Glimpses of a Century-Old Story

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1 Glimpses of a Century-Old Story Agrobacterium, a Pathogen Deployed for Genetic Engineering Jasmine M Shah Jasmine M Shah is a postdoctoral fellow, Department of Biotechnology, Indian Institute of Technology-Madras. She is interested in plantmicrobial interactions and works on influence of bacterial infection on plant genome fidelity. Keywords Agrobacterium, T-DNA, crown gall, transgenic. Figure 1. Areas developed due to leads from plant Agrobacterium interaction. The story of Agrobacterium has been widely discussed for over a century. Fridiano Cavara in 1897 first described the occurrence of plant-derived tumors with bacterial origin. Detailed molecular analysis of this microbe accelerated in the last 30 years and it gained worldwide acceptance as a natural genetic engineering tool. This tool has an interesting history starting from the tumors that it makes, nomenclature, genome, pathogenicity and DNA transfer, to its economic importance. This review is a glimpse of some basic and interesting facts of the unique Agrobacterium. Introduction Inter-kingdom gene transfer, a technique that sounds like a manmade development, is nothing new for Agrobacterium. Someof the Agrobacterium species are masters of inter-kingdom gene transfer and they practice this to enslave plants or parts of plants for preparing a special food for them. This natural genetic engineering technique has tactfully been used to generate a wide array of transgenic plants with improved nutritional quality, taste, shelf life, disease resistance, salinity and drought tolerance. Close observation of the association between Agrobacterium and plants has also paved the way to the discovery of many other scientific facts (Figure 1). 336 RESONANCE April 2013

2 What is Agrobacterium? Genus Agrobacterium comprises soil-borne, gram-negative, plantpathogenic or non-pathogenic, bacteria. A. tumefaciens, A. rhizogenes, A. rubi and A. vitis are some members belonging to this genus. Erwin Smith and C O Townsend in 1907 reported that Bacterium tumefaciens (now Agrobacterium tumefaciens) was capable of inducing tumors in plants (which mimic tumors in mammals) because of uncontrolled cell proliferation, at wound sites. Such tumours are known as crown gall (Figure 2). Some species of Agrobacterium induce uncontrolled adventitious roots (also known as hairy roots ). Agrobacterium infects a broad range of plants, comprising largely dicotyledonous plants and a few monocots but rarely gymnosperms 1. What s in a Name? The history of A. tumefaciens s nomenclature is quite interesting. In the initial period since its discovery, it was known by various names such as: Pseudomonas tumefaciens, Rhizobiumradiobacter, Agrobacterium biovar 1, Polymonas tumefaciens, Phytomonas tumefaciens, Bacterium tumefaciens, Pseudomonas radiobacter, Alcaligenes radiobacter, Achromobacter radiobacter, Bacterium radiobacter and Bacillus radiobacter. However, based on phenotypic and genetic evidences including 16S rdna analyses, all Agrobacterium species are now circumscribed in the Rhizobium genus; for instance, A. tumefaciens is renamed as Rhizobium radiobacter. However, in this article and in other recent publications, Agrobacterium is not denoted by the new nomenclature, probably for the sake of convenience. Multiple Genome of Agrobacterium Figure 2. Photograph of a tree with crown gall disease. (Taken at the Indian Institute of Technology-Madras campus, Chennai, Tamil Nadu, India.) 1 The recent developments have extended Agrobacterium transformationtomanymonocotsand even non-plant eukaryotic organisms including fungi and human cells. Most of the bacteria harbor only one bacterial chromosome. However, Agrobacterium has two of them, one circular bacterial chromosome and an unusual linear bacterial chromosome (Figure 3). Besides, two large plasmids are also present, and the four replicons together encode approximately 5,400 genes. RESONANCE April

3 Crown Gall, Cane Gall and Hairy Root Diseases A. tumefaciens and A. vitis are known to cause the crown gall disease and A. rubi causes cane gall disease; both the diseases result in gall leading to tumor in plants. A. rhizogenes causes the hairy root disease. The principal agent in all these cases is an exceptionally large plasmid (>200 kb), which carries a bit of DNA (10 20 kb) called T- DNA (transfer DNA) and a set of genes called vir (virulence) genes.agrobacterium turns pathogenic only if it harbors either the tumor-inducing (Ti) or root-inducing (Ri) plasmid 2. Figure 3. Representation of genome (5.67 Mbp) of Agrobacterium tumefaciens C58. 2 If Rhizobium, the bacteria known for nitrogen fixation, is transformed with Ti plasmid, it induces tumors and when Agrobacterium is transformed with psym1 (carrying genes for root nodulation and nitrogen fixation), it induces nodules in plants. Two Rhizobium strains 163C and ATCC11325T have the psym plasmid. However, since they harbor pri and pti plasmids, respectively, they induce hairy roots or tumors respectively. T-DNA is flanked by specialized borders (left and right borders), about 25-bp long, directly repeated sequences. T-DNA comprises genes required for either inducing tumor or hairy root. These genes cause tumors or adventitious rooting simply because they encode proteins involved in the synthesis of the plant growth hormones auxin (iaam and iaah) and cytokinin (ipt). The collective effort of vir gene products is to help transfer T-DNA from bacteria to plant cell. What is the reason for these bacteria to harbor this strange plasmid? Interestingly, the theory behind the occurrence of these plasmids in all disease-causing strains of Agrobacterium is the same. T-DNA also carries with it genes coding for synthesis of opines (amino acid sugar conjugates), the favorite food of Agrobacterium, often not favored by other microflora. Hence, the tumor or bunches of roots serve as a factory for large-scale opine production. Moreover, opines are opined to stimulate the induction of vir genes also. Based on the type of opines synthesized, Ti plasmids are classified as octopine, nopaline, agropine, and succinamopine types. Also, genes required for the dissimilation of these opines are also located on the same Ti plasmid. Hence, the Agrobacterium from which Ti plasmid is removed, cannot utilize opine. The Ti plasmid is lost when Agrobacterium cultures are subjected to temperatures above 36 C. 338 RESONANCE April 2013

4 Transformation, an Orchestra of Events The transfer of T-DNA from Agrobacterium to plant cell (Figure 4) and its integration into the plant genome is an elaborate orchestra-like event comprising numerous Vir proteins (located on Ti or Ri plasmid), chromosomal proteins, T-DNA and even plant proteins [1,2]. The trigger for transformation-initiation is a wound on plants. Upon injury, plants produce phenolic chemicals such as acetosyringone and related compounds, which are recognized by Agrobacterium. This recognition enables Agrobacterium to get attracted towards the wounded area, make an attachment and transfer the T-DNA. The details of the transformation are described here. Figure 4. Steps involved in T-DNA transfer. (1) Travel of T-DNA from Agrobacterium attached to a plant cell. (2) Factors involved in its travel in protoplasm. (3) Entry into nucleus. Recognition A signal transduction cascade initiates when the protein VirA, located in the inner membrane, recognizes phenolic compounds. VirA is the sensory protein of a two-component regulatory system and it gets auto-phosphorylated upon interaction with phenolics. Phosphorylated VirA in turn activates the cytoplasmic protein VirG, the transcriptional activator of all other vir genes RESONANCE April

5 (virb, virc, vird, vire, virf, virh and virj). High levels of homology does exist between the vir regions of octopine and nopaline Ti plasmids, between octopine and succinamopine Ti plasmids and even between octopine Ti and Ri plasmids. However, efficient induction followed by functioning of vir genes occurs only when vira, virg, andvir box are from the same Ti plasmid. The chromosomally located chvgi, chve, andros gene products are also involved in the regulation of the vir genes. Attachment 3 Attachment is not a prerequisite for T-DNA transfer and incorporation because successful transformation is achieved when the non-attaching mutant bacteria are micro-injected into the plant cells. 4 T-DNA border sequences are similar to those found at origins of transfer (orit) of some conjugative plasmids and the nuclease activity of VirD1 and VirD2 resembles some conjugal DNArelaxing enzymes. This is mediated by a bacterial cell-associated, capsular polysaccharide. The bacterium extensively synthesizes cellulose fibrils and these ensnarl more and more bacteria on the wounded surface. These components are synthesized from different loci situated on different genomes. Polysaccharide synthesis requires the attr locus (situated on the Ti plasmid); chromosomal virulence genes chva, chvb, and psca are involved in the synthesis, processing and export of cyclic -1,2-glucans and other sugars. Further, bacterial attachment is favored by two plant cell-wall proteins also, vitronectin-like protein and rhicadhesin-binding protein. Bacterial attachment 3 is immediately succeeded by T- DNA transfer from Agrobacterium to the plant cell, the overall process requiring about 2 4 hours. T-DNA Transfer and Integration Processing of T-DNA from Ti plasmid starts when VirD2, with the help of VirD1, nicks Ti plasmid at the 25-bp borders on either side 4. VirD2 now covalently attaches to the 5 -end of T-DNA, generating a single-stranded DNA (ssdna) molecule called the T-strand. VirD2 ( pilot protein) guides the T-DNA from Agrobacterium to plant cell nucleus. T-DNA-VirD2-complex (DNA-protein-complex) is known as the T-complex. The movement of T-complex from bacteria to host is via a pilus, which is made up of 11 VirB proteins (VirB1 to VirB11) and Vir D4. VirB2 protein is the major constituent of the T-pilus. Along with T-complex, VirE2, VirE3, VirD5 and VirF also pass through 340 RESONANCE April 2013

6 the pilus into the plant cell and subsequently associate with the T- complex, offering protection from plant nucleases to the T-DNA. Transport of VirE2 is facilitated by VirE1, which is its specific chaperone, inhibiting the binding of VirE2 to T-strands in the bacterial cell. VirE2 is the most plenteous Vir protein accumulated in Agrobacterium cells that have been subjected to acetosyringone. VirE2 is also the largest known prokaryotic ssdna-binding protein (60 kda). VirE2 is thought to form a channel spanning the plant plasma membrane, thereby facilitating T-complex transfer 5. In order to reach its destination, that is the plant nucleus, the T- DNA has to cross the nuclear membrane via the nuclear pore. For any molecule to pass through a nuclear pore, nuclear localization signal (NLS) is often mandatory and the Agrobacterium-derived VirD2 and VirE2 possess NLS for plant nucleus. NLS of VirD2 functions in animals cells also and the VirE2 NLS allows entry into the animal nucleus after small alterations. In order to facilitate the localization of T-complex to the nucleus, VirD2 interacts with the plant protein phosphatase (PP2C) and nine Arabidopsis importins. Similarly, VirE2 interacts with plant protein importin IMPa-4 and two VirE2-interacting proteins (VIP1 and VIP2). VirE3 is also localized to the nucleus and interestingly, it functions as a plant transcription factor, and can also substitute the duty of plant-encoded VIP1. VirF plays the role of proteolysing VirE2. This facilitates the removal of VirE2 from T-complex for easy T-DNA integration. Integration of T-DNA into plant genome is thought to be facilitated by VirD2 and plant proteins. However, it is still not resolved whether bacterial effectors play a direct role in the T-DNA integration 6. Some A. rhizogenes Strains Lack VirE2 Complete nucleotide sequence of the Ri plasmid in A. rhizogenes has revealed that Ri and Ti plasmids are almost superposable, including in the organization of the vir operons. Surprisingly, the Ri plasmid of some A. rhizogenes strains (like strain 1724) lack VirE1 and VirE2 genes, but still efficiently transfer the T-strand 5 VirE2 interacts with membrane lipids of the plant cell. It forms channels or pores that traverse through the artificial, plant plasma-like membranes. These channels preferably permit only negatively charged molecules like single-stranded DNA. Since VirE2 is a protein gatekeeper that selectively facilitates the transport of DNA into cell, even mammalian cell, its potential may be utilized as a tool in gene therapy. 6 Transfer of T-DNA in Agrobacterium is independent of its location on plasmid. When the T-region was cloned into the chromosomal DNA of A. tumefaciens and vir genes were placed in trans, T-DNA transfer into plant cell was observed. RESONANCE April

7 Philip R White and Attain C Braun (1942) showed that crown gall tumors required bacteria only for initiation and their proliferation was bacteriaindependent. with the aid of GALLS-gene products, GALLS-FL and GALLS- CT. These complement A. tumefaciens-vire functions, but do not share any sequence homology with either of the VirE proteins. Like VirE2, GALLS-FL contains NLS and unlike VirE2, it has ATP-binding and helicase motifs. Tumor Proliferation is Independent of Bacteria Philip R White and Attain C Braun [3] showed that crown gall tumors required bacteria only for initiation and their proliferation was bacteria-independent. This indicated the most important requirement for tumor formation the plant cells were genetically transformed. Genetic Engineering Tool What if the tumor causing genes are removed from T-DNA? Can it still infect plants? Finally, what if these genes are replaced by genes of economic interest? It is, in fact, the left and right borders that give T-DNA its transfer property because, it is these borders that the bacterial Vir proteins recognize, attach and lead the DNA bit to plant cell. Hence, it just means that any stretch of nucleotide within these borders will be transported and it need not be tumoror root-inducing genes. This fact is indeed the trump card for all the successful Agrobacterium-based genetic engineering experiments till date and rightly denoted as the gene jockeying tool by Stanton B Gelvin [4]. The first transgenic plants with T-DNA harboring gene of interest and not oncogenes were generated by Jeff Schell s group and they produced kanamycin-resistant tobacco plants. The year 1983 witnessed a spate of reports on transgenic plants. The first transgenic plants with T-DNA harboring gene of interest and not oncogenes were generated by Jeff Schell s group and they produced kanamycin-resistant tobacco plants. Following this were reports by Robert Fraley, Stephen Rogers and Robert Horsch at Monsanto (developed kanamycin-resistant petunia plants) and Timothy Hall s group (inserted a bean gene into sunflower plants). In these initial transformation experiments, genes of interest were incorporated in cis in the T-DNA of Ti plasmids, replacing oncogenes. Soon binary systems were developed where Vir 342 RESONANCE April 2013

8 factors were provided in trans as T-DNA harboring plasmids, and virulent helper plasmids (harboring vir genes) were separated into two different repilcons. This increased the utility of Agrobacterium as a vehicle for gene transfer and Lee and Gelvin [5] have reviewed an array of vector systems that can be opted. Placing the genes of interest on different replicons soon paved the way for the elimination of unwanted marker genes. Ceaseless Complications Transfer of the DNA region within the borders is what Agrobacterium requires. However, insertion of Ti plasmid fragments apart from the T-DNA, into plant genomes, has also been reported. Surprisingly, Agrobacterium can sometimes transfer bits of its chromosomal DNA also to plants. Detailed sequence analysis of transgenic Arabidopsis and rice plants revealed that bits (up to 18 kb) of Agrobacterium chromosomal DNA were integrated in at least one in every 250 transgenic plants. Conclusions Apart from being used as a routine genetic engineering tool, Agrobacterium is used to study a wide range of molecular and biological processes. Knocking-out of genes for functional genomics is performed by incorporating the desired knock-out cassette into a T-DNA, followed by transformation. However, many plants are recalcitrant to knock-out strategy and this hinders functional analysis at the genomic level. Interestingly, plant functional genomics is expanding exponentially mainly due to the availability of T-DNA insertion mutants. For example, the Arabidopsis Biological Resource Centre (ABRC) has an extremely large collection of Arabidopsis T-DNA insertion mutants, deposited by various research groups across the world. In each of these mutants, a gene or a regulatory portion of the plant genome is knocked out due to T-DNA insertion. The unique relation of plant Agrobacterium has revealed many facts about plant microbial interaction and plant s defense processes. Unlike other bacterial pathogens, which induce hypersensitive Apart from being used as a routine genetic engineering tool, Agrobacterium is used to study a wide range of molecular and biological processes. RESONANCE April

9 response (HR) and systemic acquired resistance (SAR) in plants, Agrobacterium association leads to induced systemic resistance (ISR). Microarray studies by Gelvin s group have revealed that Agrobacterium is capable of first triggering plant defense response and unlike other pathogens, it starts suppressing plant defense response two hours post infection. The concept of gene silencing was understood better after the observation that transgenes got silenced when two T-DNAs integrated adjacent to each other, as inverted repeats. The process of T-DNA integration into the plant genome has elaborated the study on various concepts of DNA repair including non-homologous end joining and homologous recombination [6]. The study of chromosomal recombination was facilitated as multiple reporter genes could be easily integrated into plant genomes via T-DNA transfer. Address for Correspondence Jasmine M Shah Post Doctoral Fellow Department of Biotechnology Indian Institute of Technology- Madras Chennai , India. jas109@gmail.com Many facts related to T-DNA integration are yet to be excavated. For example, it is still not known as to which proteins exactly participate in the breaking and joining of DNA during T-DNA integration; it is not known whether those are only plant proteins, or only Agrobacterium-derived proteins or both. Generally pathogen stress alters the overall plant genome stability by increasing the somatic homologous recombination frequency. It is yet to be investigated as to how the overall plant genome stability is affected when this natural genetic engineering tool infects the plant. Suggested Reading [1] S B Gelvin, Plant proteins involved in Agrobacterium-mediated genetic transformation, Annual Review of Phytopathology, Vol.48,pp.45 68, [2] B Lacroix, J Li, T Tzfira and V Citovsky, Willyou let me use your nucleus? How Agrobacterium gets its T-DNA expressed in the host plant cell, Canadian Journal of Physiology and Pharmacology, Vol.84,pp , [3] P R White, A C Braun, A cancerous neoplasm of plants, Autonomous bacteria-free crown-gall tissue, Cancer Research, Vol.2p , [4] S B Gelvin, Agrobacterium-mediated plant transformation: The biology behind the gene-jockeying tool, Microbiology and Molecular Biology Reviews, Vol.67,pp.16 37, [5] L Y Lee, and S B Gelvin, T-DNA binary vectors and systems, Plant Physiology, Vol.146, pp , [6] A Pitzschke, H Hirt, New insights into an old story:agrobacterium-induced tumour formation in plants byplant transformation, The EMBO Journal, Vol.29pp , RESONANCE April 2013