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Crown gall is a neoplastic disease caused by the infection of dicotyledonous plants by virulent strains of the Gram-negative soil bacterium Agrobacterium tumefaciens. During the process of infection, part of a bacterial plasmid, called the tumor inducing (Ti) plasmid, is transferred from the bacterium to the plant, where it stably integrates into the nuclear DNA. This transferred, or T-DNA, can be expressed as mRNAs which are translated. Tumorous lesions result, as well as the production of rare compounds called opines which the bacterium can utilize as an energy source.

In our laboratory we have been interested in the molecular mechanism of Ti-plasmid transfer, integration, and expression. Projects related to these areas include molecular and genetic analyses of the T-DNA transfer machinery in A. tumefaciens, regulation of the vir genes of A. tumefaciens that direct this transfer, and a molecular analysis of the form of T-DNA that is transferred. We have recently initiated experiments to examine the process of T-DNA transfer to plant cells, including the targeting of the T-DNA to the plant nucleus and the very early events of T-DNA expression and integration. After the T-DNA integrates into plant chromosomal DNA, T-DNA genes are transcribed. We are investigating promoters and transcriptional activators of T-DNA genes, and have made recombinant promoters with a very high level of transcriptional activity. These promoters will be useful for the genetic engineering of plants. Other projects in the lab involve an investigation, using various ecotypes of Arabidopsis thaliana that are susceptible or resistant to crown gall tumorigenesis, of molecular aspects of plant defense responses to pathogen attack. We have cloned several Arabidopsis genes necessary for susceptibility to Agrobacterium-mediated transformation.

Professor:    Stanton B. Gelvin

Research Associates:    B. Balaji, Burgund Bassuner, Sudip Chattapadahyay, Jaime Menendez Humara, C.T. Ranjith Kumar, Jyothi Rajagopal, Veena, Ho Chul Yi

Assistant Research Scientist:    Lan-Ying Lee

Research Assistants:    Hongbin Cao, Masha Kononova

Instructor:    Susan J. Karcher

Graduate Students:    Saikat Bhattacharjee, Hau-Hsuan Hwang, Kiran Mysore, and Praveen Rao

Undergraduate Students:    Tony Kaiser, Andrea Kopecki, Aneeza Salim, and Ben Svarczkopf

PUBLICATIONS

Gelvin, S.B. 1998. The introduction and expression of transgenes in plants. Curr. Opin. Biotechnol. Curr. Opin. Biotechnol. 9:227-232.

Mysore, K.S., Bassuner, B., Deng, X-b. Darbinian, N.S., Motchoulski, A., Ream, W., and Gelvin, S.B. 1998. Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration. Mol. Plant-Microbe Interact. 11:668-683.

Nam, J., Mysore, K.S., and Gelvin, S.B. 1998. Agrobacterium transformation of the radiation hypersensitive Arabidopsis mutants uvh1 and rad5. Mol. Plant-Microbe Interact. 11:1136-1141.

Gelvin, S.B. 1998. Agrobacterium VirE2 proteins can form a complex with T-strands in the plant cytoplasm. J. Bacteriol. 180:4300-4302.

Gelvin, S.B. 1998. Multigene plant transformation: More is better! Nature Biotechnol. 16:1009-1010.

Lee, L.-Y., Gelvin, S.B., and Kado, C.I. 1999. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export. J. Bacteriol. 181:186-196.

Nam, J., Mysore, K.S., Zheng, C., Knue, M., Matthysse, A.G., and Gelvin, S.B. 1999. Identification of T-DNA tagged Arabidopsis mutants that are resistant to Agrobacterium transformation. Mol. Gen. Genet. 261:429-438.

Mysore, K.S., Nam, J., and Gelvin, S.B. 2000. An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration PNAS 2000 97: 948-953.

Mysore, K.S., and Gelvin, S.B. 2000. Agrobacterium germ-line transformation bypasses some of the steps involved during conventional root transformation of Arabidopsis. Plant J. In press.

Gelvin, S.B. 2000. Agrobacterium and plant proteins involved in T-DNA transfer and integration. Annu. Rev. Plant Physiol. Plant Mol. Biol. In press.

Kononov, M.E., and Gelvin, S.B. 1999. New and improved plant transformation vectors containing the super-promoter. Plant Mol. Biol. Submitted.

Tao, Y., Rao, P., and Gelvin, S.B. 2000. A plant phosphatase is involved in nuclear import of the Agrobacterium VirD2/T-DNA complex. Submitted.

Tao, Y., Rao, P., and Gelvin, S.B. 2000. Ser³⁹⁴, a potentially phosphorylated residue of VirD2 protein, plays a role in nuclear import of T-DNA and in crown gall tumorigenesis. In preparation.

Mysore, K.S., Yi, H.-C., and Gelvin, S.B. 2000. Molecular cloning, characterization, and structural organization of histone H2A genes in Arabidopsis. In preparation.

Mysore, K.S., and Gelvin, S.B. 2000. Transgenic Arabidopsis plants expressing Agrobacterium VirD2 protein are resistant to Agrobacterium transformation. In preparation.

Kononov, M.E., Bassuner, B., Wang, K., and Gelvin, S.B. 2000. A comparison of the super-promoter with other promoters in maize. In preparation.

BOOK CHAPTERS

Nam, J., and Gelvin, S.B. 1998. Arabidopsis ecotypes resistant to crown gall tumorigenesis. in Horizontal Gene Transfer. (M. Syvanen and C. Kado, eds.) Chapman-Hall, London.

GENERAL SUMMARY

          Crown gall is a neoplastic disease caused by the infection of dicotyledonous plants by virulent strains of the Gram-negative soil bacterium Agrobacterium tumefaciens. During the process of infection, part of a bacterial plasmid, called the tumor inducing (Ti) plasmid, is transferred from the bacterium to the plant, where it stably integrates into the nuclear DNA. This transferred, or T-DNA, can be expressed as mRNAs which are translated. Tumorous lesions result, as well as the production of rare compounds called opines which the bacterium can utilize as an energy source.

          In our laboratory we have been interested in the molecular mechanism of Ti-plasmid transfer, integration, and expression. To define these mechanisms, we are investigating both Agrobacterium and plant genes required for transformation. The following projects are currently underway to investigate these phenomena. In addition, we are interested in using the Ti-plasmid as a vector for delivering foreign DNA into plant cells, and for using Agrobacterium to effect homologous and site-directed integration of foreign genes into the plant genome.

SUMMARIES

I.    INITIAL EVENTS IN THE TRANSFER AND INTEGRATION OF T-DNA TO PLANT CELLS

Investigator: Lan-Ying Lee

          We have investigated the role that the osa gene of the plasmid pSa plays in oncogenic suppression. When pSa (or a plasmid containing just the osa gene) is in an otherwise tumorigenic Agrobacterium cell, tumorigenesis is inhibited. However, vir gene induction and T-DNA processing are normal. The results of transient versus stable expression assays (GUS activity, tumorigenesis, and transformation to kanamycin-resistance) indicate that pSa inhibits the transformation of the plant in an early step of the infection process. This inhibition appears to result from a block in transfer of VirE2 protein, but not T-DNA, to plant cells. This result was verified by the inability of osa to inhibit T-DNA transfer, but its ability to inhibit VirE2 protein transfer, in "extracellular" complementation experiments. Unlike the situation of oncogenic suppression by the plasmid RSF1010, the inhibition of tumorigenesis by pSa cannot be mitigated by overexpression of the VirB9, 10, and 11 protein in Agrobacterium. osa oncogenic inhibition is also stronger than that effected by RSF1010. Finally, we determined that osa blocks VirE2 protein export from Agrobacterium to plant cells by blocking transport through the VirB-encoded pilus.

II.    T-DNA TRANSPORT TO THE NUCLEUS OF TOBACCO CELLS

Investigators: Stanton B. Gelvin, Hau-Hsuan Hwang, and Praveen Rao

          We have used two approaches to investigate the targeting of the T-DNA to the plant nucleus. We are using the yeast two-hybrid system to identify tomato and Arabidopsis cDNAs encoding proteins that interact with the VirD2 NLS. After screening more than five million such cDNAs, we have identified one tomato and seven Arabidopsis cDNA clones that interact strongly with the NLS. DNA sequence analysis and enzymatic assays indicated that the tomato cDNA encodes a phosphatase type IIC (PP2C). When overexpressed in tobacco protoplasts, this PP2C can inhibit nuclear targeting of a GUS-NLS fusion protein. Furthermore, an Arabidopsis mutant in this PP2C homologue (abi1 mutant) is more susceptible to Agrobacterium-mediated transformation than is a wild-type Arabidopsis line. These data suggest that the PP2C (and possibly phosphorylation of the NLS region of VirD2 protein) may regulate VirD2/T-DNA nuclear transport. We have shown that VirD2 protein can be phosphorylated, both in vitro by protein kinase C and in vivo by tobacco protoplasts, at a serine residue two amino acids upstream of the NLS region. Alteration of serine to either alanine or to aspartic acid reduces nuclear targeting of a GUS-NLS fusion protein. This alteration additionally reduces tumorigenesis by bacteria harboring these mutant virD2 genes. In addition, we are sequencing the seven Arabidopsis cDNA clones that encode proteins that specifically interact with the VirD2 NLS.

          In addition to the nuclear targeting activity of VirD2 protein, the Agrobacterium T-DNA associates with another virulence protein, VirE2, that also contains a NLS. Although a popular model of T-DNA transfer posits that a T-strand/VirD2/VirE2 complex (the T-complex) is formed in the bacterium and exported to the plant, recent evidence from several laboratories, including ours, suggests that VirE2 protein is separately exported from the bacterium. If this were true, then VirE2 protein would have to be able to meet the T-strand in the plant cell. We investigated whether this happens in the plant cytoplasm or in the nucleus. We made an Agrobacterium strain that lacks the VirD2 NLS region and also lacks virE2. Thus, the T-strand/mutant VirD2 complex has no ability to target to the plant nucleus. We infected VirE2-producing transgenic tobacco plants and asked whether VirE2 protein produced in the plant could interact with the T-strand in the cytoplasm and guide it to the nucleus. We found that it could. This experiment lends support to the hypothesis that VirE2 protein and the T-strand/VirD2 complex are separately exported from the bacterium and form a complex in the plant cell.

          Both VirD2 and VirE2 proteins contain nuclear targeting sequences that may help direct the T-strand to the plant cell nucleus. We are currently conducting experiments to determine the relative rolls of these two NLSs in T-DNA nuclear targeting. We have synthesized fluorescently labeled DNA that can be cleaved in vitro by VirD2. These fluorescent complexes will be microinjected into plant cells, and the rate and extent of DNA nuclear localization determined. The experiments will be repeated with the T-strand coated with VirE2 protein, and with specific mutations in VirD2 that delete the NLS or change the phosphorylatable serine to an alanine.

          Finally, we have noted that transgenic Arabidopsis plants that express wild-type VirD2 protein are recalcitrant to further infection by Agrobacterium. We are determining the mechanism of this oncogenic suppression by generating transgenic Arabidopsis plants that express mutant versions of VirD2.

III.     CHARACTERIZATION OF THE EXPRESSION OF NOVEL PROMOTERS IN MONOCOT PLANTS

Investigator: Burgund Bassuner and Masha Kononova

          We have initiated a study in which we have "mixed and matched" the promoters and upstream activating elements of the octopine synthase and mannopine synthase genes and linked them to a GUS reporter gene. These chimaeric genes have been introduced into tobacco and plants regenerated. Fluorimetric GUS analyses of the stems, roots, and leaves of these plants indicate that various combinations of activator and promoter elements stimulate transcription in these tissues differently. In particular, very strong promoters are generated by combining both the octopine synthase and mannopine synthase activators with the promoters from either of these genes. A novel promoter consisting of a trimer of the ocs activator affixed to the mas activator plus promoter is approximately 70-fold stronger that is the CaMV promoter in the leaves of transgenic tobacco plants. We have performed histochemical analyses of sections of transgenic stems, roots, and leaves to determine the cell specificity conferred upon the mannopine synthase and octopine synthase promoters by different combinations of activating elements from these genes.

          We have expanded these experiments to determine the relative strengths of these chimaeric promoters in monocot plant species. We have constructed a series of plasmids that contain the "super-promoter", the mas2´ promoter, the CaMV 35S promoter, or the rice ubiquitin promoter affixed to a gusA reporter gene. In addition, some constructions contain a potato ST-LS1 intron in the gusA structural gene, or a maize ubiquitin intron in the 5´ leader region of the gene. We have electroporated these plasmids into maize BMS cells and compared the relative strengths of these promoters, with or without introns. The results of these experiments indicate that there is no major difference in the strengths of these promoters using this transient expression assay. In addition, we have stably introduced these constructions into maize tissue and regenerated maize plants. Our analyses revealed that the "super-promoter" is as strong as are the other three promoters tested. However, when affixed to a maize ubiquitin intron, only the ubiquitin promoter responded with a large increase in expression activity.

IV.    INTERACTIONS OF AGROBACTERIUM TUMEFACIENS AND ARABIDOPSIS THALIANA

Investigators: Hongbin Cao, Sudip Chattapadahyay, Jaime Menendez Humara, C.T. Ranjith Kumar, Kiran Mysore, Jyothi Rajagopal, Veena, and Ho Chul Yi

          We have initiated a project to investigate the interactions of A. tumefaciens and Arabidopsis thaliana. To identify Arabidopsis genes involved in tumorigenesis, we have begun screening the Feldmann T-DNA insertion library of ecotype Ws for mutants that are not susceptible to tumorigenesis. We have identified more than 30 such mutants (rat mutants, for resistant to Agrobacterium transformation), and have begun to determine which steps of the tumorigenesis process are blocked in these mutants. In some cases, we have cloned T-DNA/plant DNA junction fragments, and have proven that these insertions are responsible for the recalcitrant phenotype. We have used some of these junction fragments to identify the wild-type homologs of the mutant genes in cDNA and genomic cosmid DNA libraries. We are currently concentrating on several Arabidopsis genes necessary for Agrobacterium-mediated transformation. rat1 and rat3 are required for efficient binding of Agrobacterium to plant cells. The rat1 gene product encodes an arabinogalactan protein found in plant cell walls. Incubation of wild-type Arabidopsis roots with �(beta)-glucosyl Yariv reagent (that binds to arabinogalactan proteins) inhibits transformation by Agrobacterium. DNA sequence analysis of rat3 suggests that it also encodes a cell-wall-localized protein. We have generated antibodies to recombinant Rat1 and Rat3 proteins. Rat3 protein is found predominantly in the roots and stems of wild-type plants, but is absent in the leaves. The rat3 mutation results in the abolishment of detectable Rat3 protein in all plant tissues examined. In addition, rat3 plants have short roots compared to wild-type plants. Overexpression of the rat3 gene in wild-type plants results in an elongated root phenotype. Both rat1 and rat3 mutant plants show increased susceptibility to the pathogen Pseudomonas syringae pv. tomato DC3000 and T1A compared to wild-type plants. Therat4 gene encodes a cellulose synthase-like protein.

Rat5 encodes a histone H2A protein. This gene is one of a six-member multigene family. The rat5 mutant is deficient in T-DNA integration. The mutant phenotype can be complemented by introducing a wild-type rat5 gene into the mutant plant by vacuum infiltration. We have recently identified, using a reverse genetics approach, mutant Arabidopsis plants containing T-DNA insertions in or near three of the other histone H2A genes. We are currently investigating whether these plants are also deficient in T-DNA integration. Furthermore, we are attempting to complement the rat5 phenotype using other members (both cDNA and genomic clones) of the histone H2A gene family.

The rat17 gene encodes a myb-like transcription factor, an allele of the "caprice" gene. rat17 mutant plants are deficient in T-DNA integration.

V.     USE OF MUTANT AGROBACTERIUM STRAINS TO EFFECT HOMOLOGOUS AND SITE-DIRECTED RECOMBINATION IN PLANTS

Investigators: Saikat Bhattacharjee and Maria Kononova

          We have initiated experiments to determine whether mutant Agrobacterium strains can be used to effect homologous and site-directed recombination (using the FLP/FRT system) in plants. We are using a bacterial strain that encodes a mutant virD2 gene. This strain efficiently delivers T-DNA to the plant nucleus, but is severely impaired in its ability to integrate T-DNA. Our hope is that this mutant bacterial strain will favor homologous and site-directed integration over illegitimate recombination.