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 in the past 3 years, we have screened approximately 10,000 additional lines from the Feldmann collection, resulting in approximately 14,000 total lines screened. Of the 64 pools of 100 individually mutagenized plants in this collection, we have 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: 50
Total rat mutants from forward genetic screening: 71
rat mutants from reverse genetic screening (see below): 29
Total rat mutants from all screenings: 100

      We have recently 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 have initiated screening of the available mutant lines from this collection. Much of the screening of this library is being undertaken by the Citovsky laboratory. We have screened 2,200 plants corresponding to 50 pools of the INRA T-DNA mutant collection. Seven putative mutants have been identified in the first round of screening and three of them have been 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 recently 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 have generated a new T-DNA disruption library (approximately 70,000 members) in ecotype Ws. We have been screening of this library for rat mutants. To date, approximately 2,000 plants from this line have been screened in the Gelvin and Hohn laboratories. Of the 600 plants screened in the Hohn laboratory, 53 putative mutants were identified in the first round. Six mutants were confirmed in the second round of screening (although some of these may be sibs).

      In all, the Gelvin laboratory has screened more than 12,000 lines from the three T-DNA insertion libraries. We are currently concentrating our efforts on screening the Bressan library.

      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 has 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. They 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 are continuing this work in Dr. Prakash's laboratory during this academic year. Salaries for these two students were funded by a special supplement to this grant.

       We have 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 23 rat mutants. Many of these junctions lie in or near "unknown" or "hypothetical" proteins. Among the junctions that we have identified are:

rat1	Arabinogalactan protein
rat3	Likely cell wall protein
rat4	Xylan synthase
rat5	Histone H2A
rat7	Unknown protein
rat9	Unknown protein
rat17	myb-like transcription factor (caprice gene; this does not co-segregate with the rat phenotype)
rat19	Unknown protein
ratJ1	Importin Beta-3
ratJ2	Cleavage stimulation factor subunit 1-like protein
ratJ3	Hypothetical protein
ratJ6	Adenosine kinase/3-isopropylmalate dehydrogenase
ratJ7	�DEAD box� RNA helicase
ratJ9	Hypothetical protein
ratT3	Near a putative rac GTPase activating protein
ratT4	Between an unknown protein and an ethylene responsive element binding factor-like protein
ratT5	DREB2A transcription factor
ratT8/T9	Between a APC-like-binding protein EB1 and a receptor-like protein kinase
ratT10	Between an unknown protein and a squamosa 2 binding-like protein
ratT13	Between an unknown protein and a CND41 chloroplast nucleoid DNA binding-like protein
ratT15	Between two unknown proteins
ratT16	Unknown protein
ratT17	Between two replacement histone H3 genes
ratT18	Beta-expansin protein
ratA2	Type 2A protein phosphatase
ratA4	Kinesin protein
ratA5	Unknown protein

      For these rat mutants, we have successfully complemented rat1, rat3, rat4, rat5, ratA2, ratJI, ratT5, ratT16 and ratT17.

      We have also use 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 (we are subcontractors from the University of Arizona). Among the T-DNA insertional mutants that we have identified as rat mutants are insertions in or near the following genes:

Histone H2A-1	(HTA1) [This is an additional insertion into the promoter region of the rat5 gene]
Histone H2A-2	(HTA2)
Histone H2A-3	(HTA3)
Histone H2A-7	(HTA7)
Histone H2A-10	(HTA10)
Histone H2A-11	(HTA11)
Histone H2A-13	(HTA13)
Unknown gene next to Histone H2B-5/6
Histone H3-4/5 (between these two genes)	(HTR4/HTR5)
Histone H4-1	(HFR1)
Histone H4-3	(HFR3)
Histone H4-4	(HFR4)
Histone acetyltransferase-6	(HAC6)
Histone acetyltransferase-9	(HAC9)
Histone acetyltransferase-10	(HAC10)
Histone acetyltransferase-11	(HAC11)
Histone deacetylase-1	(HDA1)
Histone deacetylase-2	(HDA2)
Histone deacetylase-6	(HDA6)

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

Importin-alpha1 [This mutant has been complemented]
Importin-alpha7 [This mutant has been complemented]
Actin 2 (ACT2) [This mutant has been complemented]
Actin 7 (ACT7) [This mutant has been complemented]
Myosin 2 (MYO2)
A rab GTPase (this mutation was made using anti-sense RNA and RNAi technologies)
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)

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

      a. Efforts of the Gelvin laboratory:

      We have been using 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 two million colonies containing the VirB2 bait and the prey cDNA library and have identified two classes of strongly interacting colonies 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, and preliminary localization experiments of GFP fusions with the proteins suggest that they are localized to the plant wall or plasma membrane. In addition, many Arabidopsis plants harboring anti-sense or RNAi constructions to these proteins show a rat phenotype. We are further characterizing these genes and proteins.

      We have initiated screening the same cDNA library using as a bait the C-terminal half of VirD2 protein. We shall specifically be looking for proteins that interact with the nuclear localization and omega domains of VirD2.

      Finally, we plan to screen a cDNA library using as a bait the VirE1 protein. Although this protein has been proposed to function only in Agrobacterium, recent data in our laboratory indicates that VirE1 interacts with the same importin-alpha family members as do VirD2 and VirE2.

      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 plant pools with potential VIP1 mutants have also been identified and their screen continues to isalte 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 antisense 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 overexpression 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).

      Using the CytoTrap yeast two-hybrid system, we additionally isolated 14, 6, and 5 different cDNA clones coding for proteins that bind VirB2, VirB1, and VirB1*, respectively. Presently, these interactions are being verified by independent approaches.

      Besides FIP1, we discovered that VIP1, a cellular protein that interacts with VirE2 and assists its nuclear import, also interacts with VirF. Thus, VirF may function in a multiprotein complex with VIF1, VIP1, and VirE2; this complex may associate with the transported T-strand due to the ssDNA binding activity of VirE2. Presently, we are investigating the role of VirF in this complex. We hypothesize that, because VirF is an F-box protein, it may function (similarly to other known F-box proteins) to direct its specific ligand(s) for targeted proteolysis. The experiments to test whether or not expression of VirF triggers proteolytic degradation of FIP1 and/or VIP1 are currently underway.

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:

Putative Functions of Identified Clones	#Clones type (Total 83)
Glutathione S-Transferase	eight
Proteins responds to agents that induce systemic acquired resistance and TMV-inducible	five
Sucrose synthase	three
Extensin like protein	three
Osmotin-like protein	two
Non-symbiotic hemoglobin	two
Nodulin	two
Short-chain type dehydrogenase/reductase	one
Putative translationally controlled tumor protein (TCTP1)	one
Tumor related protein, expansin like proteins or pollen allergen like proteins	one
Glucosyl transferase	one
Glucan beta-1,3-glucosidase	one
Beta -1,3-glucanase	one
Beta-xylosidase	one
Glyceraldehyde-3-phosphophate dehydrogenase	one
Monodehydroascorbate reductase	one
Metal-transporting ATPase,	one
H+-transporting ATP synthase	one
HCF106 Required for the Delta pH pathwayin vivo	one
DNAJ protein-like	one
Alcohol dehydrogenase	one
Caffeoyl-CoA O-methyltransferase	one
Cyclin-dependent kinase inhibitor	one
Cinnamyl-alcohol dehydrogenase	one
Apoptosis related protein	one
Hypothetical protein	one
*6 to be characterized	one

       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 tumefaciensAt804 (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 infecti0n (30-36 hr), and only by the transfer-competent strain A.tumefaciensAt804.

      Finally, we have 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.

      In the coming year, we plan to repeat these experiments using infected Arabidopsis suspension cell cultures and "whole genome" oligonucleotides microarrays.

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 which could be inhibited.

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 K_D's for these interactions are in the range of 4-6 uM. 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. The actin-interacting domain of VirD2, when fused to GST, confers microfilament interaction upon the GST fusion protein.

      b.    Arabidopsismutants 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 laboratory).

      This project was proposed not to start until the fourth year of the project. As a first step, we have (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 have additionally generated transgenic plants containing an �empty vector� lacking the RAT5 gene. We have evaluated, using Northern blots, the expression of the RAT5 gene in leaves of these plants. We plan this coming year to test whether transgenic maize plants over-expressing the Arabidopsis RAT5 gene have increased susceptibility to Agrobacterium infection.

      Additionally, we have determined that over-expression of the RAT5 gene in several recalcitrant Arabidopsis ecotypes increases their susceptibility to Agrobacterium-mediated transformation. We are currently testing whether the RAT5 gene increases transformation of Brassica (oilseed rape) plants.