Research Interests Molecular mechanisms of plant-pathogen interactions
I.Studies of Agrobacterium-plant interactions
One of the research interests in my laboratory is to understand the molecular mechanisms of Agrobacterium.-plant interactions. A. tumefaciens is a bacterial plant pathogen that causes crown gall disease on a wide range of dicot plants (Fig.1). The transformation of plant cells is incited by transferring its one segment of DNA (T-DNA) via a T-pilus associated type IV secretion system from bacteria into the plant nuclear genome. Due to the nature of this interkingdom DNA transfer, A. tumefaciens has also been the most popular genetic transformation tool in plant biology studies.
Figure 1. Crown gall tumor on an elm tree at UC Davis campus. (Photo taken by Jer-Ming Hu)
The proteome analysis of Agrobacterium virulence has led to the identifications of several new proteins involved in virulence.The recent complete Agrobacterium genome sequences have open a new avenue to study the molecular mechanisms of Agrobacterium-plant interactions. We have initiated a proteomic project to determine the differentially expressed or distributed proteins of A. tumefaciens in response to plant signals. Two-dimensional gel electrophoresis (2-DE) was employed to determine the differentially expressed proteins of A. tumefaciens in response to acetosyringone (AS), a known virulence gene inducer released from wounded plant cells. Several known AS-induced proteins including VirB4, VirB8, VirB9, VirB10, VirB11, VirE2, VirH1, VirK, and Tzs were identified. One Ti-plasmid encoded hypothetical protein Y4mC and one chromosomally encoded small heat shock proteins HspL were newly discovered AS-induced proteins (Fig. 2). Apart from the examination of proteome expressed inside the bacterial cells, the secretome of A. tumefaciens were also analyzed to identify the secretory proteins that may play roles in virulence. By 2-DE, SDS-PAGE, and shotgun proteomics analysis of secretome, a total of 13 proteins were consistently identified in the culture medium of A. tumefaciens grown in the presence and/or absence of AS. The proteome analysis of Agrobacterium virulence has led to the identifications of several new proteins involved in virulence. We are currently investigating the roles of these new virulence factors in Agrobacterium virulence that shall lead to further understanding of Agroabacterium-plant interactions and biotechnology applications in Agrobacterium-mediated plant transformation.
Figure 2. Acetosyringone (AS)-induced proteome of A. tumefaciens
II. Functional genomic and proteomic studies of rice resistance/defense mechanisms
Another research interest in my laboratory is to understand the molecular network of disease resistance/defense pathways in rice. The initial objective is to screen and identify the genes, promoters, and proteins involved in rice resistance/defense signaling pathways in response to defense signaling molecules.
To date, the knowledge of plant resistance/defense mechanism has been largely obtained from the studies in eudicot plants Arabidopsis and tobacco. The studies on monocot model plant rice did reveal some conserved components, yet the picture of its defense signal transmission network is less clear. We have successfully employed proteomics and DNA microarray to identify several known defense-related genes and hundreds of novel genes with functions yet to be determined in rice (Fig. 3). The expression data suggest the pathways involving mitogen activated protein kinase (MAPK) cascades, calcium-dependent protein kinases (CDPK) signaling, lipid signaling, phytoalexin, and lignin biosynthesis were responsible for the chemically-induced disease resistance in rice. We are currently generating the transgenic rice plants expressing the selected novel defense-related genes to study their roles in rice disease resistance/defense mechanisms.
Figure 3. 2-DE mapping of 9 probenazole-regulated (red) and 31 abundant (green) protein spots in rice seedlings.
Lai, E. M. and Kado, C. I. (1998). Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens. J. Bacteriol. 180: 2711-2717.
Eisenbrandt, R., Kalkum, M., Lai, E. M., Lurz, R., Kado, C. I., Lanka, E. (1999). Conjugative pili of IncP plasmids and the Ti plasmid T pilus are composed of
cyclic subunits. J. Biol. Chem. 274: 22548-22555.
Lai, E. M., Chesnokova, O, Banta, L., and Kado, C. I. (2000). Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens. J. Bacteriol. 182: 3705-3716.
Lai, E. M. and Kado, C. I. (2000). The T-pilus of Agrobacterium tumefaciens.
Trends in Microbiol. 8: 361-369.
Lai. E. M. and Kado. C. I. (2002). The Agrobacterium tumefaciens T-pilus Composed of cyclic T-pilin is highly resilient to extreme environments. FEMS Microbiol. Letters 210: 111-114.
Lai, E. M., Eisenbrandt, R., Kalkum, M., Lanka. E., and Kado, C. I. (2002). Biogenesis of T-pili in Agrobacterium tumefaciens requires precise VirB2 propilin
cleavage and cyclization. J. Bacteriol. 184: 327-330.
Lai, E. M., Phadke, N. D., Kachman, M. T., Giorno, R., Vazquez, S., Vazquez, J. A., Maddock, J. R., and Driks, A. (2003). Proteomic analysis of the spore coats of Bacillus subtilis and Bacillus anthracis. J. Bacteriol. 185: 1443-1454.
Lai, E. M., Nair, U., Phadke, N. D., and Maddock, J. R. (2004) Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli. Mol. Microbiol. 52: 1029-1044.
Lai, E. M., Shih, H. W., Wen, S. R., Cheng, M. W., Hwang, H. H., and Chiu, S. H. (2006). Proteomic analysis of Agrobacterium tumefaciens response to the vir gene inducer acetosyringone. Proteomics 6: 4130-4136.
Lin, Y. Z., Chen, H. Y., Chang, S. P., Kao, R., Chang, S. J., and Lai, E. M. (2007). Proteomic analysis of rice defense response induced by probenazole. Phytochemistry (in press)
