1. Identification and characterization of Arabidopsis rat mutants and molecular cloning of RATgenes.

       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 (ratmutants). During the course of this grant in the past 2 years, we have screened approximately 5350 additional lines from the Feldmann collection, resulting in approximately 9350 total lines screened. Of the 64 pools of 100 individually mutagenized plants in this collection, we have screened representatives from 61 of these pools. It is therefore likely that we have approached saturation in screening of this T-DNA insertion library.

       Of the 5350 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: 48
Total confirmed rat mutants: 69
        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 be deficient in T-DNA integration. We are currently pursuing further analysis of these mutants.

        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 1,500 plants corresponding to 50 pools of the INRA T-DNA mutant collection. Four putative mutants have been identified in the first round of screening and one of them has 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 AP3gene 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 AP3gene. Therefore, in collaboration with Dr. Ray Bressan of Purdue University, we have generated a new T-DNA disruption library (approximately 60,000 members) in ecotype Ws. We have been screening of this library for ratmutants.

       In all, the Gelvin laboratory has screened more than 11,200 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 are receiving training in rat mutant screening, and will continue this work in Dr. Prakash's laboratory during the ensuing 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 beta-importin
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, and rat5. Successful co-segregation of the T-DNA with the rat phenotype has been shown for rat4 and ratJ1. We are currently conducting co-segregation analyses on the other rat mutants.

2. Identification of Arabidopsis proteins that directly interact with Agrobacterium Vir proteins.

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 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 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 w domains of VirD2.

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 are continuing to characterize VirE2 interactors, VIP1 and VIP2, identified in the previous year. 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 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 is now being characterized. The FIP1 sequence was deposited to GenBank under Accession number AF332565. We have also begun reverse genetics experiments to search for FIP1 insertional mutants (no results yet). In addition, we found that VirF is actively imported into the host cell nucleus and interacts with VIP1, resulting in formation of VirE2-VIP1-VirF ternary complexes.

3. Identification of Arabidopsis genes induced or repressed during the initial stages of Agrobacterium transformation.

       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
Putative Functions of Identified Clones #Clones type (total 33)
Histone H2A two
Histone H4 four
Histone H3 two
Histone H2b four
Ribosomal Proteins four
Polyubiquitin (Ubi) mRNA one
G protein beta-subunit-like protein one
Genomic RNA expressed in roots one
5-epimerase one
Protein homologue to human Wilm's tumor-related protein one
Unknown one

Sequence analysis of clones identified from Reverse Subtracted Library
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 pathway in vivo one
DNAJ protein-like one
Alcohol dehydrogenaseone
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 the histone H2B gene family was induced only upon infection of tobacco cells 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. We are now determining which, or both, of these transferred molecules is responsible for histone gene induction.

4. T-DNA ligation assays.

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

       These genes are described in Section 1 above.

6. Improvement of agronomically important crops using Arabidopsis genes essential for Agrobacterium infection.

       This project was proposed not to be started until the fourth year of the project. We have not initiated this part of the project yet.