1. Identification and characterization of Arabidopsis rat mutants and molecular cloning of RAT genes.

       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 year, 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 screened during the course of this grant, approximately 700 appeared to have the rat phenotype upon first screening. Approximately 190 of these putative mutants have been re-screened, and we shall re-screen the remainder of these putative mutants in the next few months. After several additional rounds of screening, we have confirmed the following number of rat mutants:

From efforts previous to this grant: 21
From efforts during the course of this grant: 16
       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.

       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 initiated the generation of a new T-DNA disruption library (approximately 60,000 members) in ecotype Ws. This library should be completed by the end of the summer,2000. We shall subsequently pursue screening of this library for rat mutants.

       Vitaly Citovsky will be sending a new postdoctoral research fellow to Stan Gelvin's laboratory in June, 2000 for a period of one month. Therefore, the rat mutant screening technology will be shared between these laboratories.

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 175,000 colonies containing the VirB2 bait and the prey cDNA library and have identified 20 strongly interacting colonies. We are currently rescuing prey plasmids from the positive interactors and will subsequently characterize the cDNAs contained in these prey plasmids.

       We have just 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.

       Reverse genetics experiments to screen for Arabidopsis insertional mutants in VIP1,VIP2, and AtKAP-alpha genes were initiated using the NSF-funded knockout facilities at the University of Wisconsin-Madison.

       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.

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 80-90% after 3 days of Agrobacterium infection. Total RNA was isolated from BY-2 cells without infection and after 0, 12 and 24 hr of infection with At793 and At804. The cDNA from these samples was prepared and used for differential display experiments. Initially we have used only two primer combinations for differential display in order to standardize the technique in our system. Using these primer combinations we were able to identify a few bands differentially expressed between infected and non-infected cells. However, the pattern of differential bands was similar for both the strains (At793 and At804) used for infection. These preliminary results suggest that the differential RNA species we see reflect Agrobacterium interaction with the plant cells, and not a response of the plant cells to the transfer of T-DNA per se. We are in process of characterizing these selected fragments showing differential expression by Northern blots and sequencing.

       We are also performing more differential display reactions using other sets of primer combinations to find out if there are genes which are differentially expressed in BY-2 cells infected with At804 strains as compared to control or At793 infected BY-2 cell suspension cultures.

       Finally, we are establishing Arabidopsis cell suspensions to use for similar experiments.

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

       Because we are now in the process of identifying genes disrupted by the T-DNA in the ratmutants, we have not had the opportunity to characterize the functions of these genes. Characterization will begin in the second year of the grant.

5. 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.