T-DNA Activation Tagging Research Articles

Homeobox transcription factor OsZHD2 promotes root meristem activity in rice by inducing ethylene biosynthesis.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

T-DNA activation tagging in rice results in a variable response to Meloidogyne graminicola infection

Plant-parasitic nematodes (PPNs) are known to be devastating pests of agricultural crops; the rice root knot nematode Meloidogyne graminicola being the most damaging in rice. Variability in response to any trait is important not only to understand plant-stress interactions, but also its utility in stress mitigation. In this direction, the focus of the study was to exploit activation tagging, a well-established functional genomics approach in rice as a plausible strategy to assess variability in the phenotypic and molecular response to M. graminicola infection. T-DNA insertional mutants were developed in rice (acc. JBT 36/14) using an apical meristem-targeted in planta transformation strategy. A panel of 30 mutants that demonstrated molecular evidences for T-DNA integration and portraying a robust phenotype were chosen for the study. Vigorous phenotyping of these mutants against M. graminicola second stage juveniles both under in vitro pluronic gel medium and soil conditions depicted variable response based on four disease scoring parameters and the derived nematode reproductive factor. Among 30 mutants that were evaluated, five showed a resistant phenotype while 25 depicted a susceptible reaction. The resistant plants clearly demonstrated a reduction in all the disease parameters both under in vitro as well as greenhouse conditions. Quantification of transcripts of 25 genes associated with host defence responses belonging to salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) pathways also indicated corroboration with the phenotype exhibited by the mutants. Distinct pattern of gene expression profiles was observed not only between the susceptible and resistant mutants, but also amongst the resistant ones, demonstrating the possibility of lesser known defense pathways being operated in these plants, apparently depending upon the T-DNA integration loci. The variability in the response to nematode challenge demonstrated in this study can be a step towards deciphering of compatible/incompatible relationships that can form a knowledge base towards not only understanding plant-nematode interactions but also extrapolation of their utility in rice improvement programmes.

Overexpression of a putative Arabidopsis BAHD acyltransferase causes dwarfism that can be rescued by brassinosteroid

Plant growth and development are ensured through networks of complex regulatory schemes. Genetic approaches have been invaluable in dissecting these regulatory pathways. This study reports the isolation of a semi-dominant dwarf mutant designated abnormal shoot1-1 dominant (abs1-1D) through an Arabidopsis T-DNA activation tagging mutant screen. It was shown that the overexpression of a novel BAHD family acyltransferase gene, ABS1/At4g15400, was the cause of the dwarf phenotype in abs1-1D. Overexpression of ABS1 led to many phenotypic features reminiscent of brassinosteroid (BR) deficient or signalling mutants, and it was shown that exogenously applied BR could effectively rescue the dwarf phenotype of abs1-1D. Furthermore, genetic analyses indicated that abs1-1D interacted, in different ways, with the BR-deficient mutant det2-1, the constitutive BR response mutant bes1-D and the photomorphogenic mutant phyB-1. Moreover, ABS1 expression was activated by BR treatment or in a bes1-D mutant background. Genome-wide transcriptome profiling of abs1-1D revealed clear reprogramming of metabolic pathways, and it was demonstrated that BR biosynthesis genes were activated in abs1-1D and that the flavonoid biosynthesis pathway was repressed in abs1-1D, as well as in det2-1. This work provides new insights into the possible involvement of BAHD acyltransferase in the regulation of plant growth and development, and indicates a possible role of ABS1 in maintaining BR homeostasis.

Overexpression of EVE1, a novel ubiquitin family protein, arrests inflorescence stem development in Arabidopsis

In Arabidopsis, inflorescence stem formation is a critical process in phase transition from the vegetative to the reproductive state. Although inflorescence stem development has been reported to depend on the expression of a variety of genes during floral induction and repression, little is known about the molecular mechanisms involved in the control of inflorescence stem formation. By activation T-DNA tagging mutagenesis of Arabidopsis, a dominant gain-of-function mutation, eve1-D (eternally vegetative phase1-Dominant), which has lost the ability to form an inflorescence stem, was isolated. The eve1-D mutation exhibited a dome-shaped primary shoot apical meristem (SAM) in the early vegetative stage, similar to that seen in the wild-type SAM. However, the SAM in the eve1-D mutation failed to transition into an inflorescence meristem (IM) and eventually reached senescence without ever leaving the vegetative phase. The eve1-D mutation also displayed pleiotropic phenotypes, including lobed and wavy rosette leaves, short petioles, and an increased number of rosette leaves. Genetic analysis indicated that the genomic location of the EVE1 gene in Arabidopsis thaliana corresponded to a bacterial artificial chromosome (BAC) F4C21 from chromosome IV at ∼17cM which encoded a novel ubiquitin family protein (At4g03350), consisting of a single exon. The EVE1 protein is composed of 263 amino acids, contains a 52 amino acid ubiquitin domain, and has no glycine residue related to ubiquitin activity at the C-terminus. The eve1-D mutation provides a way to study the regulatory mechanisms that control phase transition from the vegetative to the reproductive state.

Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T-DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, alpha-tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus-end marker GFP-EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell-to-cell movement of TMV encoding GFP-tagged movement protein (MP-GFP) is reduced in ATER2 accompanied by a reduced association of MP-GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.

Activation tagging and its application in plant functional genomics research

It is a practical strategy to screen for mutants in the research of plant functional genomics. Comparing with classical T-DNA knock-out mutagenesis technology by the loss-of-function mutation, the activation T-DNA tagging technique based on the gain-of-function mutation has its own particular advantages, mainly characterized by producing dominant mutants of genes with functional redundancy and easily cloning of the genes. First, the basic principle of activation tagging, and the progress of its application in the research on plant functional genomics was reviewed, especially in Arabidopsis and rice. The recent research progress in the mechanism of plant biotic and abiotic stress tolerance and of plant development unraveled by the method of activation tagging was then addressed. Finally, the limitation and prospects of this technique were discussed.

Activation tagging and its application in plant functional genomics research

It is a practical strategy to screen for mutants in the research of plant functional genomics. Comparing with classical T-DNA knock-out mutagenesis technology by the loss-of-function mutation, the activation T-DNA tagging technique based on the gain-of-function mutation has its own particular advantages, mainly characterized by producing dominant mutants of genes with functional redundancy and easily cloning of the genes. First, the basic principle of activation tagging, and the progress of its application in the research on plant functional genomics was reviewed, especially in Arabidopsis and rice. The recent research progress in the mechanism of plant biotic and abiotic stress tolerance and of plant development unraveled by the method of activation tagging was then addressed. Finally, the limitation and prospects of this technique were discussed.

Actin is bundled in activation-tagged tobacco mutants that tolerate aluminum

A panel of aluminum-tolerant (AlRes) mutants was isolated by protoplast-based T-DNA activation tagging in the tobacco cultivar SR1. The mutants fell into two phenotypic classes: a minority of the mutants were fertile and developed similarly to the wild type (type I), the majority was male-sterile and grew as semi-dwarfs (type II). These traits, along with the aluminum tolerance, were inherited in a monogenic dominant manner. Both types of mutants were characterized by excessive bundling of actin microfilaments and by a strongly increased abundance of actin, a phenotype that could be partially phenocopied in the wild type by treatment with aluminum chloride. The actin bundles could be dissociated into finer strands by addition of exogenous auxin in both types of mutants. However, actin microfilaments and leaf expansion were sensitive to blockers of actin assembly in the wild type and in the mutants of type I, whereas they were more tolerant in the mutants of type II. The mutants of type II displayed a hypertrophic development of vasculature, manifest in form of supernumerary leaf veins and extended xylem layers in stems and petioles. Whereas mutants of type I were characterized by a normal, but aluminum-tolerant polar auxin-transport, auxin-transport was strongly promoted in the mutants of type II. The phenotype of these mutants is discussed in terms of reduced endocytosis leading, concomitantly with aluminum tolerance, to changes in polar auxin transport.

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.

T-DNA activation tagging.

T-DNA activation tagging is a method to generate dominant mutations in plants or plant cells by random insertion of a T-DNA carrying constitutive enhancer elements, which can cause transcriptional activation of flanking plant genes. The method consists of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype. Subsequently, the activated plant gene is rescued from selected mutant transformants for further functional analysis. Since the exact procedure depends on the plant material and the selected phenotype, this chapter describes one specific example of T-DNA activation tagging of suspension-cultured cells, including, where possible, cross-references to more general applications of the technique.

Activation-tagged tobacco mutants that are tolerant to antimicrotubular herbicides are cross-resistant to chilling stress.

T-DNA activation tagging was used to generate tobacco mutants with increased tolerance to antimicrotubular herbicides and chilling stress. After transformation, protoplast-derived calli were screened for tolerance to treatments that affect microtubule assembly. In one screen mutants with tolerance to aryl carbamates (a blocker of microtubule assembly) were selected, the second screen was targeted to chilling-tolerant mutants that could survive for several months at 3 degrees C, a third screen combined both factors. The resistance of these mutants to aryl carbamates or chilling was accompanied by resistance of microtubules to these factors. The carbamate tolerant mutants were cross-resistant to chilling stress. This was mirrored by an adaptive reorganization of microtubules and a reduction of microtubule dynamics in response to chilling. The analysis of these mutants suggests (1) that microtubule dynamics limit the tolerance to chilling and EPC, and (2) that the cold sensitivity of microtubules limits chilling tolerance in tobacco.

Activation tagging using the En-I maize transposon system in Arabidopsis.

A method for the generation of stable activation tag inserts was developed in Arabidopsis using the maize (Zea mays) En-I transposon system. The method employs greenhouse selectable marker genes that are useful to efficiently generate large populations of insertions. A population of about 8,300 independent stable activation tag inserts has been produced. Greenhouse-based screens for mutants in a group of plants containing about 2,900 insertions revealed about 31 dominant mutants, suggesting a dominant mutant frequency of about 1%. From the first batch of about 400 stable insertions screened in the greenhouse, four gain-in-function, dominant activation-tagged, morphological mutants were identified. A novel gain-in-function mutant called thread is described, in which the target gene belongs to the same family as the YUCCA flavin-mono-oxygenase that was identified by T-DNA activation tagging. The high frequency of identified gain-in-function mutants in the population suggests that the En-I system described here is an efficient strategy to saturate plant genomes with activation tag inserts. Because only a small number of primary transformants are required to generate an activation tag population, the En-I system appears to be an attractive alternative to study plant species where the present transformation methods have low efficiencies.

Cloning of an Arabidopsis Patatin-Like Gene, STURDY, by Activation T-DNA Tagging

Activation T-DNA tagging can generate dominant gain-of-function mutants by overexpression of a particular endogenous gene. We identified an activation-tagged mutant, sturdy, exhibiting a stiff inflorescence stem, thicker leaves, shorter siliques, larger seeds, round-shaped flowers, and delayed growth. It is most important that unlike its wild-type counterpart, this mutant is less prone to lodging. Cloning of STURDY revealed that in sturdy, there is an open reading frame containing a single intron encoding a patatin-like homolog. The T-DNA is inserted into the 3' region of the second exon. The mutant phenotype was shown to be the result of overexpression of STURDY by mRNA analysis and transgenic studies. Preliminary histological studies have revealed an increase in cell number in the inflorescence stem of mutant plants; however, additional studies are needed to better understand the overexpression phenotype.

T-DNA activation tagging as a tool to isolate regulators of a metabolic pathway from a genetically non-tractable plant species.

T-DNA activation tagging is a method used to generate dominant mutations in plants or plant cells by the insertion of a T-DNA which carries constitutive enhancer elements that can cause transcriptional activation of flanking plant genes. We applied this approach to the species Catharanthus roseus (L.) G. Don (Madagascar periwinkle), in an attempt to isolate regulators of genes that are involved in the biosynthesis of secondary metabolites of the terpenoid indole alkaloid (TIA) class. Several TIAs have pharmaceutically interesting activities, including the anti-tumour agents vincristine and vinblastine. The use of suspension-cultured cells enabled us to screen in a relatively easy way hundreds of thousands of T-DNA-tagged cells for resistance to a toxic substrate of one of the TIA biosynthetic enzymes: tryptophan decarboxylase. This screening yielded several interesting tagged cell lines. Further characterisation of one of the tagged cell lines led to the isolation of Orca3, a gene encoding an AP2/ERF-domain transcription factor that acts as a master regulator of primary and secondary metabolism. The T-DNA activation tagging results described in detail in this paper illustrate the usefulness of this approach to isolate regulators of a complex metabolic pathway from a genetically non-tractable plant species.

Identification and role of adenylyl cyclase in auxin signalling in higher plants.

Cyclic AMP is an important signalling molecule in prokaryotes and eukaryotes1, but its significance in higher plants has been generally doubted2 because they have low adenylyl cyclase activity and barely detectable amounts of cAMP3. Here we used activation T-DNA tagging to create tobacco cell lines that can proliferate in the absence of the phytohormone auxin in the culture media4,5. The sequence tagged in one line, axi 141, was used to isolate a complementary DNA encoding adenylyl cyclase, the first from a higher plant. Sequence analysis reveals that the tobacco adenylyl cyclase is probably soluble, contains characteristic leucine-rich repeats, and bears similarity with adenylyl cyclase from the yeast Schizosaccharomyces pombe. Expression of the cDNA in Escherichia coli results in an increase in endogenous cAMP levels, and in yeast its expression functionally complements the cry1 mutation. Tobacco protoplasts treated with cAMP, or the adenylyl cyclase activator forskolin, no longer require auxin to divide. This finding, together with the observation that the adenylyl cyclase inhibitor dideoxyadenosine inhibits cell proliferation in the presence of auxin, suggests that cAMP is involved in auxin-triggered cell division in higher plants.

Identification and role of adenylyl cyclase in auxin signalling in higher plants.

Cyclic AMP is an important signalling molecule in prokaryotes and eukaryotes, but its significance in higher plants has been generally doubted because they have low adenylyl cyclase activity and barely detectable amounts of cAMP. Here we used activation T-DNA tagging to create tobacco cell lines that can proliferate in the absence of the phytohormone auxin in the culture media. The sequence tagged in one line, axi 141, was used to isolate a complementary DNA encoding adenylyl cyclase, the first from a higher plant. Sequence analysis reveals that the tobacco adenylyl cyclase is probably soluble, contains characteristic leucine-rich repeats, and bears similarity with adenylyl cyclase from the yeast Schizosaccharomyces pombe. Expression of the cDNA in Escherichia coli results in an increase in endogenous cAMP levels, and in yeast its expression functionally complements the cry1 mutation. Tobacco protoplasts treated with cAMP, or the adenylyl cyclase activator forskolin, no longer require auxin to divide. This finding, together with the observation that the adenylyl cyclase inhibitor dideoxyadenosine inhibits cell proliferation in the presence of auxin, suggests that cAMP is involved in auxin-triggered cell division in higher plants.

A plant cation-chloride co-transporter promoting auxin-independent tobacco protoplast division.

Although auxin plays a central role in plant development, little is known about the signal transduction pathways triggered by auxin regulating cell elongation, division and differentiation. We describe the molecular analysis of the mutant tobacco line axi 4/1, which was regenerated from an auxin-independent callus created by activation T-DNA tagging. Transcriptional enhancer-mediated deregulated expression of the tagged plant gene axi 4 uncouples division of axi 4/1 protoplasts from external auxin stimuli, whereas in untransformed protoplasts expression of axi 4 and cell division are auxin dependent. axi 4 encodes a 109 kDa protein with significant homology to a family of electroneutral cation-chloride co-transporters (CCCs). We show that overexpression of AXI 4 or another member of the CCC family, the Na+/K+/2Cl--co-transporter from shark, triggers auxin-independent growth of tobacco protoplasts. We suggest that Na+/K+/2Cl--co-transporters may play a role in signalling cell division and that this function is highly conserved between AXI 4 and the shark Na+/K+/2Cl--co-transporter. We also demonstrate that a C-terminal fragment of AXI 4 is sufficient to promote auxin-independent cell division, showing that the C-termini of CCCs are functional subunits triggering cell division. This may allow a molecular dissection of this process not only in plants but also in animal cells.

T-DNA tagging reveals a novel cDNA triggering cytokinin- and auxin-independent protoplast division.

Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.

High level transcription of a member of a repeated gene family confers dehydration tolerance to callus tissue of Craterostigma plantagineum.

An experimental system has been developed which allows the identification of intermediates in the abscisic acid (ABA) signal transduction pathway leading to desiccation tolerance in plants. Desiccation tolerance in callus of the resurrection plant Craterostigma plantagineum is mediated via the plant hormone ABA, which induces the expression of gene products related to desiccation tolerance. Based on T-DNA activation tagging, a gene (CDT-1) was isolated which encodes a signalling molecule in the ABA transduction pathway. Constitutive overexpression of CDT-1 leads to desiccation tolerance in the absence of ABA and to the constitutive expression of characteristic transcripts. CDT-1 represents a novel gene with unusual features in its primary sequence. The CDT-1 gene resembles in several features SINE retrotransposons. Mechanisms by which CDT-1 activates the pathway could be via a regulatory RNA or via a short polypeptide.

Isolation of Axi (auxin independent groowth)1 Gene by Activation T-DNA Tagging

Isolation of Axi (auxin independent groowth)1 Gene by Activation T-DNA Tagging