Oxalate decarboxylase from Agrobacterium tumefaciens C58 is translocated by a twin arginine translocation system

Oxalate decarboxylases (OXDCs) (E.C. 4.1.1.2) are enzymes catalyzing the conversion of oxalate to formate and CO2. The OXDCs found in fungi and bacteria belong to a functionally diverse protein superfamily known as the cupins. Fungi-originated OXDCs are secretory enzymes. However, most bacterial OXDCs are localized in the cytosol, and may be involved in energy metabolism. In Agrobacterium tumefaciens C58, a locus for a putative oxalate decarboxylase is present. In the study reported here, an enzyme was overexpressed in Escherichia coli and showed oxalate decarboxylase activity. Computational analysis revealed the A. tumefaciens C58 OXDC contains a signal peptide mediating translocation of the enzyme into the periplasm that was supported by expression of signal-peptideless and full-length versions of the enzyme in A. tumefaciens C58. Further site-directed mutagenesis experiment demonstrated that the A. tumefaciens C58 OXDC is most likely translocated by a twin-arginine translocation (TAT) system.

棉花抗虫基因工程研究

本文通过根农杆菌（Agrobacterium tumfaciens）介导法分别将Signal和KDEL修饰的豇豆胰蛋白酶抑制剂（Cowpea trypsin inhibitor, CpTI）基因、豌豆外源凝集素（Pea lectin, P-Lec）和大豆Kunitz型胰蛋白酶抑制剂（Soybean Kunitz typsin inhibitor, SKTI）双价抗虫基因、雪花莲外源凝集素（Galanthus nivals agglutinin, GNA）基因以及高效复合启动子OM控制的苏云金杆菌（Bacillus thuringiensis, B.t.）杀虫毒蛋白基因导入了陆地棉（Gossypium hirsutum L.）栽培品种新陆早1号、新陆中2号、晋棉7号、冀合321、辽9和晋棉12号，并获得了大批转基因再生植株。 实验中对影响棉花转化和再生的一些条件进行了研究，从根农杆菌培养、棉花无菌苗的制备、转化操作和共培养等方面对转化条件进行了探讨;从激素配化、植物表达载体、外植体类型、基因型等方面对抗性愈伤组织的诱导进行了摸索;从激素、从碳源、培养容器、pH值、抗褐化剂及固化剂的选择等方面对影响植株再生的条件进行了优化。 本文开创性地采用嫁接代替移栽，从而极大地提高了转化植株定植成活率，缩短了缓苗时间并增加了转化植株当代的繁殖系数。 在建立了一套较为高效的陆地棉转化及再生系统基础上，本文还进行了其它转化方式和转化体系的初步探讨。利用棉花幼嫩种子无菌苗下胚轴作为外植体，通过改变愈伤组织诱导培养基配方面提高胚性愈伤组织的诱导频率，进而得到更多的体细胞胚状和再生植株，缩短再生周期;尝试用胚性愈伤组织作为外植体的根农杆菌介导法转化，确定了一些与转化有关的条件;建立了一套棉花茎尖培养程序，为运用基因枪法轰击棉花茎尖分生组织或用根农杆菌直接转化茎尖分生组织，以克服根农杆菌转化棉花时体胚发生的基因型局限开辟了一条新途径。 本文还建立了一种快速鉴定转化植株后代的方法。这一简便方法还有助于进行转基因棉纯合系的筛选以及外源基因的遗传稳定性研究。 转基因植株经Npt-II ELISA、PCR、PCR Southern 检测证明外源抗虫基因CpTI、SKTI、P-lec、GNA以及B.t.基因已存在于转化植株基因组内。修饰的CpTI转基因植株抗棉铃虫（Heliothis armigera Hubner）试验结果表明，其杀虫效果显著优于前期未修饰的CpTI转化植株。P－lec和SKTI双价转基因植株抗棉铃虫试验结果表明，转基因植株对棉铃虫幼虫具有较强的杀虫活力。 目前，已获得转以上抗虫基因棉花T1代植株。为今后进一步将植物基因工程技术应用于棉花遗传改良打下了基础。

Enhancing agrobacterium-mediated transformation efficiency of sugarcane : progress towards an efficient, genotype-independent method

Several key issues need to be resolved before an efficient and reproducible Agrobacterium-mediated sugarcane transformation method can be developed for a wider range of sugarcane cultivars. These include loss of morphogenetic potential in sugarcane cells after Agrobacterium-mediated transformation, effect of exposure to abiotic stresses during in vitro selection, and most importantly the hypersensitive cell death response of sugarcane (and other nonhost plants) to Agrobacterium tumefaciens. Eight sugarcane cultivars (Q117, Q151, Q177, Q200, Q208, KQ228, QS94-2329, and QS94-2174) were evaluated for loss of morphogenetic potential in response to the age of the culture, exposure to Agrobacterium strains, and exposure to abiotic stresses during selection. Corresponding changes in the polyamine profiles of these cultures were also assessed. Strategies were then designed to minimize the negative effects of these factors on the cell survival and callus proliferation following Agrobacterium-mediated transformation. Some of these strategies, including the use of cell death protector genes and regulation of intracellular polyamine levels, will be discussed.

Agrobacterium-mediated transformation of indica rice cultivars using binary and superbinary vectors

The Agrobacterium-mediated transformation system was extended to two indica cultivars: a widely cultivated breeding line IR-64 and an elite basmati cultivar Karnal Local. Root tips and shoot tips of seedlings, and scutellar-calli derived from mature seeds showed high-efficiency Agrobacterium tumefaciens infection and stable transformation. In addition to the superbinary vector pTOK233 in Agrobacterium strain LBA4404, almost equally high levels of transformation were achieved with a relatively much smaller (13.1 kb) binary vector (pCAMBIA1301) in a supervirulent host strain AGL1. In both cases, as well as in both cultivars, while 60–90% of the infected explants produced calli resistant to the selectable agent hygromycin, 59–75% of such calli tested positive for GUS. A high level (400 μM) of acetosyringone in the preinduction medium for Agrobacterium and a higher level (500 μM) in the cocultivation medium was necessary for an enhancement in transformation frequency of the binary vector to levels comparable to a superbinary. Hygromycin-resistant calli could be produced from all the explants used. Transformants could be regenerated for both cultivars using the superbinary and binary vector, but only for calli of scutellar origin. In addition to the molecular confirmation of hpt and gus gene transfer and transcription, absence of gene sequences outside the transferred DNA (T-DNA) region confirmed absence of any long T-DNA transfer.

Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants

A series of improved vectors have been constructed that are suitable for use in Agrobacterium tumefaciens-mediated monocot transformation. These binary vectors have several useful features, including the selectable marker genes bar (phosphinothricin resistance) or hph (hygromycin resistance) driven by either the cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin promoter, a high-copy-number replication origin that allows reliable mini-prep DNA isolation from Escherichia coli, and a polylinker sequence into which target genes can be easily inserted. A significant improvement has been made to the hph gene by the introduction of an intron into its coding region. The presence of the intron abolishes hph expression in A. tumefaciens, rendering the bacterium susceptible to the selective agent hygromycin B. The use of such an intron-hph vector thus enables the antibiotic in plant culture media to function as both a selective agent for transformed tissue and as a contraselective agent for A. tumefaciens growth, thus minimising the overgrowth of A. tumefaciens on plant tissues during transformation. Furthermore, the intron appears to be correctly spliced in plant cells and significantly enhances hph expression in transformed rice tissue. In our experiments, the use of the intron-hph vector increased the frequency of rice transformation and has enabled the production of transgenic barley.

Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes

Efficient transformation of barley cv. Schooner was achieved using Agrobacterium delivery, hygromycin or bialaphos selection and embryogenic callus. Using this system, transgenic plants were generated that contained either the green fluorescent protein gene, or transgenes derived from barley yellow dwarf (BYDV) and cereal yellow dwarf (CYDV) viruses. Many of these plants contained 1-3 transgene copies that were inherited in a simple Mendelian manner. Some plants containing BYDV and/or CYDV derived transgenes showed reduced virus symptoms and rates of viral replication when challenged with the appropriate virus. The ability to transform Schooner is a significant advance for the Australian barley industry, as this elite malting variety is, and has for the last 15 years been, the most widely grown barley variety in eastern Australia.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens

In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.

pGreen : a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation

Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols. The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species. This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids. Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures. pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain. pSoup provides replication functions in trans for pGreen. The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum. Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors. Information on the pGreen plasmid system is supplemented by an Internet site (http://www.pgreen.ac.uk) through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found.

A guide to Agrobacterium binary Ti vectors

Agrobacterium-based plasmid vectors allow the transformation of a wide range of plant species by capitalizing on a natural bacterial system to introduce DNA into the nuclear genome of plants. It is often a complex task to consider fully all the possible plasmid vectors and Agrobacterium strains available, and it can thus be difficult to take full advantage of these research tools. This practical guide is a survey of the many binary Ti plasmid vectors and Agrobacterium strains available, and aims to help researchers to make an informed decision about the system that is best suited to their needs...

The Introduction of Transgenes to Control Blackheart in Pineapple: Biolistics Vs Agrobacterium Transformation, in 'Contributing to a Sustainable Future'

Techniques for the introduction of transgenes to control blackheart by particle bombardment and Agrobacterium co-transformation have been developed for pineapple cv. Smooth Cayenne. Polyphenol oxidase (PPO) is the enzyme responsible for blackheart development in pineapple fruit following chilling injury. Sense, anti-sense and hairpin constructs were used as a means to suppress PPO expression in plants. Average transformation efficiency for biolistics was approximately 1% and for Agrobacterium was approximately 1.5%. These results were considered acceptable given the high regeneration potential of between 80-90% from callus cultures. Southern blot analysis revealed stable integration of transgenes with lower copy number found in plants transformed with Agrobacterium compared to those transformed by biolistics. Over 5000 plants from 55 transgenic lines are now undergoing field evaluation in Australia

Characterization of small GTPase Cdc42 from the ectomycorrhizal fungus Suillus bovinus and Agrobacterium tumefaciens-mediated transformation of fungi

Ectomycorrhizal formation between the host tree, Pinus sylvestris and fungal symbiont, Suillus bovinus was investigated at the molecular level by isolating genes regulating the organization of the actin cytoskeleton in the fungal partner S. bovinus. An Agrobacterium tumefaciens mediated transformation (ATMT) system was developed for the ectomycorrhizal fungi in order to assign specific functions to the cloned molecules. The developed ATMT system was also used to transform a plant pathogenic fungus, Helminthosporium turcicum, to hygromycin B resistance. Small GTPases Cdc42 and Rac1, the regulators of actin cytoskeleton in eukaryotes were isolated from S. bovinus. Sbcdc42 and Sbrac1, are both expressed in vegetative and in the symbiotic hyphae of S. bovinus . Using IIF microscopy, Cdc42 and actin were co-localized at the tips of vegetative hyphae and were visualized in association with the plasma membrane in swollen cells typical to the symbiotic hyphae. These results suggest that the small GTPases Cdc42 may play a significant role in the polarized growth of S. bovinus hyphae and regulate fungal morphogenesis during ectomycorrhiza formation through reorganization of the actin cytoskeleton. The functional equality of Cdc42 was tested in yeast complementation experiments using a Saccharomyces cerevisiae temperature sensitive mutant, cdc42-1ts. The genomic clone of CDC42 was isolated from S. bovinus genomic DNA via specific primers for Cdc42. The analogous S. cerevisiae cdc42 mutations, dominant active G12V and dominant negative D118A, were generated in the Sbcdc42 gene by in-vitro mutagenesis. The ectomycorrhizal fungi, S. bovinus, P. involutus and H. cylindroporum were transformed using ATMT and phleomycin as a selectable marker. PCR screeing suggested that the T-DNA was inserted in all the three fungal genomes but the fate of integration could not be proved by Southern blot analysis. An alternative Agrobacterium strain, AGL-1 and selection marker, hygromycin was used to transform our model fungus S. bovinus. PCR and Southern analysis suggested an improved efficiency of transformation. All the transformed fungal colonies selected for hygromycin gave positives in PCR and the Southerns showed multiple or single copy T-DNA integrations into the S. bovinus genome. Using the same Agrobacterium strain and the selectable marker, a maize pathogen, H. turcicum was also subjected to ATMT. The H. turcicum transformation data suggested the single copy T-DNA integrations into the genome of the screened transformants that further confirms wider applicability of the ATMT. The plasmids carrying the wild-type (pHGCDC42) and the mutated Sbcdc42 alleles (pHGGV; pHGDA) under Agaricus bisporus gpd promoter were constructed in an A. tumefaciens vector. ATMT was used to transform S. bovinus with the plasmids carrying the wild-type and mutated Sbcdc42 alleles. The isolation of Sbcdc42 and Sbrac1 genes and some other functionally related genes from ectomycorrhizal fungus, S. bovinus will form the basis of future work to resolve the signalling pathway leading to ectomycorrhizal symbiosis. The development of ATMT system will be a valuable tool in analysing the exact function of signalling pathway components in ectomycorrhizal symbiosis or in plant pathogenic interactions. The transformation frequency and broad applicability along with the simplicity of T-DNA integration make Agrobacterium a valuable, new and a powerfull tool for targeted and insertional mutagenesis in these plant associated fungi. The developed ATMT systems should therefore make it possible to generate large number of transformants with tagged genes which could then be screened for their specific roles in symbiosis and pathogenecity, respectively.