Exquisite specificity of mitogenic lectin from <Emphasis Type="Italic">Cephalosporium curvulum</Emphasis> to core fucosylated N-glycans

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.     

Heterologous expression of a <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> late embryogenesis abundant protein gene (<Emphasis Type="Italic">AmLEA</Emphasis>) enhances <Emphasis Type="Italic">Escherichia coli</Emphasis> viability under cold and heat stress

A late embryogenesis abundant protein gene, AmLEA from Ammopiptanthus mongolicus, was introduced into Escherichia coli using the IMPACT™-TWIN system to analyze the possible function of AmLEA under heat and cold stresses. A fusion protein about 38 kD was expressed in E.coli cells harboring pTWIN-LEA after the induction of IPTG by SDS–PAGE analysis and the accumulation of the fusion protein peaked 3 h after IPTG addition when cultured at 37°C. Compared with control cells, the E. coli cells expressing AmLEA fusion protein showed improved chilling and heat resistence, illuminating the protein may play a protective role in cells under stress conditions. These results suggested the natively unstructured protein, similar to other members of LEA proteins, has high capacity for binding water and potential protective function against dehydration or action similar to the cold shock chaperones.     

Lectin genes in the <Emphasis Type="Italic">Frankia alni</Emphasis> genome

Frankia alni strain ACN14a’s genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind specifically to sugar motifs. The genome of F. alni was found to contain 7 such lectin-coding genes, five of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations.     

Complex <Emphasis Type="Italic">N</Emphasis>-glycans or core 1-derived <Emphasis Type="Italic">O</Emphasis>-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le<Superscript>Y</Superscript> in the preimplantation mouse embryo

The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and Le^Y was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or Le^Y on mouse embryos.     

The ammonia-catalyzed release of glycoprotein <Emphasis Type="Italic">N</Emphasis>-glycans

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.     

A genetic analysis of in vivo selenate reduction by <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium LT2 and <Emphasis Type="Italic">Escherichia coli</Emphasis> K12

The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Salmonella enterica serovar Typhimurium and Escherichia coli many Tat substrates are known or predicted to bind a molybdenum cofactor in the cytoplasm prior to export. In the case of N- and S-oxide reductases, co-ordination of molybdenum cofactor insertion with protein export involves a ‘Tat proofreading’ process where chaperones of the TorD family bind the signal peptides, thus preventing premature export. Here, a genetic approach was taken to determine factors required for selenate reductase activity in Salmonella and E. coli. It is reported for both biological systems that an active Tat translocase and a TorD-like chaperone (DmsD) are required for complete in vivo reduction of selenate to elemental red selenium. Further mutagenesis and in vitro biophysical experiments implicate the Salmonella ynfE gene product, and the E. coli YnfE and YnfF proteins, as putative Tat-targeted selenate reductases.     

Identification and Expression of a <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> Collagenase in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Extracellular protein profiles were compared for broth-grown cultures of Burkholderia pseudomallei and its avirulent close relative Burkholderia thailandensis. A number of protein bands were present in the B. pseudomallei profile but absent or less abundant in the B. thailandensis profile. Four such prominent secreted proteins were identified by using N-terminal sequencing coupled to searches of the B. pseudomallei genome sequence database. The genes for two proteins with similarity to carbohydrate-binding proteins, and a further protein homologous to known bacterial collagenases, were present in both B. pseudomallei and B. thailandensis. The putative collagenase gene was cloned and expressed as a fusion protein in Escherichia coli. Cell lysates from Escherichia coli containing the recombinant protein exhibited detectable gelatinase and collagenase activities.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Expression of <Emphasis Type="Italic">recA</Emphasis> gene of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

Plasmids pKS5 and pKSrec30 carrying normal and mutant alleles of the Deinococcus recA gene controlled by the lactose promoter slightly increase radioresistance of Escherichia coli cells with mutations in genes recA and ssb. The RecA protein of D. radiodurans is expressed in E. coli cells, and its synthesis can be supplementary induced. The radioprotective effect of the xenologic protein does not exceed 1.5 fold and yields essentially to the contribution of plasmid pUC19-recA1.1 harboring the E. coli recA ⁺ gene in the recovery of resistance of the ΔrecA deletion mutant. These data suggest that the expression of D. radiodurans recA gene in E. coli cells does not complement mutations at gene recA in the chromosome possibly due to structural and functional peculiarities of the D. radiodurans RecA protein.     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Characterization of the <Emphasis Type="Italic">N</Emphasis>-glycosylation phenotype of erythrocyte membrane proteins in congenital dyserythropoietic anemia type II (CDA II/HEMPAS)

Congenital dyserythropoetic anemia type II (CDA II) is characterized by bi- and multinucleated erythroblasts and an impaired N-glycosylation of erythrocyte membrane proteins. Several enzyme defects have been proposed to cause CDA II based on the investigation of erythrocyte membrane glycans pinpointing to defects of early Golgi processing steps. Hitherto no molecular defect could be elucidated. In the present study, N-glycosylation of erythrocyte membrane proteins of CDA II patients and controls was investigated by SDS-Page, lectin binding studies, and MALDI-TOF/MS mapping in order to allow an embracing view on the glycosylation defect in CDA II. Decreased binding of tomato lectin was a consistent finding in all typical CDA II patients. New insights into tomato lectin binding properties were found indicating that branched polylactosamines are the main target. The binding of Aleuria aurantia, a lectin preferentially binding to α1-6 core-fucose, was reduced in western blots of CDA II erythrocyte membranes. MALDI-TOF analysis of band 3 derived N-glycans revealed a broad spectrum of truncated structures showing the presence of high mannose and hybrid glycans and mainly a strong decrease of large N-glycans suggesting impairment of cis, medial and trans Golgi processing. Conclusion: Truncation of N-glycans is a consistent finding in CDA II erythrocytes indicating the diagnostic value of tomato-lectin studies. However, structural data of erythrocyte N-glycans implicate that CDA II is not a distinct glycosylation disorder but caused by a defect disturbing Golgi processing in erythroblasts.     

Molecular cloning,expression in <Emphasis Type="Italic">Escherichia</Emphasis><Emphasis Type="Italic">coli</Emphasis> of Attacin A gene from <Emphasis Type="Italic">Drosophila</Emphasis> and detection of biological activity

Attacin, a 20 kDa antibacterial peptide, plays an important role in immunity. To understand this gene better, gene cloning, expression and biological activity detection of Attacin A was carried out in present study. The full-length open reading frame (ORF) coding for Attacin A gene was generated using RT-PCR which takes total RNA extracted from Drosophila as the template. The gene was inserted directionally into the prokaryotic expression vector pET-32a (+). The resulting recombinant plasmid was transformed into E. coli Rosetta. SDS–PAGE was carried out to detect the expression product which was induced by IPTG. The antimicrobial activity and hemolysis activity were tested in vitro after purification. Agarose gel electrophoresis indicated that the complete ORF of Attacin A gene has been cloned successfully from Drosophila stimulated by E. coli which includes 666 bp and encodes 221 AA. The gene encoding mature Attacin A protein was amplified by PCR from the recombinant plasmid containing Attacin A, which includes 570 bp in all. SDS–PAGE analysis demonstrated that the fusion protein expressed was approximately 39.2 kDa. Biological activities detection showed that this peptide exhibited certain antibacterial activity to several G− bacteria, as well as minor hemolysis activity for porcine red blood cells. In conclusion, Attacin A gene was cloned and expressed successfully. It was the basis for further study of Attacin.     

Transgenic tobacco plants containing <Emphasis Type="Italic">Bt</Emphasis> and <Emphasis Type="Italic">GNA</Emphasis> genes

A new plant expression vector (pBSbtCry1Ac-GNA) containing two insect resistant genes, a synthetic chimeric gene SbtCry1Ac encoding the insecticidal protein CrylAc and a gene GNA encoding snowdrop lectin (Galanthus nivalis agglutinin) was constructed. Transgenic tobacco plants containing these two genes were obtained through Agrobacterium-mediated transformation of tobacco leaf discs. Results from PCR detection and genomic DNA Southern blot analysis indicated that both SbtCrylAc gene and GNA gene were integrated into the genome of these plants. Results of Western blot analysis indicated that these two proteins were expressed in the analyzed plants. Bioassays of Myzus persicae and Helicoverpa assulta on detached leaves of transformed tobacco plants were carried out. The average aphid inhibition rate of these plants tested at 12 d post-infestation was 71.9 %. The average H. assulta mortality of these plants tested at 6 d post-infestation was up to 89.8 %. The kanamycin resistance of the T₁ progeny of these transgenic plants was analyzed and a typical 3:1 segregation was observed.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Monosaccharide profiling of silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis> L.) nervous system during development and aging

Glycoconjugates have various functions in differentiation, development, aging and in all aspects of normal functioning of organisms. The reason for increased research on this topic is that glycoconjugates locate mostly on the cell surface and play crucial biological roles in the nervous system including brain development, synaptic plasticity, learning, and memory. Considering their roles in the nervous system, information about their existence in the insect nervous system is rather sparse. Therefore, in order to detect monosaccharide content of N- and O-glycans, we carried out capLC–ESI–MS/MS analysis to determine the concentration changes of glucose, mannose, galactose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, xylose, arabinose, and ribose monosaccharides in the nervous system of Bombyx mori during development and aging processes. In addition to LC–MS, lectin blotting was done to detect quantitative changes in N- and O-glycans. Developmental stages were selected as 3rd (the youngest sample), 5th (young) larval instar, motionless prepupa (the oldest sample), and pupa (adult development). Derivatization of monosaccharides was performed with a solution of PMP agent and analyzed with capLC–ESI–MS/MS. For lectin blotting, determination of glycan types was carried out with Galanthus nivalis agglutinin and Peanut agglutinin lectins. In all stages, the most abundant monosaccharide was glucose. Although all monosaccharides were present most abundantly in the youngest stage (3rd instar), they are generally reduced gradually during the aging process. It was observed that amounts of monosaccharides increased again in the pupa stage. According to lectin blotting, N- and O-linked glycoproteins expressions were different and there were some specific glycoprotein expression differences between stages. These findings suggest that the glycosylation state of proteins in the nervous system changes during development and aging in insects in a similar fashion to that reported for vertebrates.     

Biological synthesis of quercetin 3-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine conjugate using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing <Emphasis Type="Italic">UGT78D2</Emphasis>

Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide sugar metabolic pathway in the host strain.     

Heterologous expression of the <Emphasis Type="Italic">msp2 gene</Emphasis> from <Emphasis Type="Italic">Marasmius scorodonius</Emphasis>

For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca²⁺, and hemin.     

Stable expression and secretion of polyhydroxybutyrate depolymerase of <Emphasis Type="Italic">Paucimonas lemoignei</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An efficient strategy for the expression and secretion of extracellular polyhydroxybutyrate depolymerase (PhaZ1) of Paucimonas lemoignei in Escherichia coli was developed by employing the signal peptide of PhaZ1 and a truncated ice nucleation protein anchoring motif (INPNC). Directly synthesized mature form of Phaz1 was present in the cytoplasm of host cells as inclusion bodies, while a construct containing Phaz1 and its own N-terminal signal peptide (PrePhaz1) enabled the secretion of active Phaz1 into the extracellular medium. However, the PrePhaz1 construct was harmful to the host cell and resulted in atypical growth and instability of the plasmid during the cultivation. In contrast, INPNC-Phaz1 and INPNC-PrePhaz1 fusion constructs did not affect growth of host cells. INPNC-Phaz1 was successfully displayed on the cell surface with its fusion form, but did not retain Phaz1 activity. In the case of INPNC-PrePhaz1, the initially synthesized fusion form was separated by precise cleavage of the signal peptide, and active Phaz1 was consequently released into the culture medium. The amount of Phaz1 derived from E. coli (INPNC-PrePhaz1) was almost twice as great as that directly expressed from E. coli (PrePhaz1), and was predominantly (approximately 85%) located in the periplasm when cultivated at 22°C but was efficiently secreted into the extracellular medium when cultivated at 37°C.     

Vanillin production using <Emphasis Type="Italic">Escherichia coli</Emphasis> cells over-expressing isoeugenol monooxygenase of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.     

Colonization of the <Emphasis Type="Italic">Arabidopsis</Emphasis> rhizosphere by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. activates a root-specific,ethylene-responsive <Emphasis Type="Italic">PR-5</Emphasis> 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.