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Agrobacterium in the Genomics Age

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Agrobacterium in the Genomics Age Update on Genomics of Agrobacterium-Mediated Plant Transformation Agrobacterium in the Genomics Age Stanton B. Gelvin* Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907–1392 Members of the genus Agrobacterium cause the neo- Korber et al., 1991), or may be involved in chromatin plastic diseases crown gall, hairy root, and cane gall on remodeling (gene6b; Terakura et al., 2007). Expression numerous plant species. Extensive genetic analyses of these genes results in tumorigenic or rhizogenic conducted in the 1980s identified key bacterial genes growth. A second set of genes directs the synthesis of involved in virulence. During the past decade, how- various low Mr compounds, opines, that can serve as ever, genomic technologies have revealed numerous energy sources for the inciting bacterial strain and can additional bacterial genes that more subtly influence perhaps affect virulence (Veluthambi et al., 1989). For transformation. The results of these genomic analyses reviews, the reader should see Gelvin (2000, 2003), allowed scientists to develop a more integrated view Tzfira and Citovsky (2001, 2003), McCullen and Binns of how Agrobacterium interacts with host plants. In a (2006), and Citovsky et al. (2007). In addition, the similar manner, genomic technologies have identified reader is directed to an excellent new book on Agro- numerous plant genes important for Agrobacterium- bacterium biology (Tzfira and Citovsky, 2008). mediated genetic transformation. Knowledge of these Most plant biologists, however, best know Agro- genes and their roles in transformation has revealed bacterium as an agent of horizontal gene transfer that how Agrobacterium manipulates its hosts to increase plays an essential role in basic scientific research and the probability of a successful transformation out- in agricultural biotechnology. In the 1980s, scientists come. In this article, I review our current knowledge of learned to disarm (delete the oncogenes and, usually, Agrobacterium-plant interactions and how genomic the opine synthase genes) virulent Agrobacterium and proteomic technologies have increased our un- strains such that tissues infected by the bacteria could derstanding of this unique plant-microbe interaction. regenerate into normal plants (Bevan et al., 1983; Agrobacterium species are phytopathogens that Fraley et al., 1983; Herrera-Estrella et al., 1983). cause a variety of neoplastic diseases, including crown Substituting genes of interest for oncogenes and opine gall (Agrobacterium tumefaciens and Agrobacterium vi- synthase genes resulted in plants expressing these tis), hairy root (Agrobacterium rhizogenes), and cane gall novel transgenes and, thus, novel phenotypes. Al- (Agrobacterium rubi). Virulent strains of Agrobacterium though transgene substitution for oncogenes within contain tumor-inducing (Ti) or root-inducing (Ri) plas- T-DNA was initially conducted in cis (i.e. novel trans- mids. During infection, enzymes encoded by plasmid- genes were placed within T-DNA of Ti-plasmids; localized virulence (vir) genes process the T-DNA Caplan et al., 1983; Fraley et al., 1985), the develop- region of these plasmids. The resulting single-strand ment of binary systems, in which T-DNA and viru- DNA (T-strand) linked to VirD2 protein exits the lence helper plasmids were separated into two bacterium via a type IV protein secretion system and different replicons (de Framond et al., 1983; Hoekema enters the plant cell. Within the plant, T-strands likely et al., 1983), greatly increased the utility of Agrobacte- form complexes with other secreted virulence effector rium as a vehicle for gene transfer in plant biology proteins, including VirE2, VirE3, VirD5, and VirF, and laboratories. supercomplexes with plant proteins as they traverse Throughout its development as a gene jockeying the cytoplasm and target the nucleus. Once inside the tool, genomic studies on Agrobacterium and its plant nucleus, T-strands integrate randomly into the plant hosts guided scientists in basic science and agricul- genome and express T-DNA-encoded transgenes. Two tural biotechnology developments. In this article, I classes of T-DNA genes mediate the pathology of review some of the key genomic methodologies and Agrobacterium infection. The first group, the onco- findings that have contributed to our knowledge of genes, either effect phytohormone production (iaa how Agrobacterium works and will contribute in the and ipt; Akiyoshi et al., 1984; Schroder et al., 1984), future better to utilize Agrobacterium’s amazing gene sensitize the plant to endogenous hormone levels (rol transfer abilities in the laboratory and in the agricul- and other genes of pRi, gene5 and gene6 of pTi; Shen tural biotechnology industry. et al., 1988; Spanier et al., 1989; Tinland et al., 1990; GENOMICS OF AGROBACTERIUM * E-mail [email protected]. The author responsible for distribution of materials integral to the Whole-Genome Mutagenesis findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Although not frequently considered genomics, im- Stanton B. Gelvin ([email protected]). portant early studies on A. tumefaciens and A. rhizo- www.plantphysiol.org/cgi/doi/10.1104/pp.109.139873 genes utilized whole-genome mutagenesis and mass Ò Plant Physiology , August 2009, Vol. 150, pp. 1665–1676, www.plantphysiol.org Ó 2009 American Society of Plant Biologists 1665 Gelvin phenotypic screening to define Agrobacterium genes important phytopathogen. A. tumefaciens C58 contains important for transformation (i.e. T-DNA and Vir four replicons: a circular and a linear chromosome and protein transfer) and tumorigenesis. Transposon mu- two plasmids (pTiC58 and pAtC58). The genome tagenesis was generally the method of choice because contains approximately 30 insertion sequence elements of the relatively random integration pattern of trans- and encodes an unusually large number of trans- posons in the bacterial genome and because the posi- porters (at least 153) and two-component regulatory tions of transposon insertions could easily be systems (at least 25). Recently, Ulker et al. (2008) de- determined by restriction endonuclease mapping. scribed the surprising observation that Agrobacterium Thus, scientists localized genes involved in opine can transfer its chromosomal DNA to plants. Interest- catabolism to a specific region of the Ti-plasmid and ingly, particular insertion sequence elements and identified genes involved in crown gall tumorigenesis transporter gene sequences are hot spots for chromo- in the T-DNA, in regions of the Ti-plasmid not within somal DNA transfer. The preferential appearance of the T-DNA (later to be identified as the virulence these chromosomal sequences associated with T-DNA region), and in the bacterial chromosome (chromo- in Arabidopsis (Arabidopsis thaliana) and rice (Oryza somal virulence [chv] genes; Garfinkel and Nester, sativa) T-DNA/plant DNA junctions suggests multiple 1980; Holsters et al., 1980; Ooms et al., 1980; De Greve mechanisms for chromosomal DNA mobilization dur- et al., 1981). More directed mutagenesis studies iden- ing T-DNA transfer (Gelvin, 2008). tified opine synthase genes and oncogenes in T-DNA The complete genome sequence and annotation of A. regions of Ti- and Ri-plasmids and specific virulence tumefaciens C58 is posted on http://depts.washington. genes within the vir gene region (Garfinkel et al., 1981; edu/agro/. In addition to this biovar I A. tumefaciens Leemans et al., 1981; Ooms et al., 1981; Ream et al., strain, DNA sequence analysis of the Agrobacterium 1983; Inze et al., 1984; White et al., 1985; Stachel and radiobacter biovar II strain K84 and the A. vitis biovar III Nester, 1986; Stachel and Zambryski, 1986). These strain S4 has recently appeared (Slater et al., 2009). A. studies involved testing of hundreds or thousands of radiobacter K84 is an especially important strain be- individually mutagenized Agrobacterium strains for cause it and its derivatives are widely used as biocon- virulence, opine catabolism and synthesis, and tumor trol agents against tumorigenic Agrobacterium strains morphology and may thus be categorized as early (Kerr and Panagopoulos, 1977; Jones et al., 1988). Agrobacterium genomic studies. Comparative analysis of the sequences of the three Rong et al. (1990) conducted a second type of genetic Agrobacterium strains and several other species of the screening, using the promoter-less lacZ-containing family Rhizobiaceae indicate a complex genome evolu- transposon MuDI-1681, to identify plant-inducible tion, including the migration of gene blocks among Agrobacterium genes on the chromosome of A. tumefa- replicons within and between species. Sequencing of ciens A136 (C58 chromosomal background lacking a other Agrobacterium strains (the biovar III strain A. vitis Ti-plasmid). These authors assayed several thousand F5R19 and the biovar II strain A. rhizogenes A4) is in randomly mutagenized Agrobacterium strains for in- progress. duced gene expression on plates containing carrot root extract and X-gal. Insertion of the transposon into the Agrobacterium Transcriptional Profiling picA gene revealed that this gene was .10-fold induc- ible by the root extract. The picA gene, currently Based upon the A. tumefaciens C58 sequence, scien- identified as encoding a polygalacturonase-like pro- tists have generated microarrays to probe the response tein (Atu3129), was the first identified plant-inducible of bacterial genes
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