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THE IPKb VECTOR SET: MODULAR BINARY PLASMIDS FOR CEREAL TRANSFORMATION The systematic collection of plant genetic resources, along with the acquisition of plentiful genomic sequence and gene expression data and the elaboration of methods allowing for both transient and stable transgene expression, have encouraged a series of novel experimental approaches to understand the genetical and physiological bases of plant phenotype. Genetic transformation, a critical technology in this process, remains, however, cumbersome and time-consuming in the cereals, which supply the bulk of the global requirement for food and feed. The vector systems that work well as transformation agents for many dicotyledonous species are, unfortunately, of only limited utility in the monocotyledons, largely because commonly used promoter sequences and/or selectable markers are ineffective in a monocotyledonous host. In addition, Agrobacterium-mediated transformation, which operates very efficiently in many dicotyledonous hosts, remains a demanding technology in the monocotyledons. In a recent paper, Himmelbach and co-workers described a novel set of modular binary vectors (the IPKb series), specifically tailored for cereal transformation and targeted to either over-expression or RNA-interference (RNAi)-mediated gene knock-down1. In both types, the insertion of effector sequences is facilitated by the exploitation of GATEWAY destination cassettes, which permit the efficient, site-specific and reliable exchange of DNA fragments between plasmids (Fig. 1). Any DNA sequence can be readily transferred from an easily cloned entry vector to the binary destination vector via an LR reaction, a procedure that avoids the need for the digestion and ligation-based cloning of the typically rather large binary vectors. This is particularly advantageous in the context of RNAi vectors, in which two inverted DNA repeats need to be connected by a spacer or intron sequence. The IPKb RNAi vectors pIPKb006 through pIPKb010 contain an inverted repeat of GATEWAY destination cassettes, separated by the wheat RGA2 intron2. The IPKb vector set also includes derivatives both of the over-expression and RNAi types in which various promoters, which are fully functional in monocotyledonous species, have been inserted to drive ubiquitous or epidermis-specific transgene expression (Table 1). Any other established or de novo isolated promoter sequence can be readily inserted upstream of the GATEWAY destination cassette of either the over-expression vector pIPKb001, or the RNAi vector pIPKb006, thereby increasing the versatility of the IPKb vector set. 
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Figure 1. Schematic representation of the IPKb over-expression and RNAi-mediated gene knock-down binary plasmid types. A gene sequence of interest (GOI) can be exchanged between an appropriate entry vector and an IPKb destination vector using the GATEWAY recombination system, as indicated by the attL and attR recombination sites (L1, L2, R1 and R2) and the dashed arrows. Derivatives are available for both plasmid types, involving any of the four promoters shown, or with a multiple cloning site (MCS), allowing the integration of further promoters. CmR = chloramphenicol resistance gene for selection of bacteria; ccdB = toxin gene for negative selection of bacteria; RGA2Int = intron of wheat RGA2 gene; RB and LB = right and left T-DNA borders; ColE1 = E. coli origin of replication; pVS1 = Agrobacterium origin of replication; SpecR = spectinomycin resistance gene for selection of bacteria; T = transcription termination sequence; HptR = hygromycin resistance gene for plant selection; ZmUbi1P = maize ubiquitin 1 promoter; OsAct1P = rice actin 1 promoter; d35SP = doubled enhanced CaMV 35S promoter; GstA1P = wheat glutathione-S-transferase 1 promoter; SfiIA and SfiIB = SfiI restriction sites. |
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Since specific selection markers may be preferred for some target hosts, or may be necessary for certain gene stacking strategies, the IPKb vectors have been constructed to allow for the ready introduction of further marker gene expression cassettes. This can be accomplished via the exchange of the selectable marker-containing SfiI fragment with that of compatible vectors such as 5U, 7U, and 9U provided by the DNA Cloning Service, Hamburg, Germany (http://www.dna-cloning-service.com). For example, this approach enables access to the dihydrofolate reductase gene (dhfr, resistance to metothrexate), the phosphinothricin-N-acetyl transferase gene (pat, resistance to phosphinothricin) and the neomycin phosphotransferase II gene (nptII, resistance to kanamycin) in which the selectable marker genes are under control of the strong ubiquitous maize ubiquitin 1 (ZmUbi1) promoter (Fig. 2). Other combinations of promoters and selectable marker genes are also feasible. The swapping of plasmid fragments is achieved in a single recombination step, using the rare cutter SfiI, which has a 13nt recognition site including five variable nucleotides in central position. Directed ligation of plasmid fragments is accomplished between appropriate pairs of single-strand overhangs derived from either SfiIA or SfiIB. As a further option, any of the available binary vectors can be engineered to lack a plant selectable marker expression cassette, which is unnecessary where the efficiency of transformation is known to be high, and which allows for the production of instantly marker-free transgenic plants. To achieve this, the SfiI fragment containing the plant selection marker is simply replaced by the compatible marker-free fragment of the B-BA binary vector (DNA Cloning Service, Hamburg, Germany). The IPKb series was based on the generic binary vector 6U (DNA Cloning Service Hamburg, Germany), in which the hygromycin phosphotransferase (hpt) coding sequence fused to the ZmUbi1 promoter acts as a very effective selectable marker gene cassette. A further important feature of 6U and its derivatives is that they harbor the pVS1 origin of replication, which ensures good plasmid persistency in agrobacteria even under non-selective conditions3, and thereby maintains the transformation competency of the Agrobacterium population during the entire co-cultivation period. In conjunction with various promoter-reporter and promoter-effector constructs, 6U produces a stable and high transformation efficiency in both barley4,5 and wheat (Hensel and Kumlehn, unpublished). The functionality of pIPKb002 to pIPKb005 was tested by introducing the gus gene into the GATEWAY destination site, followed by Agrobacterium-mediated transformation of barley and the subsequent expression analysis of stable transgenic plants. The transformation efficiency of the IPKb plasmids was on a par with that of conventional 6U-based binary vectors without a GATEWAY cassette5. The resultant T1 seedlings expressed GUS, with the strongest expression present in the leaves of lines transformed with pIPKb002_GUS (driven by the ZmUbi1 promoter), followed by pIPKb003_GUS (OsAct1 promoter), pIPKb005_GUS (TaGstA1 promoter), and pIPKb004_GUS (d35S promoter). For the transgenic pIPKb005_GUS lines, fluorescence spectroscopy revealed that GUS activity in isolated abaxial epidermis was, on average, ten times stronger than in remaining leaf tissue. This result not only provides an example of tissue-specific transgene expression achieved through the use of an IPKb vector, but also opens the way to their use as a tool for studying and manipulating the interaction between barley and many of its pathogens. The binary destination vectors pIPKb007 through pIPKb010 drive the expression of RNAi sequences, with transcriptional regulation provided by the same promoters used for the IPKb over-expression vectors. The functionality of the RNAi-vectors was verified via the biolistic delivery into barley leaf tissue of vector derivatives targeted against Mlo, a negative regulator of resistance against the causal pathogen of barley powdery mildew6. All of the plasmids tested (pIPKb007_Mlo to pIPKb010_Mlo) produced a phenocopy of the loss-of-function mlo resistance, indicating that the presence in planta of the Mlo-RNAi constructs acted to reduce the transcription of MLO, and hence increased the level of resistance to powdery mildew. In addition to the generation of numerous stable transgenic barley and wheat plants using various derivatives of the IPKb destination vectors, pIPKb002_GUS and pIPKb004_GUS proved also effective for the stable transformation of tobacco, where fluorescence spectroscopy was able to demonstrate the ubiquitous expression of GUS. Thus the IPKb vector set provides an appropriate vehicle to compare transgene expression in mono- and dicotyledonous species using an identical binary vector. The IPKb vector set provides a framework for the development of derivatives with further promoters, plant selection markers, sequences suited for homologous recombination-mediated marker deletion strategies, affinity or screenable tags that can be N- or C-translationally attached to the coding sequence, and for the development of systems permitting T-DNA insertion mutagenesis in cereals. The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) will supply pIPKb001 through pIPKb010 gratis on request for non-commercial research use. References 1. Himmelbach A, Zierold U, Hensel G, Riechen J, Douchkov D, Schweizer P and Kumlehn J (2007) A set of modular binary vectors for the transformation of cereals. Plant Physiology 145, 1192-1200 2. Douchkov D, Nowara D, Zierold U, Schweizer P (2005) A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Molecular Plant Microbe Interactions 18, 755-761 3. Itoh Y, Watson JM, Haas D, Leisinger T (1984) Genetic and molecular characterization of the Pseudomonas plasmid pVS1. Plasmid 11, 206-220 4. Kumlehn J, Serazetdinova L, Hensel G, Becker D and Loerz H (2006) Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnology Journal 4, 251-261 5. Hensel G, Valkov V, Middlefell-Williams J and Kumlehn J (2008) Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. Journal of Plant Physiology 165, 71-82 6. Bueschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, Van Daelen R, Van der Lee T, Diergaarde P, Groenendijk J, Toepsch S, Vos P, Salamini F, Schulze-Lefert P (1997) The barley Mlo gene: A novel control element of plant pathogen resistance. Cell 88, 695-705 Jochen Kumlehn |