wang at Iowa State University) generated numerous transgenic maize plants containing the Arabidopsis rat5 histone H2A cDNA under the control of a maize ubiquitin promoter and intron. We additionally generated transgenic plants containing an 'empty vector' lacking the RAT5 gene. We evaluated, using Northern blots, the expression of the RAT5 gene in leaves of these plants. When RAT5-expressing plants were re-transformed by Agrobacterium, their re-transformation efficiencies were 2- to 3-fold higher than control maize plants containing an empty vector.

Additionally, we have determined that over-expression of the RAT5 gene in several recalcitrant Arabidopsis ecotypes increases their susceptibility to Agrobacterium-mediated transformation. Transfer of the RAT5 gene (or protein as a VirF fusion) also increases the transient transformation frequency of Brassica napus. Over-expression of the RAT5 gene also increases stable transformation of Brassica napus.

As described above, over-expression of the Arabidopsis VIP1 gene increases the transformation frequency of transgenic tobacco plants. With the help of Dr. Tom Clemente (University of Nebraska), we are assessing the effect of over-expression of VIP1 on the transformation frequency of soybean.

Over-expression of the VirB2 interacting protein BTI1 increases transformation of Arabidopsis. Over-expression of the arabinogalactan protein AtAGP17 increases transformation of the recalcitrant Arabidopsis ecotype Bl-1.

8. Specific importin-alpha genes play a role in Agrobacterium-mediated transformation (Gelvin laboratory).

Successful transformation of plants by Agrobacterium requires active nuclear import of T-DNA complexed with several proteins (the T-complex). T-complex constituent proteins VirD2 and VirE2 have plant-functional nuclear localization signal (NLS) sequences that may employ importin a proteins for nuclear import. In Arabidopsis thaliana, nine members constitute the importin a family. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that multiple Arabidopsis importin a members can interact with VirD2 and VirE2. However, disruption of only importin AtImpa-4, but not some of the other importin a members, inhibited transformation (rat [resistant to Agrobacterium transformation] phenotype). Over-expression of three importin a members, as well as AtImpa-4, in the AtImpa-4 mutant background increased transformation susceptibility of many of the derived transgenic lines. Roots of transgenic Arabidopsis plants over-expressing YFP-VirD2 in AtImpa-4 mutant plants displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the AtImpa-4 mutant. Somewhat surprisingly, VirE2-YFP localized to the cytoplasm of transgenic Arabidopsis or tobacco cells. Over-expression of VirE2 rescued the rat phenotype of AtImpa-4 mutant plants, suggesting that some function linked to nuclear targeting of VirE2 may be compromised in the AtImpa-4 mutant.

9. Identification of T-DNA/plant DNA junctions using non-selective conditions indicates that T-DNA integration is more random than previously thought (Gelvin laboratory)

We performed an analysis of T-DNA insertion sites in the Arabidopsis genome using a library of T-DNA/plant DNA junctions generated under non-selective conditions. The frequency with which T-DNA insertions in this library mapped to genic and intergenic regions closely resembled their respective proportions of the Arabidopsis genome. Contrary to what others previously found using selective conditions, we found a relatively high frequency of T-DNA insertions in repetitive sequences. Analysis using a randomly generated in silico Arabidopsis library indicated that T-DNA insertion sites generated under non-selective conditions were not significantly biased toward any chromosomal region, whereas T-DNA insertion sites recovered using selective conditions were significantly enriched in 5� upstream regions. Transcriptional profiling indicated that expression levels of T-DNA targets recovered using selective conditions were significantly higher than those of random Arabidopsis sequences, whereas expression levels of genomic sequences targeted by T-DNA under non-selective conditions were similar to those of random Arabidopsis sequences. Our results indicate that T-DNA insertion may occur more randomly than previously indicated, and that selection pressure shifts the recovery of T-DNA insertions into gene rich or transcriptionally active regions of chromatin.

Findings:

1. From amongst approximately ~20,000 newly screened T-DNA mutagenized and RNAi Arabidopsis lines, we have identified and confirmed 126 new rat mutants. Many additional putative rat mutants are currently being re-screened. A complete listing of the mutants to date was published in June, 2003: Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.-Y., Cao, H., Valentine, L., Li, J., Kaiser, A.D., Kopecky, A.L., Hwang, H.-H., Bhattacharjee, S., Rao, P.K., Tzfira, T., Rajagopal, J., Yi, H., Veena, Yadav, B.S., Crane, Y.M., Lin, K., Larcher, Y., Gelvin, M.J.K., Knue, M., Ramos-Oliva, C., Zhao, X., Davis, S.J., Kim, S.-I., Ranjith-Kumar, C.T., Choi, Y.-J., Hallan, V.K., Chattopadhyay, S., Sui, X., Ziemienowicz, A., Matthysse, A.G., Citovsky, V., Hohn, B., and Gelvin, S.B. 2003. Identification of Arabidopsis rat mutants. Plant Physiol. 132: 494-505. This list can also be found on the project web site.

2. We have identified 2 classes of genes that demonstrate interaction between Arabidopsis proteins and Agrobacterium VirB2 protein. One class encodes a membrane-bound rab GTPase. The second class contains 3 related family members. Analyses indicate that anti-sense inhibition of these genes reduces transformation. Some members are induced upon Agrobacterium infection, and over-expression of one member (BTI1) enhances transformation.

3. We have identified two Arabidopsis proteins, VIP1 and VIP2, that interact with Agrobacterium VirE2 protein in a yeast two-hybrid system. As well as being involved in nuclear import of VirE2, VIP1 also plays a role in T-DNA integration, perhaps by interacting with histone proteins.

4. We have identified, using suppressive subtractive hybridization and macroarray analyses, tobacco genes that are rapidly upregulated following Agrobacterium transformation. Most of these genes encode histones and ribosomal proteins. We have additionally identified tobacco genes that are rapidly downregulated following Agrobacterium transformation. Most of these genes are involved in plant stress and defense responses. Mutations in Arabidopsis genes involved in defense responses result in hyper-susceptibility to Agrobacterium transformation, whereas mutations that result in constitutive over-expression of defense genes result in the rat phenotype.

5. We have identified a nuclear-localized Arabidopsis ligase activity that will join a synthetic T-DNA to a model plant DNA in vitro.

6. We have determined that the plant actin cytoskeleton is involved in Agrobacterium-mediated transformation.

7. We have determined that VirF protein interacts in yeast with a SKP1 protein, and that this interaction results in degradation of VirE2 protein.

8. Using Affymetrix microarrays, we have determined that numerous Arabidopsis genes are up- or down-regulated following co-cultivation of suspension culture cells with various Agrobacterium strains.

9. We have investigated the role of various importin a family members in Agrobacterium-mediated transformation. Only AtImpa-4 is essential for transformation. Furthermore, imaging of VirE2-YFP indicates that this protein, which is biologically active, remains cytoplasmic and is not taken to the nucleus. Bimolecular fluorescence complementation indicates that VirD2 interacts with many importin a isoforms, and in all instance is localized to the nucleus. VirE2 also interacts with all importin a isoforms tested, but remains in the cytoplasm.

10. Agrobacterium-mediated transformation is affected by plant defense responses. Arabidopsis mutants which constitutively express PR genes are resistant to transformation, whereas mutants with debilitated defense responses are hyper-susceptible to transformation. Elicitation of plant defense responses by treatment of Arabidopsis with salicylic acid, methyljasmonate, ethephon, or BTH results in resistance to transformation.

11. Using non-selective transformation conditions, we have shown that T-DNA integrates into the Arabidopsis genome randomly. Integration does not prefer gene-rich or transcriptionally active regions of the genome.

Training and Development:

The project has to date trained four graduate students, twelve technicians, and fourteen postdoctoral research associates in techniques involved with plant tissue culture, Agrobacterium transformation assays, RNA differential screening, ligase assays, transcriptional profiling,and use of yeast two-hybrid systems to identify interacting proteins. In addition, the project has supported the training of fourteen undergraduate students and one technical school student in areas of plant tissue culture, plant genetics, and the use of PCR-based reverse genetics to identify mutations in specific plant genes. For one year, a Supplemental Award by the NSF funded a program to bring two minority students from Tuskegee University to Purdue University for a summer to learn to screen for and characterize Arabidopsis rat mutants in Dr. Gelvin�s laboratory. The students were then to return to Tuskegee for their senior year and continue this research in the laboratory of Dr. C.S. Prakash. There were several purposes of this program: To train minority students in plant genetic and molecular biology research, to encourage minority students to seek a career in science, to establish a close link between laboratories at Purdue University and Tuskegee University, and to serve as a recruitment tool for minority students to the graduate program at Purdue University. The initial program was highly successful. Two excellent students (Carla Reese and Emerald Screws) visited Dr. Gelvin�s laboratory, learned the Agrobacterium/rat mutant screening system, and returned to Tuskegee University where they continued their research throughout their senior year. Dr. Gelvin is very pleased that both of these students are still involved in science, and are seeking advanced degrees; he still maintains contact with these students and is involved in writing recommendation letters for them. Unfortunately, after the first year of this program, Dr. Prakash told Dr. Gelvin that he no longer had the time to help train the students, and that he personally would have to drop out of this program. For the last two years of this project, the grant also supported a minority undergraduate student, Esan Wilkinson, to come to Dr. Gelvin's laboratory during the summer. Esan learned techniques of Agrobacterium growth and plant tissue culture, and was responsible for a project involving the AtAGP17 gene, which this project determined to be involved in Agrobacterium-mediated transformation. After each summer in Dr. Gelvin's lab, Esan returned to his 'home' lab (Ted Muth, Brooklyn College, NY) where he continued is research during the academic year. Esan served as the 'bond' to link the Gelvin and Muth laboratories, which are presently conducting collaborative research on the role of the AtAGP17 gene in transformation.

Journal Publications:

Mysore, K.S., Kumar, C.T.R., and Gelvin, S.B., "Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformation.", Plant J., vol. 21, (2000), p. 9. Published

Mysore, K.S., Nam, J., and Gelvin, S.B., "An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration.", Proc. Natl. Acad. Sci. USA , vol. 97, (2000), p. 948. Published

Gelvin, S.B., "Agrobacterium and plant proteins involved in T-DNA transfer and integration.", Annu. Rev. Plant Physiol. Plant Mol. Biol., vol. 51, (2000), p. 223. Accepted

Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C., & Citovsky, V., "A genetic system for detection of protein nuclear import and export.", Nature Biotechnol., vol. 18, (2000), p. 433. Published

Tzfira, T., Rhee, Y., Chen, M.-H., Kunik, T., and Citovsky, V., "Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls.", Annu. Rev. Microbiol., vol. 54, (2000), p. 187. Published

Tzfira, T. and Citovsky, V., "From host recognition to T-DNA integration: the function of bacterial and plant genes in the Agrobacterium-plant cell interaction.", Mol. Plant Pathol. , vol. 1, (2000), p. 201. Published

Rhee, Y., Gurel, F., Gafni, Y., Dingwall, C., & Citovsky, V., "A genetic system for detection of protein nuclear import and export.", Nature Biotechnol., vol. 18, (2000), p. 433. Published

Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, D., Dingwall, C., & Citovsky, V., "Genetic transformation of HeLa cells by Agrobacterium.", Proc. Natl. Acad. Sci. USA, vol. 98, (2001), p. 1871. Published

Tzfira, T., Vaidya, M., Triger, A., and Citovsky, V., "VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.", EMBO Journal, vol. 20, (2001), p. 3596. Published

Tzfira, T. and Citovsky, V., "Comparison between nuclear import of nopaline- and octopine-specific VirE2 protein of Agrobacterium in plant and animal cells.", Mol. Plant Pathol., vol. 2, (2001), p. 171. Published

Shen, W.-H., Escudero, J., and Hohn, B. , "T-DNA transfer to maize plants.", Mol. Biotechnol., vol. 44, (2000), p. 346. Published

Ziemienowicz, A., Tinland, B., Bryant, J., Gloeckler, V., and Hohn, B. , "Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro.", Mol. Cell Biochem. , vol. 20, (2000), p. 6317. Published

Dumas, F., Duckely, M., Pelzcar, P., van Gelder, P., and Hohn, B., "An Agrobacterium VirE2 channel for T-DNA transport into plant cells.", Proc. Natl. Acad. Sci. USA, vol. 98, (2001), p. 485. Published

Ziemienowicz, A., Merkle, T., Schoumacher, F., Hohn, B., and Rossi, L., "Import of Agrobacterium T-DNA into plant nuclei: Two distinct functions of VirD2 and VirE2 proteins.", Plant Cell, vol. 13, (2001), p. 369. Published

Wu, Y.-Q., Hohn, B., and Ziemienowicz, A., "Characterization of an ATP-dependent type I DNA ligase from Arabidopsis thaliana.", Plant Mol. Biol., vol. , (2001), p. 161. Published

Tzfira, T. & Citovsky, V., "Partners-in-infection: host proteins involved in genetic transformation of plant cells by Agrobacterium.", Trends Cell Biol., vol. 12, (2002), p. 121. Published

Tzfira, T., Vaidya, M., & Citovsky, V., "Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis VIP1 gene.", Proc. Natl. Acad. Sci. USA, vol. 99, (2002), p. 10435. Published

Tzfira, T., Mayda, E., & Citovsky, V., "Nuclear import of VirF, a host range factor of Agrobacterium, in plant cells.", Proc. Natl. Acad. Sci. USA, vol. , (2002), p. . In preparation.

Yi, HC, Mysore, K.S. and Gelvin, S.B. , "Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation.", Plant J., vol. 32, (2002), p. 285. Published

Lee, L.-Y., Humara, J.M., and Gelvin, S.B., "Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome.", Mol. Plant-Microbe Interact., vol. 14, (2001), p. 577. Published

Gelvin, S.B., "Agrobacterium and plant transformation: The biology behind the "gene-jockeying" tool.", Microbiol. Mol. Biol. Rev., vol. 67, (2003), p. 16. Published

Gelvin, S.B., "Improving plant genetic engineering by manipulating the host.", Trends Biotechnol., vol. 21, (2003), p. 95. Published

Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.-Y., Cao, H., et al., "Identification of Arabidopsis rat mutants.", Plant Physiol., vol. 132, (2003), p. 494. Published

Veena, Jiang, H., Doerge, R.W., and Gelvin, S. B., "Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression.", Plant J., vol. 35, (2003), p. 219. Published

Tao, Y., Rao, P., Bhattacharjee, S.K., and Gelvin, S.B., "A plant phosphatase is involved in nuclear import of the Agrobacterium VirD2/T-DNA complex.", Proc. Natl. Acad. Sci. USA, vol. , (2003), p. . Submitted

Tian, L., Chen, M., Fong, P., Cao, H., Gelvin, S.B., and Chen, Z.J. 2003. , "Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression.", Genetics, vol. , (2003), p. . Accepted

Gaspar, Y.M., Nam, J., Schultz, C.J., Lee, L.-Y., Gilson, P., Gelvin, S.B., and Bacic, A., "Reduced expression of an Arabidopsis lysine-rich arabinogalactan-protein, AtAGP17, results in a decreased efficiency of Agrobacterium transformation.", Plant Cell, vol. , (2003), p. . Submitted

Zhu, Y., Nam, J., Carpita, N.C., Matthysse, A.G. and Gelvin, S.B. , "Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene.", Plant Physiol., vol. , (2003), p. . Submitted by June, 2003

Eul�lio, A., Nunes-Correia, I., Carvalho, A.L., Faro, C., Citovsky, V., Sim�es, S., & Pedroso de Lima, M. C., "Multiple sequences mediate the nuclear export of the p37 structural protein of African swine fever virus.", Virology, vol. , (2003), p. . Submitted

Tzfira, T., Vaidya, M., & Citovsky, V., "VirF, a host range factor of Agrobacterium, mediates targeted proteolysis of VirE2 and its plant cell interactor, VIP1.", Proc. Natl. Acad. Sci. USA, vol. , (2003), p. . Submitted

Duckely, M., and Hohn, B. , "The VirE2 protein of Agrobacterium tumefaciens: the Yin and Yang of T-DNA transfer", FEMS Microbiology Letters, vol. , (2003), p. . Accepted

Tzfira, T. & Citovsky, V., "The Agrobacterium-plant cell interaction: taking biology lessons from a bug.", Plant Physiol., vol. 133, (2003), p. 943. Published

Tzfira, T., Frankman, L., Vaidya, M., & Citovsky, V., " (2003) Site-specific integration of Agrobacterium T-DNA via double-stranded intermediates.", Plant Physiol., vol. 133, (2003), p. 1011. Published

Tzfira, T., Li, J., Lacroix, B., & Citovsky, V., "Agrobacterium T-DNA integration: molecules and models.", Trends Genet., vol. 20, (2004), p. 375. Published

Citovsky, V., Kapelnikov, A., Oliel, S., Zakai, N., Rojas, M.R., Gilbertson, R.L., Tzfira, T., & Loyter, A., "Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro.", J. Biol. Chem., vol. 279, (2004), p. 29528. Published

Tzfira, T., Vaidya, M., & Citovsky, V., "(2004) Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium.", Nature, vol. 431, (2004), p. 87. Published

Lacroix, B., Li, J., Tzfira, T., & Citovsky, V., "Will you let me use your nucleus? How Agrobacterium gets its T-DNA expressed in the host plant cell.", Can. J. Physiol. Pharmacol., vol. 84, (2005), p. . Accepted

Loyter, A., Rosenbluh, J., Zakai, N., Li, J., Kozlovsky, S.V., Tzfira, T., & Citovsky, V., "The plant VirE2 interacting protein 1. A molecular link between the Agrobacterium T-complex and the host cell chromatin?", Plant Physiol., vol. 138, (2005), p. 1318. Published

Tzfira, T., Tian, G.W., Lacroix, B., Vyas, S., Li, J., Leitner-Dagan, Y., Krichevsky, A., Taylor, T., Vainstein, A., & Citovsky, V., "pSAT vectors: a modular series of plasmids for fluorescent protein tagging and expression of multiple genes in plants.", Plant Mol. Biol., vol. 57, (2005), p. 503. Published

Lacroix, B., Tzfira, T., Vaidya, M., & Citovsky, V., "The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation.", EMBO J., vol. 24, (2005), p. 428. Published

Li, J., Krichevsky, A., Vaidya, M., Tzfira, T., & Citovsky, V., "Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium.", Proc. Natl. Acad. Sci. USA, vol. 102, (2005), p. 5733. Published

Lacroix, B., Tzfira, T., Vainstein, A., & Citovsky, V., "A case of promiscuity: Agrobacterium?s endless search for partners.", Trends Genet., vol. 22, (2006), p. . Accepted

Tao, Y., Rao, P.K., Bhattacharjee, S. and Gelvin, S.B., "Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2.", Proc. Natl. Acad. Sci. USA, vol. 101, (2004), p. 5164. Published

Gaspar, Y.M., Nam, J., Schultz, C.J., Lee, L.-Y., Gilson, P., Gelvin, S.B., and Bacic, A., " Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of Agrobacterium transformation. ", Plant Physiol., vol. 135, (2004), p. 2162. Published

Lee, L.-Y., and Gelvin, S.B., "Osa protein constitutes a strong oncogenic suppression system that can block vir-dependent transfer of IncQ plasmids between Agrobacterium cells, and the transfer of T-DNA and IncQ plasmids to plant cells.", J. Bacteriol., vol. 186, (204), p. 7254. Published

Hwang, H.-H, and Gelvin, S.B., "Plant proteins that interact with VirB2, the Agrobacterium pilin protein, are required for plant transformation.", Plant Cell, vol. 16, (2004), p. 3148. Published

Gelvin, S.B., "Gene exchange by design.", Nature, vol. 433, (2005), p. 583. Published

Gelvin, S.B., "Viral-mediated plant transformation gets a boost.", Nature Biotechnol., vol. 23, (2005), p. 684. Published

Hwang, H.-H., Mysore, K.S. and Gelvin, S.B., "Transgenic Arabidopsis plants expressing Agrobacterium tumefaciens VirD2 protein are resistant to Agrobacterium transformation. ", Nature Biotechnol., vol. , (2005), p. . Submitted

Yi, H.-C., Fujinuma, T. Chan, C.-W., Veena, and Gelvin, S.B., "Functional redundancy among Arabidopsis histone H2A family members for Agrobacterium-mediated plant transformation.", Plant Cell, vol. , (2005), p. . Submitted

Bhattacharjee, S., Cao, H., Lee, L.-Y., Veena, and Gelvin, S.B., "AtImpa-4, an Arabidopsis importin a isoform, is preferentially involved in Agrobacterium-mediated plant transformation.", Plant Cell, vol. , (2005), p. . Submitted

Kim, S.-I., Veena, and Gelvin, S.B., "Genome-wide analysis of Agrobacterium T-DNA insertion sites in the Arabidopsis genome generated under non-selective conditions.", Proc. Natl. Acad. Sci. USA., vol. , (2005), p. . Submitted

Book(s) of other one-time publications(s):

Crouzet, P., and Hohn, B., "Transgenic Plants" , bibl. MacMillan www.els.net, (2001). Book Published of Collection: , "Encyclopedia of Life Sciences"

Ziemienowicz, A., Gorlich, D., rossi, L., Merkle, T., Tinland, B., and Hohn. B., "Nuclear import and integration of Agrobacterium T-DNA in vitro." , bibl. Vol. 2 International Society of Molecular Plant--Microbe Interactions, St. Paul, Minn USA pp. 164-169., (2000).

Book Published of Collection: P. de Wit, T. Bisseling, W. Stiekma, "Proceedings of the MPMI Congress on the Biology of Plant-Microbe Interactions "

Wu, Y.-Q., and Hohn, B., "Transfer to plants and integration into chromosomal DNA of T-DNA of Agrobacterium tumefaciens" , bibl. pp. 1-18, (2003). Book Published of Collection: G. Stacey and N.T. Keen, "Plant-Microbe Interactions"

Other Specific Products:

Physical collection (samples, etc.)

We are creating a library of Arabidopsis rat mutants. We are also creating a library of genes that are required for Arabidopsis root transformation, and a library of genes that interact with Agrobacterium VirE2, VirD2, VirF, and VirB2 proteins.

The first library will eventually be deposited in the Ohio State University Arabidopsis stock center. The second library will be available from the individual laboratories.

Internet Dissemination:

This site has been designed specifically for this award. It currently contains the names and c.v."s of the P.I.s, and a copy of the proposal. The site will eventually contain detailed data generated by this proposal, as well as a bibliography of Agrobacterium/crown gall disease, and links to related sites.

Contributions:

Contributions within Discipline:

Our project deals with the identification of plant genes involved in the Agrobacterium T-DNA transfer process. Very little is currently known about the role plant genes and proteins play in this process. Thus, for the purposes of both plant basic research and plant genetic transformation, it is important to identify and define the roles played by plant genes. Our work has begun to fill in the gaps in this area. Specifically, work in identifying Arabidopsis rat mutants has revealed that there are likely to be several hundred plant genes involved in Agrobacterium transformation. Among these are cell wall structural and biosynthetic genes (represented by Rat1, Rat3, and Rat4) that play a role in Agrobacterium attachment to plant walls. Genes encoded by Rat5, Rat17, Rat18, Rat20, and Rat22 are likely involved in T-DNA integration into the plant chromosome. Several genes involved in the actin cytoskeleton (act2 and act7) appear to be involved in T-DNA nuclear transfer, as are several alpha- and beta-importin genes. We have also identified several chromatin genes, including histones H2A, H2B, H3, H4, histone acetyltransferases, and histone deacetylases, as involved in Agrobacterium transformation, probably at the T-DNA integration step. Additionally, protein interaction experiments using the yeast two-hybrid system were conducted to identify plant proteins that interact with the Agrobacterium Virulence (Vir) proteins VirB2, VirD2, VirE2, and VirF. These proteins are involved in the T-DNA transfer and nuclear targeting processes. In particular, we have identified a protein, VIP1, that interacts with VirE2 and several Arabidopsis proteins that interact with VirB2, the major Agrobacterium pilin protein. Finally, we have identified a number of plant genes that are up- and down-regulated rapidly after Agrobacterium infection.

Materials generated from this project have been and will continue to be distributed to all interested parties. A recent publication describing some of our work (Zhu, Y., Nam, J., Humara, J.M., Mysore, K.S., Lee, L.-Y., Cao, H., Valentine, L., Li, J., Kaiser, A.D., Kopecky, A.L., Hwang, H.-H., Bhattacharjee, S., Rao, P.K., Tzfira, T., Rajagopal, J., Yi, H., Veena, Yadav, B.S., Crane, Y.M., Lin, K., Larcher, Y., Gelvin, M.J.K., Knue, M., Ramos-Oliva, C., Zhao, X., Davis, S.J., Kim, S.-I., Ranjith-Kumar, C.T., Choi, Y.-J., Hallan, V.K., Chattopadhyay, S., Sui, X., Ziemienowicz, A., Matthysse, A.G., Citovsky, V., Hohn, B., and Gelvin, S.B. 2003. Identification of Arabidopsis rat mutants. Plant Physiol. Plant Physiol. 132: 494-505) specifically informs readers that all rat mutants are available upon request from Dr. Gelvin. To date, we have distributed marterial generated during this grant to a number of scientists, including Dr. Mary-Dell Chilton, Dr. Walt Ream, Dr. Holger Puchta, Dr. Jersey Paszcowsky, Dr. Anthony Bacic, Dr. Maureen McCann, Dr. Nick Carpita, Drs. Shauna and Chris Sommerville, Dr. Jeffrey Chen, and Dr. Ken Keegstra. In addition, mutants generated during the course of this grant have been distributed to members of the University of Arizona-led chromatin research group for use in their NSF Plant Genome Award project, and to members of the Iowa State University-led maize transformation research group for use in their NSF Plant Genome Award project.

Contributions to Other Disciplines:

Our work on plant genes involved in Agrobacterium-mediated transformation is important for the field of plant genetic transformation in general. Many agriculturally important plant species remain recalcitrant to Agrobacterium transformation. By identifying plant genes required for the transformation process, we hope to manipulate and utilize these genes to improve transformation. In addition, the identification of plant genes involved in bacterial attachment will be useful to increase our knowledge of how bacteria conjugate DNA between cells. Our work showing that overexpression of VIP1, BTI1, AtAGP17, and histone H2A-1 increases transformation of plant cells indicates that our approach has been successful.

Contributions to Education and Human Resources:

Our project has involved training of numerous undergraduate and graduate students, postdoctoral research fellows, and technicians. Many of these people had no previous experience in Agrobacterium-mediated plant transformation. Because this method of transformation remains a major technique for generating transgenic plants, the training provided by this grant will increase the pool of highly skilled scientists required by the growing agricultural biotechnology industry. In addition, a special supplement to our grant has now allowed us specifically to train two under-represented minority undergraduate students from Tuskegee University per year.

The following personnel trained on this grant are from under-represented minority categories:

Luis Zapata
Carla Reese
Emerald Screws
Cynthia Ramos
Esan Wilkinson

The following relevant lectures were presented by Dr. Gelvin during the fourth year (2002-2003) of this grant:

23rd Annual Crown Gall meeting (University of Minnesota, Nov. 1-3, 2002). Posters and oral presentations by members of the Gelvin laboratory:
'Role of the plant cytoskeleton in Agrobacterium-mediated transformation'
'Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediated transformation and suppresses host defense gene expression'
'Host factors involved in the stable integration of T-DNA into the plant genome'
'The pattern of histone H2A-1 expression affects Agrobacterium-mediated transformation'
'Over-expression of the Arabidopsis histone H2A gene HTA1 can improve Agrobacterium-mediated transformation of plants'
'Identification of a putative plant receptor for the Agrobacterium T-pilus'
'The importin part of the T-complex nuclear import process�One t(w)o many?'
'Identification and characterization of Arabidopsis rat mutants'

Institute of Botany, Academia Sinica and Department of Botany, National Taiwan University, Taipei, Taiwan (Nov. 16-24, 2002): 'Plant genes involved in Agrobacterium-mediated transformation'

Maize Transformation Workshop, University of Wisconsin, Madison, March 11-12, 2003: 'Manipulation of plant genes to effect better transformation of dicots: Will this work in maize?'

Indiana University, Bloomington, April 17-18, 2003: 'Plant genes involved in Agrobacterium-mediated transformation: Can they be used to improve transformation?'

Biotechnology Research and Development Corporation Annual meeting, Chicago, IL May 19-20, 2003: 'Using plant genes to increase Agrobacterium-mediated genetic transformation'

The following relevant lectures were presented by Dr. Citovsky during the fourth year (2002-2003) of this grant:

Department of Botany and Plant Sciences, University of California, Riverside, CA, May 1, 2002: 'Intercellular and Nuclear Transport of Proteins and Nucleic Acids: The Molecules That Walk Through the Walls.'

Department of Biochemistry and Cell Biology, SUNY Stony Brook, NY January 30, 2003: 'Transport of Proteins and Nucleic Acids Through Plasmodesmata and Nuclear Pores: The Molecules That Walk Through the Walls.'

Graduate Student Seminar Series Department of Biology, Queens College, CUNY, Flushing, NY, April 30, 2003: 'Nucleic Acid Transport in Plant-Microbe Interactions: The Molecules That Walk Through the Walls.'

The following relevant lectures were presented by Dr. Gelvin during the fifth, and no-cost extension, period of the grant (2004-2005):

BASF Corp., Research Triangle Park, NC, March 7-8, 2004: 'Using plant genes to increase Agrobacterium-mediated transformation'

Presidents's lecture keynote address, Midwest ASPB meeting, The Ohio State University, Columbus, OH, March 19-21, 2004: 'Plant genes involved in Agrobacterium-mediated transformation'

NSF EPSCoR conference, Oklahoma State University, Stillwater, OK, May 12-14, 2004: 'Using transcriptional profiling to understand Agrobacterium-mediated plant transformation'

Society of In Vitro Biology annual meeting, San Francisco, CA, May 22-25, 2004: 'Integration of the Agrobacterium T-DNA into the plant genome'

University of Minnesota, Minneapolis, MN, June 6-8, 2004: 'Plant genes involved in Agrobacterium-mediated plant transformation'

Institute of Botany, Academia Sinica, Taipei, Taiwan, June 17-July 2, 2004. Taught a course entitled 'Agrobacterium biology and plant transformation', and presented seminars 'Integration of Agrobactrium T-DNA into the plant genome'

National Chung-Tsing University, Taichung, Taiwan, June 30, 2004: 'Plant genes involved in Agrobacterium-mediated plant genetic transformation'

25th Annual Crown Gall Conference, University of Illinois, Champaign, IL, Aug. 13-17, 2004:

'Plant processes involved i Agrobacterium-0mediated genetic transformation: More (genes and collaborators) are better'

Members of the Gelvin lab presented the following talks/posters:

'Genome-wide analysis of T-DNA target sites in the Arabidopsis genome under non-selective conditions'

'The preference of Arabiodpsis thaliana AtImpa-4 for Agrobacterium-mediated transformation--best of the rest'

'Arabidopsis histone H2A-1 increases Agrobacterium-mediatd transformation efficiency through increased transgne expression'

'The host plant defense response influences Agrobacterium-mediated transformation'

'RNAi-mediated gene silencing reveals involvement of host chromatin genes in Agrobacterium-mediated transformation of Arabidopsis'

'The N-terminal 39 amino acids of histone H2A-1 are suffiecient to complement the rat5 mutant'

'Identification of Arabidopsis mutants that are hyper-susceptible to Agrobacterium-mediated transformation (hat mutants)'

'Additional copies of several different histone genes can increase Agrobacterium-mediated transformation of Arabidopsis'

PIPRA workshop, Danforth Plant Science Center, St. Louis, MO: 'Gene delivery'

NSF Maize Transformation Workshop, University of Wisconsin, Madison, WI, March 9-10, 2005: 'Manipulation of plant genes to effect better transformation of dicots: Will this work for maize?'

Starbucks Corp., Seattle, WA, April 11, 2005: 'Using Agrobacterium-mediated transformation to improve plants'

Institute of Botany, Academia Sinica, Taipei, Taiwan, May 27-June 15, 2005: Taught course on 'Protein routing through the cell' and presented a seminar 'The role of plant genes in Agrobacterim-mediated transformation'

APS meeting, Austin, TX, July 31-Aug. 2, 2005: 'Mechanisms of Agrobacterium-mediated transformation: Agrobacterium as a plant pathogen'

26th Annual Crown Gall conference, Indiana University, Bloomington, IN, Aug. 5-7, 2005: The following presentations were made by the Gelvin laboratory:

'Genome-wide analysis of T-DNA target sites in the Arabidopsis genome under non-selective conditions: How random really is T-DNA integration?'

'Arabidopsis importin alpha proteins interact with Agrobacterium virulence proteins in plants as deteced by bimolecular fluorescence complementation (BiFC)'

'Crucial role of the host plant defense response in determining the efficiency of Agrobacterium-mediated transformation'

'Increased biofilm fomration on the root surfaces of attachment-deficient Arabidopsis rat mutants is not sufficient to permit Agrobacterium-mediated transformation'

'Effect of Agrobacterium strain and binary vector replication origin on Agrobacterium-mediated transformation frequency and transgene copy number'

'Characterization of T-DNA activation-tagged Arabidopsis mutants that are hyper-susceptible to Agrobacterium transformation (hat mutants)'

'Over-expression of various Arabidopsis histone genes increases the Agrobacterium-mediated transformation frequency of Arabidopsis'

Contributions Beyond Science and Engineering: