Agrobacterium-Mediated DNA Transfer, and Then Some
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NEWS AND VieWS Uncloned sequencing Enrichment with DNA- No assembly stable isotopic probes No enrichment and isopycnic centrifuge Substrate enrichment BrdU probe Microbes with labeled DNA identifying those Entire microbial community utilizing specific substrates Community actively growing Functional overview in association with substrate Specific functional guilds comparing differences enabling novel pathways across environments to be identified Subdividing the microbes into functional groups enabling within-environment comparison http://www.nature.com/naturebiotechnology Community Specific capacities metabolic tasks Figure 1 Different methods for analyzing microbial communities provide different levels of selectivity and functional description. These tools enable researchers to describe microbes from community capacities to individual metabolic tasks. once again demonstrates that microbes incor- DNA-SIP) (Fig. 1)1. These metagenomic tools 3. Breitbart, M. et al. PNAS 22, 14250 (2002). 4. Edwards, R.A. et al. BMC Genomics 7, 57 (2006). porate new DNA when exposed to different will provide deeper insights into the produc- 5. Dinsdale, E.A. et al. PLoS ONE 3, e1584 (2008). nutrient sources and that these new acquisi- tivity of microbial communities and the speci- 6. DeLong, E.F. et al. Science 311, 496 (2006). Nature Publishing Group Group Nature Publishing 8 tions allow them to use multiple chemicals. fication of individual metabolic tasks. 7. Tringe, S.G. et al. Science 308, 544 (2005). 8. Dinsdale, E.A. et al. Nature 452, 629 (2008). M. mobilis displayed remarkable flexibility: for 200 1. Kalyuzhnaya, M.G. et al. Nat. Biotechnol. 26, 1029– 9. Mou, X. et al. Nature 451, 708 (2008). 1034 (2008). 10. McHardy, A.C. et al. Nat. Methods 4, 63 (2007). © example, it used the formaldehyde-activation proteins to convert methylamine when grown 2. Beja, O. et al. Science 289, 1902 (2000). 11. Sharon, I. et al. ISME J. 1, 492 (2007). on that substrate and incorporated the gene cluster RuBisCo in the presence of methanol. The movement and incorporation of DNA appears to be a fundamental strategy of micro- Agrobacterium-mediated DNA bial adaptation, and phage may hold the key to DNA transmission. As with all microbial transfer, and then some metagenomes11, Chistoserdova and colleagues1 identified high coverage of bacteriophage; other Stanton B Gelvin studies have shown that viromes encode exten- 7 sive metabolic capabilities . This finding cor- In addition to its plasmid DNA, Agrobacterium tumefaciens can transfer its roborates the notion that phage may act as a store of potentially useful DNA that enhances chromosomal DNA to plant genomes. microbial growth and activity. The new high-resolution technology The emergence of today’s agricultural bio- on the tumor-inducing (Ti) plasmid into the described by Chistoserdova and colleagues1 technology industry stems largely from the genomes of most crops. In the late 1990s, sci- enables novel microbial functions to be iden- discovery, ~30 years ago, that the pathogenic entists found that the transferred sequences can tified without the need for culturing. By com- soil bacterium Agrobacterium tumefaciens can include regions of the Ti plasmid, or T-DNA bining different metagenomic techniques, integrate so-called transfer DNA (T-DNA) ‘binary vectors’, outside the T-DNA borders1. microbial function can now be described In this issue, Ülker et al.2 show that the plant from the community level (by whole-commu- Stanton B. Gelvin is in the Department of genome may also incorporate bacterial chro- nity sequencing), to the individual level (by Biological Sciences, Purdue University, mosomal DNA. extracting active species via BrdU labeling9), West Lafayette, IN 47907-1392, USA. In the past decade, Agrobacterium-mediated to the level of single metabolic pathways (by e-mail: [email protected] transformation has been used to generate hun- 998 VOLUME 26 NUMBER 9 SEPTEMBER 2008 NATURE BIOTECHNOLOGY NEWS AND VieWS dreds of thousands of T-DNA insertion muta- unimportant for mutant gene identification or integrated Agrobacterium chromosomal DNA tions in the genomes of model plant species, as experimental artifacts of the junction iden- was more frequently associated with T-DNA such as Arabidopsis thaliana (thale cress) and tification process. right borders than with left borders, and could Oryza sativa (rice). These T-DNA insertion Prompted by the serendipitous discovery be >18 kb long. Of two ~18-kb inserts, one libraries, intended to saturate the genome with that an Arabidopsis T-DNA insertion mutant contained 16 complete bacterial open reading mutations, are important tools for forward contained T-DNA linked to Agrobacterium frames, and the other 18 Agrobacterium chro- and reverse genetic studies to understand chromosomal DNA, Ülker et al.2 set out to mosomal genes. A. tumefaciens C58 (the base plant gene function. As Agrobacterium can assess the frequency of T-DNA/Agrobacterium strain used to construct the T-DNA insertion survive and reproduce in infected plants, many chromosomal (AchrDNA) junctions in pri- libraries) contains both a linear and a circular databases used to identify plants containing mary, unfiltered data from many FST data- chromosome3,4. Sequences from both of these T-DNA flanking sequence tags (FST) in genes bases. They identified T-DNA/AchrDNA chromosomes associated with T-DNA inte- of interest are ‘scrubbed’ of T-DNA junctions junctions at a frequency of ~0.4% of the grate into the plant genome, with certain ‘hot with other T-DNAs, vector backbone sequences sequenced junctions in each of these data- spots’, such as the bacterial element IS426 and and Agrobacterium chromosomal DNA. These bases. Cloning and characterization of several an ABC transporter gene, appearing at higher junction sequences are generally considered of these junctions from plants revealed that frequency. Figure 1 Models for various mechanisms to link T-DNA to Agrobacterium chromosomal DNA. In ab each of these models, the VirD2 endonuclease nicks the T-DNA region or bacterial DNA at + + a border sequence and covalently attaches VirD2 VirD2 to the 5′ end of the resulting single-stranded T-strand. (a) The T-strand integrates into a double-stranded break site in chromosomal http://www.nature.com/naturebiotechnology DNA (perhaps generated by transposition of an IS element), is replicated to a double-stranded form and regenerates a border sequence. VirD2 VirD2 subsequently nicks this new border, linking VirD2 T-DNA with chromosomal DNA. (b) The T-strand directly links to a nick in the chromosomal DNA, generating a single-stranded T-DNA/ VirD2 chromosomal DNA hybrid molecule. (c) Homologous sequences within the T-DNA region c d and bacterial chromosomal DNA recombine. Subsequent nicking of the T-DNA border by VirD2 generates an Hfr-like molecule, which Nature Publishing Group Group Nature Publishing 8 transfers to the plant. (d) Homologous sequences within the T-DNA region and 200 bacterial chromosomal DNA recombine. © Additional homologous sequences flanking the first region of homology recombine, forming an F′-like molecule. Nicking of the T-DNA border by VirD2 generates a hybrid molecule consisting of T-DNA linked to chromosomal DNA. (e) Two single-stranded molecules formed by VirD2 VirD2 border nicking in the T-DNA region and VirD2 in chromosomal DNA integrate into the same site in the plant genome. (f) An IS element on the chromosome inserts into the T-DNA region ef by replicative transposition, resulting in co- integration of the chromosome with the T-DNA + region. A T-strand is generated by VirD2 border + cleavage before resolution of the co-integrate. Parallel double lines, double-stranded DNA; single lines, single-stranded DNA; red lines, T-DNA region or T-strands; black lines, bacterial chromosomal DNA; light green lines, vector backbone sequences flanking the T-DNA region; VirD2 orange lines, plant DNA; brown regions, T-DNA border sequence; light blue boxes, regions of VirD2 homology; gray boxes, other regions of homology. The thin crossed lines in c and d (including the wide X between the two gray boxes in d) indicate recombination events. The thin single line in f indicates co-integration of the bacterial genome VirD2 with the T-DNA region resulting from IS element replicative transposition. NATURE BIOTECHNOLOGY VOLUME 26 NUMBER 9 SEPTEMBER 2008 999 NEWS AND VieWS Ancient T-DNA integration events have been Alternatively, IS elements within the bacterial unlinked to the selected T-DNA would likely found in plant genomes5,6, and expression of chromosome may link bacterial DNA with be eliminated during this stage of the breeding these T-DNA sequences may have contributed T-region DNA during a process similar to that process. to plant evolution7,8. Similarly, the presence of of replicative transposition (Fig. 1f). These two High-throughput sequencing technolo- Agrobacterium chromosomal DNA in plants latter models do not require homology between gies should allow the rapid identification of suggests intriguing evolutionary possibilities the T-region and the bacterial chromosome. bacterial chromosomal DNA integrated into and expands our knowledge of horizontal gene Whatever the mechanism, the introduction the plant genome and the elimination of such flow between species. of bacterial chromosomal DNA into transgenic transgenic plants from breeding programs. In Whereas plasmid backbone transfer was plants is not welcome news for regulators. The addition, mutant Agrobacterium strains that somewhat predictable