Ds Element Research Articles

Expression analysis of gene trap lines and mapping of donor loci for Dissociation transposition in Arabidopsis

In order to establish a system for characterizing gene function utilizing information obtained from genome sequences, we generated T-DNA insertion lines using a newly constructed binary vector. The vector carries a uidA (b- glucuronidase (GUS)) reporter gene which allows the promoter activity of the inserted genes to be monitored, a transposable element Dissociation (Ds) for targeted insertional mutagenesis, and the cis sequences required for Agrobacterium-mediated transformation. Approximately 8% of the 20,000 lines tested for GUS activity exhibited positive staining. Staining was detected in various organs including roots, leaves, stems, flowers, and siliques. These lines are therefore useful resources for analyzing tissue or organ specific gene expression. We have included the GUS expression patterns on our web site (http://www.kazusa.or.jp/ja/plant/GUS/). To establish a system for targeted insertional mutagenesis, integration sites of the T-DNAs in 140 lines with a single T-DNA insertion were mapped on the genome. The T-DNA contains the Ds element; therefore these 140 lines could be used as donor loci for Ds transposition. The integration sites were almost evenly distributed on all five chromosomes except for the nuclear organizer regions of chromosomes 2 and 4 and the centromeric regions. The Ds element in one line was transposed in combination with an Activator (Ac) element. In about half of the transposed lines, the Ds elements were reinserted within about 1 M bp from the donor locus. These results indicate that the donor loci for Ds transposition that were mapped in this study are a valuable resource for targeted mutagenesis throughout the Arabidopsis genome.

The maize Activator/Dissociation system is functional in hexaploid wheat through successive generations.

The aim of the present study was to provide useful background information and evidence of the functionality of the maize Activator/Dissociation (Ac/Ds) system in hexaploid wheat. Two transgenic parental wheat lines, one harbouring the immobilised Ac element (iAc) and the other the Ds element (pUbi[Ds-uidA]bar), were crossed. Transient GUS assays confirmed that the iAc transposase is active in hexaploid wheat. Selected F1 and F2 lines were analysed by PCR using primers specific to Ac, uidA and bar genes. The primer pair Ubi/bar-tag was used to detect excision of the Ds-uidA sequence, which occurred at a frequency of 39% in the F1 generation. Lines free of Ac and showing evidence of Ds excision were subject to Southern analysis, which indicated that at least one transposition event might have occurred in these lines. Although more evidence is required to unequivocally support the reintegration of the Ds element in the wheat genome, the evidence presented here nevertheless demonstrates the effectiveness and potential value of using this system to tag genes in wheat.

The WiscDsLox T-DNA collection: an arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline

We have developed a new community resource, called the WiscDsLox collection, for performing reverse-genetic analysis in arabidopsis. This resource is composed of 10,459 T-DNA lines generated using the Arabidopsis thaliana ecotype Columbia. The flanking sequence tag for each T-DNA insertion has been deposited in public databases, and seed for each line is currently available from the Arabidopsis Biological Resource Center. The pDsLox vector used to create this new population contains a Ds transposon and Cre/Lox recombination sites. Each WiscDsLox line therefore has the potential to serve as a launch-pad for performing local saturation mutagenesis by mobilization of the Ds element. In addition, Cre-Lox recombination between the T-DNA and a transposed Ds element should enable targeted deletion of specific genomic regions. We generated the WiscDsLox collection using an improved high-throughput pipeline that streamlines analysis of large numbers of independent Arabidopsis thaliana (L.) Hyenh. lines. In this paper we describe the details of this novel method and also provide potential users of WiscDsLox T-DNA lines with useful background information about this collection. Experiments to characterize the utility of the Ds transposon and Cre/Lox elements present in the WiscDsLox lines are in progress and will be reported in the future.

Transposition-Based Plant Transformation

Agrobacterium T-DNAs were used to deliver transposable Dissociation (Ds) elements into the nuclei of potato (Solanum tuberosum) cells. A double-selection system was applied to enrich for plants that only contained a transposed Ds element. This system consisted of a positive selection for the neomycin phosphotransferase (nptII) gene positioned within Ds followed by a negative selection against stable integration of the cytosine deaminase (codA) gene-containing T-DNA. Sixteen of 29 transgenic plants were found to contain a transposed element while lacking any superfluous T-DNA sequences. The occurrence of this genotype indicates that Ds elements can transpose from relatively short extrachromosomal DNA molecules into the plant genome. The frequency of single-copy Ds transformation was determined at 0.3%, which is only about 2.5-fold lower than the potato transformation frequency for backbone-free and single-copy T-DNAs. Because of the generally high expression levels of genes positioned within transposed elements, the new transformation method may find broad applicability to crops that are accessible to Agrobacterium T-DNA transfer.

A Membrane-Bound NAC Transcription Factor Regulates Cell Division inArabidopsis

Controlled release of membrane-tethered, dormant precursors is an intriguing activation mechanism that regulates diverse cellular functions in eukaryotes. An exquisite example is the proteolytic activation of membrane-bound transcription factors. The proteolytic cleavage liberates active transcription factors from the membranes that can enter the nucleus and evokes rapid transcriptional responses to incoming stimuli. Here, we show that a membrane-bound NAC (for NAM, ATAF1/2, CUC2) transcription factor, designated NTM1 (for NAC with transmembrane motif1), is activated by proteolytic cleavage through regulated intramembrane proteolysis and mediates cytokinin signaling during cell division in Arabidopsis thaliana. Cell proliferation was greatly reduced in an Arabidopsis mutant with retarded growth and serrated leaves in which a transcriptionally active NTM1 form was constitutively expressed. Accordingly, a subset of cyclin-dependent kinase (CDK) inhibitor genes (the KIP-related proteins) was induced in this mutant with a significant reduction in histone H4 gene expression and in CDK activity. Consistent with a role for NTM1 in cell cycling, a Ds element insertional mutant was morphologically normal but displayed enhanced hypocotyl growth with accelerated cell division. Interestingly, cytokinins were found to regulate NTM1 activity by controlling its stability. These results indicate that the membrane-mediated activation of NTM1 defines a molecular mechanism by which cytokinin signaling is tightly regulated during cell cycling.

Trans-kingdom transposition of the maize dissociation element.

Transposons are very valuable tools for genetic manipulation. However, the number of transposable elements that have been suitably adapted for experimental use is insufficient and the spectrum of heterologous hosts in which they have been deployed is restricted. To date, only transposons from animal hosts have been utilized in heterologous animal species and transposons of plant origin have been used in plant genetics. There has been no experimental evidence that any of the known elements could transpose in hosts belonging to both kingdoms. Here we demonstrate that the maize Dissociation (Ds) element is capable of effective Activator (Ac) transposase-mediated transposition in the zebrafish Danio rerio, yielding remarkable germline transmission rates. In addition, mammalian cells were also found to be conducive to Ds transposition. Furthermore, we demonstrate that nuclear localization of Ac transposase is essential for genomic Ds transposition. Our results support the hypothesis that Ac/Ds elements do not rely on host-specific factors for transposition and that host factors involved in their mobility mechanism are widely conserved. Finally, even in vertebrate cells, the Ac/Ds system displays accurate transposition, large-fragment carrying capacity, high transposition frequencies, efficient germline transmission, and reporter gene expression, all of which are advantageous for various genetic applications and animal biotechnology.

Use of the transposable element Ac/Ds in conjunction with Spm/dSpm for gene tagging allows extensive genome coverage with a limited number of starter lines: functional analysis of a four-element system in Arabidopsis thaliana

We have developed a novel four-element based gene tagging system in Arabidopsis to minimize the number of starter lines required to generate genome-wide insertions for saturation mutagenesis. In this system, the non-autonomous cassette, Ds(dSpm), comprises of both Ds and dSpm elements cloned one within the other along with appropriate selection markers to allow efficient monitoring of excision and re-integration of the transposons. Trans-activation of the outer borders (Ds) and selection against the negative selection marker (iaaH) linked to the cassette ensures unlinked spread of the Ds(dSpm) cassette from the initial site of integration of the T-DNA. This creates several launch pads within the genome from where the internal element (dSpm) can be subsequently mobilized to generate secondary insertions. In this study, starting from a single T-DNA integration we could spread the Ds(dSpm) cassette to 11 different locations over all the five chromosomes of Arabidopsis. The frequency of unlinked Ds transpositions in the F2 generation varied between 0.05 and 3.35%. Three of these lines were then deployed to trans-activate the internal dSpm element which led to the selection of 29 dSpm insertions. The study conclusively shows the feasibility of deploying Ds and the dSpm elements in a single construct for insertional mutagenesis.

Mapped Ds/T‐DNA launch pads for functional genomics in barley

A system for targeted gene tagging and local saturation mutagenesis based on maize transposable elements (Ac/Ds) was developed in barley (Hordeum vulgare L.). We generated large numbers of transgenic barley lines carrying a single copy of the non-autonomous maize Ds element at defined positions in the genome. Independent Ds lines were either generated by activating Ds elements in existing single-copy lines after crossing with AcTPase-expressing plants or by Agrobacterium-mediated transformation. Genomic DNA flanking Ds and T-DNA insertion sites from over 200 independent lines was isolated and sequenced, and was used for a sequence based mapping strategy in a barley reference population. More than 100 independent Ds insertion sites were mapped and can be used as launch pads for future targeted tagging of genes in the vicinity of the insertion sites. Sequence analysis of Ds and T-DNA flanking regions revealed a sevenfold preference of both mutagens for insertion into non-redundant, gene-containing regions of the barley genome. However, whilst transposed Ds elements preferentially inserted adjacent to regions with a high number of predicted and experimentally validated matrix attachment regions (nuclear MARs), this was not the case for T-DNA integration sites. These findings and an observed high transposition frequency from mapped launch pads demonstrate the future potential of gene tagging for functional genomics and gene discovery in barley.

Targeted Ds-tagging strategy generates high allelic diversity at the Arabidopsis HY2 locus

The targeted (or directed) tagging is a strategy aimed to mobilize a tranposon into a specific gene (target). Only a very few Arabidopsis genes have been tagged by this way, thus the efficiency of the strategy, as well as the diversity of the alleles obtained are not well documented. We have used a maize Ds element in a directed tagging of HY2. The starting Ds element, located 22 kb proximal to HY2, has been remobilized in a cross with an Ac transposase source line. From the F2 progeny of 4800 F1 we phenotypically isolated seven hy2 mutants. Molecular analysis of these alleles revealed that two contained a Ds element in HY2 and were instable, three have a large deletion that partially or completely removed HY2, one has a footprint in a HY2 exon and one leaky allele consisted of a 22 kb inversion upstream the HY2 coding sequence. Thus, the transposon-based directed tagging strategy generates a wide diversity of tagged and non-tagged alleles that can be used to generate allelic series or deletion of clustered genes.

Chromosome Breakage and Repair

Chromosome Breakage and Repair

A tomato mutant that shows stunting, wilting, progressive necrosis and constitutive expression of defence genes contains a recombinant Hcr9 gene encoding an autoactive protein

The tomato Cf-9 gene confers resistance to races of the leaf mould fungus Cladosporium fulvum that carry the Avr9 avirulence gene. Cf-9 resides at a locus containing five paralogous genes and was isolated by transposon tagging using a modified maize Dissociation (Ds) element. The tagging experiment generated an allelic series of Ds-induced mutations of Cf-9, most of which were wild type in appearance. However, one mutant, designated M205, showed stunted growth, wilting, progressive leaf chlorosis and necrosis and constitutive expression of defence genes. The phenotype of M205 was caused by a semidominant, Avr9-independent mutation that co-segregated with a Ds element insertion at the Cf-9 locus. Molecular genetic analysis indicated that the Cf-9 locus of M205 had undergone recombination, generating a chimeric gene, designated Hcr9-M205, that comprised an in-frame fusion between the 5' coding region of the Cf-9 paralogue, Hcr9-9A, and the 3' coding region of Cf-9. The presence of a possible excision footprint adjacent to the junction between Hcr9-9A and Cf-9, and a Ds insertion at the homologous position in the downstream paralogue Hcr9-9D, is consistent with recombination between Hcr9-9A and Cf-9 promoted by transposition of Ds from Cf-9 into Hcr9-9D. Agrobacterium tumefaciens-mediated transient expression of Hcr9-M205 in Nicotiana tabacum caused chlorosis and the accumulation of defence gene transcripts, indicating that the protein encoded by this novel Hcr9 gene is autoactive.

Dominant‐negative interference with defence signalling by truncation mutations of the tomato Cf‐9 disease resistance gene

The tomato Cf-9 gene confers resistance to races of the leaf mould fungus Cladosporium fulvum that carry the Avr9 avirulence gene. Cf-9 was isolated by transposon tagging using a modified maize Dissociation (Ds) element. This generated an allelic series of Ds-induced mutations of Cf-9, of which two were found to confer novel phenotypes in a screen for mutants affecting wild-type Cf-9 function in trans. Genetic and molecular analysis of these mutants suggested semidominant, Avr9-dependent, negative-interfering mutations involving Ds insertions in a defined subregion of Cf-9. Interference was associated with expression of the 5'-end of Cf-9 upstream of the Ds insertions in these mutants, suggesting that truncated Cf-9 proteins were the likely cause of interference. Transgenic tomato lines harbouring Cf-9 constructs with premature stop codons in positions similar to the Ds insertions also showed interference, indicating that the presence of Ds was not required for interference to occur. Interestingly, interference in these transgenic lines was completely dominant and was associated with a pronounced developmental phenotype that was dependent on co-expression of Cf-9, Avr9 and a truncated Cf-9 transgene. However, interference with a weakly autoactive Hcr9 gene was Avr9-independent and did not cause a developmental phenotype, suggesting that localized restoration of Cf-9/Avr9-dependent cell death was responsible for the developmental phenotype. The restricted region in which truncation of Cf-9 results in dominant-negative interference suggests that leucine-rich repeats (LRR) 16-19 of Cf-9 may mediate dimerization of Cf-9 and LRRs 20-23 may mediate interactions with downstream partner proteins required for Cf-9 signalling, or vice versa.

AtUTr1, a UDP-glucose/UDP-galactose Transporter from Arabidopsis thaliana, Is Located in the Endoplasmic Reticulum and Up-regulated by the Unfolded Protein Response

The folding of glycoproteins in the endoplasmic reticulum (ER) depends on a quality control mechanism mediated by the calnexin/calreticulin cycle. During this process, continuous glucose trimming and UDP-glucose-dependent re-glucosylation of unfolded glycoproteins takes place. To ensure proper folding, increases in misfolded proteins lead to up-regulation of the components involved in quality control through a process known as the unfolded protein response (UPR). Reglucosylation is catalyzed by the ER lumenal located enzyme UDP-glucose glycoprotein glucosyltransferase, but as UDP-glucose is synthesized in the cytosol, a UDP-glucose transporter is required in the calnexin/calreticulin cycle. Even though such a transporter has been hypothesized, no protein playing this role in the ER yet has been identified. Here we provide evidence that AtUTr1, a UDP-galactose/glucose transporter from Arabidopsis thaliana, responds to stimuli that trigger the UPR increasing its expression around 9-fold. The accumulation of AtUTr1 transcript is accompanied by an increase in the level of the AtUTr1 protein. Moreover, subcellular localization studies indicate that AtUTr1 is localized in the ER of plant cells. We reasoned that an impairment in AtUTr1 expression should perturb the calnexin/calreticulin cycle leading to an increase in misfolded protein and triggering the UPR. Toward that end, we analyzed an AtUTr1 insertional mutant and found an up-regulation of the ER chaperones BiP and calnexin, suggesting that these plants may be constitutively activating the UPR. Thus, we propose that in A. thaliana, AtUTr1 is the UDP-glucose transporter involved in quality control in the ER.

Association of reversible inactivation of the maize transposable element Ds with tissue-specific processing of the 35S:TPase transcript in carrot (Daucus carota L.)

SummaryAn Ac/Ds-based two-element transposon tagging system has been introduced into carrot. F1 progeny containing both the 35S-Ac-transposase gene (35S:TPase) and the Ds element were derived from crosses between 35S:TPase- and Dsbearing parents. While excision of Ds was not detected in any F1 plants carrying both 35S:TPase and the Ds element, calli initiated from these F1 plants had the Ds element excised, indicating Ds transposition. Reverse transcriptase-PCR analysis revealed that the 35S:TPase gene was expressed in both F1 plants and calli, and that introns 1, 2, and 3 were spliced correctly. Although intron 4 was also spliced correctly in calli, incorrectly spliced intron 4 was detected in F1 plants. Sequence analysis of incorrectly spliced reverse transcriptase-PCR products demonstrated the presence of a cryptic intron donor site within intron 4 of the 35S:TPase transcript. This probably competed with the proposed intron donor site during maturation of the major 35S:TPase transcript. These results suggested that the major transcript of 35S:TPase was incorrectly processed and, consequently, that the Ds element was reversibly inactivated in the somatic tissues of carrot plants, whereas this inactive Ds element was remobilised during tissue culture, where the 35S:TPase transcript was spliced correctly. These observations point to an important role for tissue-specific 35S:TPase transcript processing for successful transposition of Ds in carrot. Therefore, successful processing of the 35S:TPase transcript in carrot callus may indicate strategies to increase Ac transposition in other tissues.

Large-scale Systematic Study on Stability of the Ds Element and Timing of Transposition in Rice

Activator/Dissociation (Ac/Ds) transposon mutagenesis is a widely used tool for gene identification; however, several reports on silencing of the Ac/Ds element in starter lines and in stable transposants question the applicability of such an approach in later generations. We have performed a systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare). High somatic and germinal transposition frequencies observed in earlier generations were maintained as late as T4 and T5 generations; thus the propagation of parental lines did not induce transposon silencing. Moreover, the stably transposed Ds element was active even at the F5 generation, since Ac could remobilize the Ds element as indicated by the footprint analysis of several revertants. Expression of the bar gene was monitored from F3 to F6 generations in >1,000 lines. Strikingly, substantial transgene silencing was not observed in any of the generations tested. We analyzed the timing of transposition during rice development and provide evidence that Ds is transposed late after tiller formation. The possibility, that the independent events could be the result of secondary transposition, was ruled out by analyzing potential footprints by reciprocal PCR. Our study validates the Ac/Ds system as a tool for large-scale mutagenesis in rice, since the Ds elements were active in the starter and insertion lines even in the later generations. We propose that harvesting rice seeds using their panicles is an alternative way to increase the number of independent transposants due to post-tillering transposition.

Amplification of Ds Flanking Sequences from Rice Genomic DNA of Hybrids of Ac × Ds Lines and the Analysis of Ds Insertions

Ac and Ds insertions among the genomic DNAs of hybrids of Ac x Ds lines were screened by PCR. The genomic DNAs, which were proved to harbour both Ac and Ds, were used as templates in TAIL-PCR to clone the Ds flanking sequences. The cloned specific fragments were sequenced, and the sequenced Ds flanking sequences were used as query sequences to perform on-line sequence comparing analysis against GenBank by employing BLAST program of NCBI. The information about the chromosome location of Ds-inserted genes, or genes immediately downstream of the inserted sites, and their functional innotations were achieved. Based on the analysis from the cloned 93 Ds-flanking sequences, it was found that 21 hybrid plants had Ds insertions in genic regions, whereas the remaining 72 samples's intergenic regions were inserted by Ds element. Moreover, among the 72 regions, 12 were inserted immediately upstream (within 3 kb) of specific genes. Also, the strategies to improve the performance in cloning the Ds flanking sequences and in screening the Ac/Ds lines were emphasized.

Trace element evidence for growth of early continental crust by melting of rutile-bearing hydrous eclogite

The early continental crust is composed dominantly of Archean tonalite-trondhjemite-granodiorite (TTG) gneisses and is generally explained as the product of melting of metabasalts in the subducted crust. However, whether the melting occurs in shallow-level amphibolite facies or in relatively deep eclogite facies is debated. Here I present experimental partition coefficients (Ds) for 27 trace elements between garnet/amphibole and tonalitic melts. They are used together with published mineral/melt trace element Ds to model the melting of metabasalt in order to delimit the conditions for TTG production. The results clearly show that model melts with trace element characteristics that completely mimic the TTG are in equilibrium with rutile-bearing anhydrous and hydrous (amphibole bearing) eclogitic residues, but not rutile-free, amphibole-dominated residues. Rutile appears to be a necessary residual phase to account for the characteristic negative Nb-Ta anomaly in the TTG. These results thus suggest that the early continental material was produced under eclogite facies conditions. Based on the modeling results obtained using appropriate partition coefficients and the Archean geotherm, the preferred process for the TTG production is the melting of rutile-bearing hydrous eclogite, triggered by the release of H2O from the progressive breakdown of amphibole. The pressure-temperature ( P - T ) conditions for TTG production via this process are constrained to 1.5–2.5 GPa (∼50– 80 km) and 850–1050 °C by the P - T stability boundaries of amphibole and rutile in the basalt system.

Efficient insertional mutagenesis in rice using the maize En/Spm elements

We have developed a novel system for insertional mutagenesis in rice (Oryza sativa) based on the maize (Zea mays) enhancer/suppressor mutator (En/Spm) element. In this system, a single T-DNA construct with Spm-transposase and the non-autonomous defective suppressor mutator (dSpm) element is used in conjunction with green fluorescent protein (GFP) and Discosoma sp. Red Fluorescence Protein (DsRed) fluorescent markers to select unlinked stable transpositions of dSpm. Using this system, we could demonstrate high frequencies of unlinked germinal transposition of dSpm in rice. Analysis of dSpm flanking sequences from 353 stable insertion lines revealed that the dSpm insertions appear to be widely distributed on rice chromosomes with a preference for genic regions (70%). The dSpm insertions appear to differ from Activator-Dissociation (Ac-Ds) elements in genomic distribution and exhibit a greater fraction of unlinked transpositions when compared with Ds elements. The results obtained in this study demonstrate that the maize En/Spm element can be used as an effective tool for functional genomics in rice and can complement efforts using other insertional mutagens. Further, the efficacy of the non-invasive fluorescence-based selection system is promising for its application to other crops.

Identification of Three Urease Accessory Proteins That Are Required for Urease Activation in Arabidopsis

Urease is a nickel-containing urea hydrolase involved in nitrogen recycling from ureide, purine, and arginine catabolism in plants. The process of urease activation by incorporation of nickel into the active site is a prime example of chaperone-mediated metal transfer to an enzyme. Four urease accessory proteins are required for activation in Klebsiella aerogenes. In plants urease accessory proteins have so far been only partially defined. Using reverse genetic tools we identified four genes that are necessary for urease activity in Arabidopsis (Arabidopsis thaliana; ecotypes Columbia and Nössen). Plants bearing T-DNA or Ds element insertions in either the structural gene for urease or in any of the three putative urease accessory genes AtureD, AtureF, and AtureG lacked the corresponding mRNAs and were defective in urease activity. In contrast to wild-type plants, the mutant lines were not able to support growth with urea as the sole nitrogen source. To investigate whether the identified accessory proteins would be sufficient to support eukaryotic urease activation, the corresponding cDNAs were introduced into urease-negative Escherichia coli. In these bacteria, urease activity was observed only when all three plant accessory genes were coexpressed together with the plant urease gene. Remarkably, plant urease activation occurred as well in cell-free E. coli extracts, but only in extracts from cells that had expressed all three accessory proteins. The future molecular dissection of the plant urease activation process may therefore be performed in vitro, providing a powerful tool to further our understanding of the biochemistry of chaperone-mediated metal transfer processes in plants.

Colonization of the Arabidopsis rhizosphere by fluorescent Pseudomonas spp. activates a root-specific, ethylene-responsive PR-5 gene in the vascular bundle

Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISR-inducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.