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 four years, we have 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 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: 21Of 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.
From efforts during the course of this grant: 63
Total rat mutants from forward genetic screening of all libraries: 84rat mutants from reverse genetic screening (see below): 42
Total rat mutants from all screenings: 126
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 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 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 have been screening this library for rat mutants. To date, approximately 3,000 plants from this line have been 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 have screened approximately 20,000 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. In the summer of 2003, another student from Alicja Ziemienowitz�s will be 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.
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 36 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 | Cellulose synthase-like (CslA9) |
| rat5 | Histone H2A |
| rat7 | Unknown protein |
| rat9 | Unknown protein |
| rat14 | Unknown protein |
| rat17 | myb-like transcription factor (caprice gene�this does not co-segregate with the rat phenotype) |
| rat19 | Unknown protein |
| rat22 | Unknown protein |
| ratJ1 | Importin beta-3 |
| ratJ2 | MADs box protein |
| ratJ3 | Hypothetical protein |
| ratJ6 | Adenosine kinase/3-isopropylmalate dehydrogenase |
| ratJ7 | 'DEAD box' RNA helicase |
| ratJ9 | Mitochondrial chaperonin hsp60 |
| ratL1 | Cyclin/cinnamoyl transferase |
| ratL2 | Unknown protein |
| ratL3 | ARID protein/METHF dehydrogenase (The rat phenotype caused by disruption of this gene has been confirmed by identification of two mutant alleles in the SALK T-DNA insertion collection) |
| ratL4 | ATP citrate lyase/glucosidase |
| ratL5 | A-T rich repeat region |
| ratL6 | Hypothetical protein |
| ratL7 | CAAT repeat region |
| 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 |
| uta1 | Voltage-dependent anion channel |
| uta2 | F box protein |
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 alpha-7.
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:
| ratH1 | Unknown protein |
| cep1 | Constitutive expression of PR 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-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 H4-3 | (HFR3) |
| Histone H4-4 | (HFR4) |
| Histone H4-6 | (HFR6) |
| Histone acetyltransferase-1 | (HAC1) |
| Histone acetyltransferase-6 | (HAC6) |
| Histone acetyltransferase-8 | (HAC8) |
| Histone acetyltransferase-9 | (HXA1) |
| Histone acetyltransferase-10 | (HXA2) |
| Histone acetyltransferase-11 | (HAC11) |
| Histone deacetylase-1 | (HDA1) |
| Histone deacetylase-2 | (HDA2) |
| Histone deaceylase-4 | (HDA4) |
| Histone deacetylase-6 | (HDA6) |
| Chromatin remodeling protein | (CHA6) |
| Nucleosome assembly factor | (NFA2) |
| Silencing group A | (SGA1) |
| DNA methyltransferase-1 | (DMT1) |
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] |
| ACT2 | Actin-2 [This mutant has been complemented] |
| ACT7 | Actin-7 [This mutant has been complemented] |
| MYO2 | Myosin 2 |
| 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) |
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:
http://www.bio.purdue.edu/courses/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 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 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 (SCFVirF). 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. 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
| Putative Functions of Identified Clones | #Clones type (total 33) |
| Histone H2A | 2 |
| Histone H4 | 4 |
| Histone H3 | 2 |
| Histone H2b | 4 |
| Ribosomal Proteins | 4 |
| Polyubiquitin (Ubi) mRNA | 1 |
| G protein beta-subunit-like protein | 1 |
| Genomic RNA expressed in roots | 1 |
| 5-epimerase | 1 |
| Protein homologue to human Wilm's tumor-related protein | 1 |
| Unknown | 1 |
Sequence analysis of clones identified from Reverse Subtracted Library
| Putative Functions of Identified Clones | #Clones type (Total 83) |
| Glutathione S-Transferase | 8 |
| Proteins responds to agents that induce systemic acquired resistance and TMV-inducible | 5 |
| Sucrose synthase | 3 |
| Extensin like protein | 3 |
| Osmotin-like protein | 2 |
| Non-symbiotic hemoglobin | 2 |
| Nodulin | 2 |
| Short-chain type dehydrogenase/reductase | 1 |
| Putative translationally controlled tumor protein (TCTP1) | 1 |
| Tumor related protein, expansin like proteins or pollen Allergen-like proteins | 1 |
| Glucosyl transferase | 1 |
| Glucan beta-1,3-glucosidase | 1 |
| Beta -1,3-glucanase | 1 |
| Beta-xylosidase | 1 |
| Glyceraldehyde-3-phosphophate dehydrogenase | 1 |
| Monodehydroascorbate reductase | 1 |
| Metal-transporting ATPase, | 1 |
| H+-transporting ATP synthase | 1 |
| HCF106 Required for the Delta pH pathway in vivo | 1 |
| DNAJ protein-like | 1 |
| Alcohol dehydrogenase | 1 |
| Caffeoyl-CoA O-methyltransferase | 1 |
| Cyclin-dependent kinase inhibitor | 1 |
| Cinnamyl-alcohol dehydrogenase | 1 |
| Apoptosis related protein | 1 |
| Hypothetical protein | 1 |
| *6 to be characterized | 1 |
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.
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.
We are currently repeating these experiments using infected Arabidopsis root segments and 'whole genome' oligonucleotides microarrays.
Because our macroarray 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. 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 the last year 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 (kanR) was always co-transfected as a control.
In order to analyze the different DNA repair pathways in the rat mutants it is important to check the validity of using this plasmid-based assay with Arabidopsis plantlets. This has been achieved by bombarding gold particles coated with linearized (ampR) and circular (kanR) plasmids, then assaying for repair of the linearized substrate. The measurement is achieved by rescuing plasmid DNA, then transforming E. coli and counting kanr versus ampr colonies. The repaired plasmids are also sequenced at the repair site. To date we have shown this to be a viable approach and have conducted a time course indicating that 2 hours after bombardment gave the maximum repair efficiency.
To analyze the two different DSB repair pathways in the rat mutants, we plan to produce two reporter gene constructions that will differentiate between the two pathways. A luciferase gene with an intron in the coding sequence that is subsequently digested in the intron will provide the substrate for non-homologous end joining due to the fact that even unfaithful repair will result in a functional reporter protein. The use of this plasmid does have limitations as large insertions or deletions may disrupt the splicing mechanism. However, because T-DNA integration frequently results in rearrangements of the plant sequences at the junctions and often also contains 'filler' DNA, this test does measure activities pertinent to T-DNA integration.
This plasmid will be co-bombarded with a second disrupted reporter gene, with homologous overlapping sequence (direct or indirect) within the gusA gene. This construction allows quantitative analysis of the homologous recombination pathway. Visualization of both reporter genes will allow direct comparison of DNA repair pathways in the rat mutants.
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 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. Transfer of the RAT5 gene (or protein as a VirF fusion) also increases the transient transformation frequency of Brassica napus. We are currently determining the affect of over-expressing the RAT5 gene on 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.