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Lois Banta
Lois Banta
Lois Banta
Associate Professor of Biology at Williams since 2000


Office: 213 TBL
Phone: (413) 597-4330
E-mail: lbanta@williams.edu
Area of Interest: Microbiology



Education
Courses Offered
Students
Biology Lab
Members of the Banta lab 2002-2003: (left to right) Bronwyn Butcher, Jessica Bauman, Susan Levin, Maywa Montenegro, Lois Banta
Banta lab at the 26th Annual Crown Mall Conference in Bloomington, Indiana (Aug. 2005).
The 27th annual conference will take place at Williams next Nov. 17-19, 2006! Please see http://www.williams.edu/Biology/crowngall27/.
Banta lab
(left to right) Jessica Davis, Esa Seegulam, Shannon Chiu, Danai Musarurwa, Meghan Giuliano
Banta lab
Meghan Giuliano
Post-doctoral Fellows
Research Interests
tumors
Figure 1: Tumors, caused by infection with Agrobacterium tumefaciens, on the stem of a tobacco plant and on the left side of a leaf of a Kalanchoe plant.

In my lab, we study the interactions between the soil pathogen Agrobacterium tumefaciens and its host plants. In particular, we are interested in the assembly and function of a membrane-associated multi-protein complex involved in the transport of a large fragment of DNA across the membrane system surrounding the bacterium. Infection of susceptible plants by A. tumefaciens results in crown gall tumor formation (Figure 1). The disease mechanism involves the transfer and integration into the plant genome of a specific DNA molecule (T-DNA) from a bacterial tumor-inducing (Ti) plasmid (Figure 2). Sequences on the T-DNA encode enzymes responsible for the biosynthesis of plant growth hormones; expression of these genes in the host plant leads to uncontrolled hormone production and hence unregulated plant cell division ("plant cancer"). A second set of genes on the transferred DNA encodes enzymes directing the synthesis of opines, which are utilized by the bacterial cell (Figure 3). T-DNA processing and transfer are mediated by a number of Ti plasmid-encoded virulence (Vir) proteins. Movement of the T-DNA requires the eleven products of the virB operon, as well as the VirD4 protein.

VirB protein complex
Figure 2
 Stem with tumor
Figure 3

Recent work from several labs, including my own, supports the notion that assembly of a membrane-associated heteroligomeric VirB protein complex is a multistep process that is sensitive both to environmental influences and to the presence of nonfunctional constituent subunits. Over the past few years, we have focused on the role of several VirB proteins, and particularly VirB10, in transport apparatus assembly. Although various lines of experimentation implicate VirB10 as an essential and perhaps rate-limiting component of the transfer process, the precise activity of the protein is unknown. We have used both genetic and biochemical strategies to characterize VirB-containing protein complexes and the interactions that mediate VirB protein function. Through our biochemical studies, we have found that VirB10 stability is dependent on growth temperature and periplasmic osmodaption. Our genetic experiments have led us to conclude that over-production of wild-type VirB10 inhibits DNA transfer and suggest a mechanism by which VirB10 accumulation is regulated in vivo. Additionally, we have isolated and characterized several avirulent virB10point mutants.

The over-arching goal of my current research is to probe the interactions between the VirB pore and the transported substrates, which include not only the T-DNA, but also a second DNA molecule derived from the broad host-range plasmid RSF1010. Significantly, the VirB transport machinery can also deliver at least two proteins, VirE2 and VirF, to plant cells independently of the T-DNA. We hypothesize that upon interaction with a substrate and/or a host cell, the VirB protein complex undergoes a conformational change, such as that depicted in the figure below, to allow delivery to the host cell. The substantial sequence homologies noted recently between VirB proteins and the functions required for pertussis toxin localization, as well as for pathogenicity in Helicobacter pylori, Legionella pneumophila, and several other bacteria, indicate that our findings regarding assembly of the VirB pore may be more broadly relevant to the assembly and functioning of multi-protein transporters responsible for the delivery of a variety of pathogenic substrates to mammalian host cells. An updated list of Type IV secretion systems is maintained by our collaborator Peter Christie at the University of Texas Medical School at Houston, and is available under T4SS-updated.pdf here.

VirB protein complex
Figure 4
 Genetically engineered
Figure 5: Agricultural biotechnology includes both genetic modification of plants and growth of plant material in tissue culture.

The objective of the research funded by our current three-year grant is to investigate the role of two proteins,VirC1 and VirC2, in the regulation of substrate delivery by the VirB/D4 secretion apparatus. The virC operon in A. tumefaciens is a host-range determinant, in that it is required for the formation of tumors on some, but not all, host plants typically infected by wild-type agrobacteria. VirC1 is a member of a superfamily of ATPases that also includes the proteins involved in active partitioning of bacterial plasmids during cell division. In light of the roles played by other members of this ATPase superfamily in tethering plasmids to bacterial membranes, we have set out to test whether the putative ATP-binding motif in VirC1 is functional and whether it is required for virulence. Furthermore, we have hypothesized that the membrane-associated VirC1 may actively facilitate T-DNA interaction with the VirB transport apparatus, located at the cell poles, by tethering the Ti plasmid to the cell membrane through its interactions with the 23-base pair Overdrive sequence located adjacent to the right border of the T-DNA.

Genomics Research in the Banta Lab: As part of a consortium that also includes several other academic institutions and Monsanto (Agrobacterium.org), students in the Banta lab, as well as in the Microbiology course and the Cell Regulatory Mechanisms course, have contributed to the annotation of one published and two recently assembled sequences, those of three Agrobacterium biovars with distinct host-range specificities, genomic organization, and phylogenetic relationships (Annotated Sequences). Hypothesizing that host range and ability to incite host defense responses might depend in part on cell surface properties, we chose to focus on cell-surface polysaccharides. The process of exopolysaccharide (EPS) biosynthesis has been well characterized, both biochemically and genetically, in Sinorhizobium meliloti. Students used the information available from the sequenced genome of S. meliloti to analyze and annotate the corresponding pathways in A. tumefaciens strain C58 and A. vitis strain S4; we are currently expanding this analysis to the genome of A. radiobacter strain K84, which was just closed. As one of my students reported at the national Agrobacterium meeting in August, 2005, we noted several gene duplications, deletions, and rearrangements in the agrobacterium gene clusters responsible for succinoglycan (EPS I) synthesis. Although neither agrobacterium strain has yet been reported to make galactoglucan (also known as EPS II), we found homologs of several exp genes, involved in galactoglucan synthesis in S. meliloti, in both A. tumefaciens and A. vitis. We are currently constructing reporter and disruption strains to investigate the contributions of several of these genes to EPS production and virulence in A. tumefaciens.

In collaboration with Stan Gelvin (Purdue University), we have also initiated a project in which macro-array analysis and bioinformatics are used to explore tobacco defense responses to A. tumefaciens. After employing standard recombinant DNA techniques to create virC disruption mutants, students in the Biology 306 course used macroarray analysis to assess whether there are differences in the response of tobacco BY-2 suspension culture cells to co-cultivation with these virC mutants as compared to wild-type A. tumefaciens. The Gelvin lab has pioneered this macroarray approach and has identified 450 tobacco genes that are differentially regulated in response to wild-type A. tumefaciens (Veena, et al., Plant J. 35:219; 2003). We anticipate that we may indeed detect differences in the expression of a variety of these host genes in response to virC -deficient bacteria; since the host-range determinants VirC1 and VirC2 appear to enhance the efficiency of T-DNA delivery to host plants, bacteria lacking virC may stay "below the radar" of some plants (leading to successful infection), while delivering insufficient T-DNA to overcome host defenses (leading to a failure to incite tumorigenesis) on other plant species. Our pilot macro-array experiments, with a small number of representative plant genes that are normally differentially expressed during Agrobacterium infection, suggested that there are indeed virC -dependent differences in host gene expression. Students in the lab are currently following up on these observations, using real-time PCR to quantify differences in gene expression.

Significance: Agrobacterium transformation of plants is the only known naturally occurring example of interkingdom DNA transfer. Over the past decade or so, researchers have taken advantage of this system to deliver foreign DNA of their choosing into an ever-increasing list of host plants. Adaptation of the Ti plasmid for use as a vector has led to the ability to produce transgenic plants, and has revolutionized commercial food production, as well as research into fundamental questions of plant development and regulation of gene expression. Applications of this method of plant transformation have been limited by the recalcitrance of many species, including several agronomically important crop plants, to Agrobacterium infection. Recent progress in transforming maize and rice has been facilitated by the recognition that differences in virulence among Agrobacterium strains are attributable at least in part to the vir region of the Ti plasmid carried by the strain. Advances in our knowledge of the mechanism on T-DNA delivery may contribute to the work of those scientists engaged in efforts to increase the efficiency and host range for transformation of hitherto poorly susceptible species. In this way, our research may someday have an impact on enhancing world food supply.

Funding
Selected Publications
*Denotes Haverford College undergraduate
**My current research project was undertaken in collaboration with Prof. Paul Hooykaas at the Institute for Molecular Plant Sciences at Leiden University in the Netherlands. For more information on my family's experiences while on sabbatical in Leiden, click here.
#Photo credits: Photographs courtesy of the Consultative Group for International Agricultural Research (http://www.cgiar.org/photos.htm) and these research centers: Centro Internacional de la Papa, International Rice Research Institute, Centro Internacional de Mejoramiento de Maiz y Trigo, and the International Crops Research Institute for the Semi-Arid Tropics. Figure 1 courtesy of Matthew Stremlau.
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