Activation tagging: a means of isolating genes implicated as playing a role in plant growth and development

Activation T-DNA tagging has been used to generate a variety of tobacco cell lines selected by their ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. The majority of the cell lines studied in detail contain single T-DNA inserts genetically co-segregating with the selected phenotype. While most of the plants regenerated from the mutant cell lines appear phenotypically normal, several display phenotypes which could be inferred to result from disturbances in the content, or the metabolism, of auxins and cytokinins, or polyamines. The tagging vector is designed to allow the isolation of tagged plant genes by plasmid rescue. Confirmation that the genomic sequence responsible for the selected phenotype has indeed isolated is provided by PEG-mediated protoplast DNA uptake of rescued plasmids followed by selection for protoplast growth under the original selective conditions. Several plasmids have been rescued from the mutant lines which confer on transfected protoplasts the ability to grow either in the absence of auxin or cytokinin in the culture media, or under selective levels of an inhibitor of polyamine biosynthesis. This review describes the background to activation tagging and our progress in characterizing the genes that have been tagged in the mutant lines we have generated.     

Hairy Root-activation Tagging: a High-throughput System for Activation Tagging in Transformed Hairy Roots

Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in “transformed hairy roots”. The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.     

Signals for local and systemic responses of plants to pathogen attack

Activation of plant defences following recognition of pathogen attack involves complex reiterative signal networks with extensive signal amplification and cross-talk. The results of two approaches that have been taken to analyse signalling in plant-microbe interactions are discussed here. Activation tagging with T-DNA harbouring multiple 35S enhancer elements was employed as a gain-of-function approach to dissect signalling related to bacterial pathogen resistance in Arabidopsis thaliana. From a screen of approximately 5000 activation tagged lines, one line was identified as harbouring a T-DNA leading to over-expression of an apoplastic aspartic protease (CDR-1), that resulted in resistance to normally virulent Pseudomonas syringae. The second approach was to screen for loss-of-function mutants in T-DNA tagged populations. From a screen of 11 000 lines, one line, defective in induced resistance-1 (dir-1) lost resistance to normally avirulent P. syringae. Models for action of the products of the CDR-1 and DIR-1 genes suggest involvement of peptide and lipid signals in systemic disease resistance responses in A. thaliana.     

Improved T-DNA vector for tagging plant promoters via high-throughput luciferase screening

Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.     

T-DNA标签在植物基因克隆和功能分析中的应用

在植物功能基因组学的研究中,插入突变已成为迅速识别和研究标签基因的一个有效遗传工具.本文介绍了T-DNA标签的概念及应用前提,详细论述了T-DNA标签在大规模植物基因功能分析中的应用以及使用启动子和增强子诱捕技术分离时空特异性启动子和表达基因,另外还分析了利用其特殊形式激活标签进行基因克隆和功能分析的优越性,并展望了T-DNA标签的应用前景.     

提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。     

Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants

As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterlessuidA (gus A) gene, encoding the -glucuronidase (GUS) reporter, was introduced into populations of tobacco,Arbidopsis and potato byAgrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of thegusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns ofgusA activation in diverse organs and cell types in those populations.gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency ofgusA activation was differentially blased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T₂ and T₃ progeny. Developmentally-regulated and environmentally-inducible expression ofgusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.     

Activation tagging in plants: a tool for gene discovery

A significant limitation of classical loss-of-function screens designed to dissect genetic pathways is that they rarely uncover genes that function redundantly, are compensated by alternative metabolic or regulatory circuits, or which have an additional role in early embryo or gametophyte development. Activation T-DNA tagging is one approach that has emerged in plants to help circumvent these potential problems. This technique utilises a T-DNA sequence that contains four tandem copies of the cauliflower mosaic virus (CaMV) 35S enhancer sequence. This element enhances the expression of neighbouring genes either side of the randomly integrated T-DNA tag, resulting in gain-of-function phenotypes. Activation tagging has identified a number of genes fundamental to plant development, metabolism and disease resistance in Arabidopsis. This review provides selected examples of these discoveries to highlight the utility of this technology. The recent development of activation tagging strategies for other model plant systems and the construction of new more sophisticated vectors for the generation of conditional alleles are also discussed. These recent advances have significantly expanded the horizons for gain-of-function genetics in plants.     

Molecular genetics using T-DNA in rice

Now that sequencing of the rice genome is nearly completed, functional analysis of its large number of genes is the next challenge. Because rice is easy to transform, T-DNA has been used successfully to generate insertional mutant lines. Collectively, several laboratories throughout the world have established at least 200,000 T-DNA insertional lines. Some of those carry the GUS or GFP reporters for either gene or enhancer traps. Others are activation tagging lines for gain-of-function mutagenesis when T-DNA is inserted in the intergenic region. A forward genetic approach showed limited success because of somaclonal variations induced during tissue culture. To utilize these resources more efficiently, tagged lines have been produced for reverse genetics approaches. DNA pools of the T-DNA-tagged lines have been prepared for polymerase chain reaction (PCR) screening of insertional mutants in a given gene. Appropriate T-DNA insertion sites are determined by sequencing the region flanking the T-DNA. This information is then used to make databases that are shared with the scientific community. International efforts on seed amplification and maintenance are needed to exploit these valuable materials efficiently.     

Extracellular enzymes ofErwinia carotovora eliminate the need for azacytidine treatment for high frequency transformation ofArabidopsis thaliana

Summary This paper reports a part of our studies on large-scale T-DNA-mediated gene tagging inArabidopsis thaliana. To enhance the chance of tagging specific stress-responsive genes of this species by monitoring the preferential insertion of the T-DNA into the actively transcribed loci, we exposed the root explants to low temperature (LT), abscisic acid (ABA), and extracellular enzymes (EXE) of the plant pathogenErwinia carotovora prior to transformation byAgrobacterium tumefaciens. Both LT and ABA reduced the frequency of transformation; with these treatments, the average transformation frequencies were 8.1% and 2.6%, respectively. However, in explants pretreated with EXE the transformation frequency was 89.0%, similar to that obtained in control materials (92.6%). Transgenic calli developed from these explants did not require any treatment with azacytidine (azaC) for efficient shoot regeneration. Furthermore, this treatment enhanced multiple insertion of the T-DNA into the plant genome; within a population of EXE-treated transgenic plants, the number of lines harboring at least three copies of the integrated T-DNA was much higher (61%) than that observed in an untreated population (34%).     

Reverse Genetic Approaches for Functional Genomics of Rice

T-DNA and transposable elements e.g., Ds and Tos17, are used to generate a large number of insertional mutant lines in rice. Some carry the GUS or GFP reporter for gene trap or enhancer trap. These reporter systems are valuable for identifying tissue- or organ-preferential genes. Activation tagging lines have also been generated for screening mutants and isolating mutagenized genes. To utilize these resources more efficiently, tagged lines have been produced for reverse genetic approaches. DNA pools of the T-DNA tagged lines and Tos17 lines have been prepared for PCR screening of insertional mutants in a given gene. Tag end sequences (TES) of the inserts have also been produced. TES databases are beneficial for analyzing the function of a large number of rice genes.     

T-DNA tagging in the model legume Medicago truncatula allows efficient gene discovery

The annual legume Medicago truncatula has been proposed as a model plant to study various aspects of legume biology including rhizobial and mycorrhizal symbiosis because it is well suited for the genetic analysis of these processes . To facilitate the characterization of M. truncatula genes participating in various developmental processes we have initiated an insertion mutagenesis program in this plant using three different T-DNAs as tags. To investigate which type of vector is the most suitable for mutagenesis we compared the behavior of these T-DNAs. One T-DNA vector was a derivative of pBin19 and plant selection was based on kanamycin resistance. The two other vectors carried T-DNA conferring Basta resistance in the transgenic plants. For each T-DNA type, we determined the copy number in the transgenic lines, the structure of the T-DNA loci and the sequences of the integration sites. The T-DNA derived from pBin19 generated complex T-DNA insertion patterns. The two others generally gave single copy T-DNA inserts that could result in gene fusions for the pGKB5 T-DNA. Analysis of the T-DNA borders revealed that several M. truncatula genes were tagged in these transgenic lines and in vivo gus fusions were also obtained. These results demonstrate that T-DNA tagging can efficiently be used in M. truncatula for gene discovery.     

The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.     

Insertional tagging of regulatory sequences in tritordeum; a hexaploid cereal species

As an approach to isolate novel cereal promoters, promoterless uidA constructs and particle bombardment were used to transform tritordeum. Five of eight transgenic lines containing uidA sequences showed evidence of promoter tagging. Expression of uidA was detected in four lines as: constitutive expression, expression in short cells of the epidermis of the spikelets, expression in pollen grains and in cells of the epidermis of the spikelet, and expression in anther primordia and pollen grains. In the fifth line, the uidA was shown by RT-PCR to be transcribed, but no GUS activity was detected. The different patterns of uidA expression indicate that different regulatory sequences were tagged in each of these lines. Analysis of the progeny resulting from self-fertilisation of the primary tagged plants, indicate that the transgenes integrated at one or two loci and the patterns of expression were stably inherited. To our knowledge, this is the first report of promoter tagging in cereals by direct gene transfer.     

T-DNA tagging in Brassica napus as an efficient tool for the isolation of new promoters for selectable marker genes

A simple strategy to identify and isolate new promoters suitable for driving the expression of selectable marker genes is described. By employing a Brassica napus hypocotyl transformation protocol and a promoterless gus::nptII tagging construct, a series of 20 kanamycin-resistant tagged lines was produced. Most of the regenerated plants showed hardly any GUS activity in leaf, stem and root tissues. However, expression was readily restored in callus tissue induced on in vitro leaf segments. Genomic sequences upstream of the gus::nptII insertions were isolated via plasmid rescue. Three clones originating from single copy T-DNA lines were selected for further evaluation. The rescued plasmids were cloned as linear fragments in binary vectors and re-transformed to Brassica napus hypocotyl and Solanum tuberosum stem segments. The new sequences maintained their promoter activity, demonstrated by transient and stable GUS activity after transformation. Furthermore, the promoters provided sufficient expression of the nptII gene to yield transgenic plants when using kanamycin as selective agent. Database searching (BLASTN) revealed that the promoters have significant homology with three Arabidopsis BAC clones, one Arabidopsis cDNA and one Brassica napus cDNA. The results presented in this paper illustrate the strength of combined methods for identification, isolation and testing of new plant promoters.