Cauliflower Mosaic Virus Promoter Research Articles

Production of tENDO1 in stably transformed tobacco cell cultures for mismatch detection

Scanning DNA sequences for polymorphisms and mutations often involve the mismatch specific cleavage by endonucleases at the mismatch sites and subsequent analysis of the digested product for mutation discovery. One of the limitations of using enzymatic mutation detection methods are the cost and availability of a mismatch specific endonuclease. We report the establishment of Nicotiana tabacum L. Cv. Bright Yellow 2 cells stably expressing the truncated ENDO1 (tENDO1) mismatch specific endonuclease. The 5′-Untranslated region of N. tabacum alcohol dehydrogenase gene (NtADH 5′-UTR) under the control of cauliflower mosaic virus (CaMV 35S) promoter was employed to improve the tENDO1 protein yield. To ease the purification process, tENDO1 was secreted into the culture medium and isolated using nickel affinity chromatography. The tENDO1 was estimated to be stably produced in an average of 0.7–0.9 % total soluble protein. Functional test on tENDO1 for mismatch detection demonstrated that tENDO1 retained mismatch specific endonuclease activity resembles its native protein. Further biochemical analysis showed that tENDO1 exhibited mismatch detection specificity and efficiency comparable to other commonly used endonucleases.

Ethanol inducible expression of a mesophilic cellulase avoids adverse effects on plant development

BackgroundPlant-produced biomass-degrading enzymes are promising tools for the processing of lignocellulose to fermentable sugars. A major limitation of in planta production is that high-level expression of such enzymes could potentially affect the structure and integrity of the plant cell wall and negatively influence plant growth and development.ResultsHere, we evaluate the impact on tobacco plant development of constitutive versus alcohol-inducible expression of the endoglucanase TrCel5A from the mesophilic fungus Trichoderma reesei. Using this system, we are able to demonstrate that constitutive expression of the enzyme, controlled by the doubled Cauliflower Mosaic Virus promoter, leads to lower cellulose content of the plant combined with severe effects on plant growth. However, using an alcohol-inducible expression of the endoglucanase in the plant leaves, we achieved similar enzymatic expression levels with no changes in the crystalline cellulose content.ConclusionWe were able to produce significant amounts of cellulase in the plant leaves without detrimental effects to plant development. These results demonstrate the potential feasibility of an inducible expression system for producing biomass degrading enzymes in plants.

Development and Interlaboratory Validation of Quantitative Polymerase Chain Reaction Method for Screening Analysis of Genetically Modified Soybeans

A novel real-time polymerase chain reaction (PCR)-based quantitative screening method was developed for three genetically modified soybeans: RRS, A2704-12, and MON89788. The 35S promoter (P35S) of cauliflower mosaic virus is introduced into RRS and A2704-12 but not MON89788. We then designed a screening method comprised of the combination of the quantification of P35S and the event-specific quantification of MON89788. The conversion factor (Cf) required to convert the amount of a genetically modified organism (GMO) from a copy number ratio to a weight ratio was determined experimentally. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDR), respectively. The determined RSDR values for the method were less than 25% for both targets. We consider that the developed method would be suitable for the simple detection and approximate quantification of GMO.

Expression of wheat expansin driven by the RD29 promoter in tobacco confers water-stress tolerance without impacting growth and development

Expansins are the key regulators of cell wall extension during plant growth. Previously, we produced transgenic tobacco plants with increased tolerance to water stress by overexpressing the wheat expansin gene TaEXPB23 driven by the constitutive 35S cauliflower mosaic virus (CaMV) promoter. However, the growth and development of 35S::TaEXPB23 transgenic tobacco plants were altered under normal growth conditions, with a faster growth rate at the seedling stage, earlier flowering and maturation, and a shorter plant height compared to WT. In the current study, we determined that cellular characteristics and carbohydrate metabolism were altered in 35S::TaEXPB23 transgenic tobacco plants. We also generated transgenic Arabidopsis plants using the same vector. The transgenic Arabidopsis plants had the same phenotype as the transgenic tobacco plants, which may have resulted from the altered expression of several flowering-related genes. We then produced TaEXPB23 transgenic tobacco plants using the stress-inducible RD29A promoter. The use of this promoter reduced the negative effects of TaEXPB23 on plant growth and development. The RD29A::TaEXPB23 transgenic tobacco plants had greater tolerance to water stress than WT, as determined by examining physiological and biochemical parameters. Therefore, the use of stress-inducible promoters, such as RD29A, may minimize the negative effects of constitutive transgene expression and improve the water-stress tolerance of plants.

Transgenic tomato plants as supersweet protein thaumatin II producers

Yalf tomato plants have been transformed with a gene for thaumatin II from Thaumatococcus daniellii Benth. The nucleotide sequence for thaumatin II cDNA was cloned in the pBI121 vector under the control of the CaMV 35S promoter of cauliflower mosaic virus. Expression of the thaumatin II gene was detected in all of the studied transgenic lines. A quantitative estimation of the thaumatin II accumulation in fruits was performed by ELISA. The highest content of thaumatin in transgenic tomato fruits (line 91) was 46.4 ± 10.5 μg/mg of total soluble protein (4.6%). In the other studied lines, the thaumatin content ranged from 17.6 ± 6.1 to 41.3 ± 12.3 μg/mg of total soluble protein (1.8–4.1%). The fruits of transgenic plants had a well-defined sweet taste with a long aftertaste typical of thaumatin II. Transgenic tomato lines with high expression levels can be potentially used as producers of thaumatin for the food and pharmaceutical industries.

Combinatory SYBR® Green Real-Time PCR Screening Approach for Tracing Materials Derived from Genetically Modified Rice

Two real-time PCR approaches for the detection of genetically modified (GM) rice were tested: (1) a combination of SYBR® Green real-time PCR methods detecting the 35S promoter (P-35S) of Cauliflower Mosaic Virus, the nopaline synthase terminator (T-nos) of Agrobacterium tumefaciens and the Bacillus thuringiensis (Bt) CryIAb/Ac toxins and (2) a P-35S/T-nos duplex TaqMan® real-time PCR system. Both systems correctly recognized their respective targets in control samples of Bt63, Kefeng6 and KMD1 insect-resistant and LLRice62 and LLRice601 herbicide-resistant rice. Due to its lesser specificity but broader genetically modified organism (GMO) coverage capacity, the SYBR® Green real-time PCR approach was further tested in more detail. Melting curve, capillary and agarose gel electrophoresis analyses indicated that the P-35S, T-nos and CryIAb/Ac targets in the GM rice events are similar to the corresponding targets present in many known GMOs. High-resolution melting analysis showed that the CryIAb/Ac targets of the GM rice events Bt63 and Kefeng6 matched best the corresponding Bt11 CryIAb sequence. Digital PCR analysis on genomic DNA from the GM rice Bt63 and Kefeng6 events indicated that both GMO contained multiple inserts. Sensitivity tests showed that all SYBR® Green real-time PCR methods could detect their targets at less than an estimated five copies per reaction. Finally, it was shown that these SYBR® Green real-time PCR methods could detect low levels of their targets in rice consignments originating from China. Together, these results demonstrated that a ‘P-35S and T-nos and CryIAb/Ac’ combinatory SYBR® Green real-time PCR screening is highly suited to trace the respective targets including the possible presence of Bt63, Kefeng6 and KMD1 GM rice materials in food products.

Functional ectodomain of the hemagglutinin-neuraminidase protein is expressed in transgenic tobacco cells as a candidate vaccine against Newcastle disease virus

Recently, the use of plants for the production of recombinant proteins has been well demonstrated with promising outcomes. In this study, an efficient Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cells system expressing the ectodomain of hemagglutinin-neuraminidase (eHN) protein from Newcastle disease virus (NDV) strain AF2240 was established. Transgenic tobacco BY-2 cell cultures expressing the immunogenic eHN protein were generated and the translation efficiency of eHN protein was enhanced using the 5′-untranslated region of Nicotiana tabacum alcohol dehydrogenase gene (NtADH 5′-UTR) under the control of strong cauliflower mosaic virus (CaMV 35S) promoter. Transgenic lines verified by real-time PCR showed high level of eHN mRNA transcripts and immunoblotting confirmed the presence of 66 kD eHN protein. The eHN protein was stably produced in an average of 0.2–0.4 % total soluble protein. Green fluorescent protein-tagged eHN protein was expressed and localized at the cytosol of BY-2 cell. All mice receiving purified eHN protein from transgenic tobacco BY-2 cells produced specific anti-NDV antibodies. We concluded that plant made eHN elicit immune response and can serve as candidate vaccine against NDV.

A sandwich-type DNA array platform for detection of GM targets in multiplex assay

Over the last decade, array detection has been developed to qualitatively assess the presence of genetically modified organisms (GMOs). To date, DNA array systems have the highest capabilities as a result of GMOs analysis. We describe the construction of an array platform in the sandwich hybridization format for the detection of transgenic promoter of Cauliflower mosaic virus (CaMV; p35S). Sequence-specific signal development has been achieved by a sandwich complex composed of a surface immobilized capture probe and a fluorescein-tagged signal probe, which are partially complementary to the p35S oligonucleotide. We used poly-l-lysine-coated glass slides as support material, on which capture probes were immobilized by a heterobifunctional cross-linker. The comparative results of optimization studies including cross-linker types probe concentrations and hybridization conditions (sequence, temperature and duration) were reported. An optimum hybridization signal was obtained with a 32.5 Ǻ cross-linker, 10 μM capture and 20 μM signal probe concentrations, respectively. A relatively short hybridization time (2.5 h) provided reproducible array signals. No significant effect of hybridization sequence on the fluorescence intensity was observed. The described platform can specifically detect label-free transgenic sequences with a target of 0.01 μM concentration, while the optimized system exhibits great potential for the application of different GMO target sequences (p35S, tNOS, bar and cry) to multiplex array formats.

Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L).

Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6- phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1(-1) mannose without sucrose. Transgenic plants were verified using PCR analysis. PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.

Immunogenicity of foot-and-mouth disease virus structural polyprotein P1 expressed in transgenic rice

Transgenic plants have become developed as bioreactors for producing heterologous proteins and may even form edible vaccines. In the present study, a transgenic rice expressing the capsid precursor polypeptide (P1) gene of foot-and-mouth disease virus (FMDV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, northern blot, western blot, and ELISA analyses confirmed that the P1 gene was integrated into the transgenic rice and the protein was expressed specifically in the leaves at levels of 0.6-1.3 μg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, FMDV-specific neutralizing antibodies were detected. The immunized mice could clear virus from their sera after FMDV challenge. In addition, FMDV-specific mucosal immune responses were detected in mice after oral immunization with protein extracts from transgenic rice plants. Partial virus clearance was obtained after FMDV challenge. These results indicate the potential of using a transgenic rice-based expression system as an alternative bioreactor for FMDV subunit vaccines.

Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

AtAGP18 is localized at the plasma membrane and functions in plant growth and development

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins (HRGPs). AtAGP17, 18 and 19 comprise the lysine-rich classical AGP subfamily in Arabidopsis. Overexpression of GFP-AtAGP17/18/19 fusion proteins in Arabidopsis revealed localization of the fusion proteins on the plant cell surface of different organs. Subcellular localization of the fusion proteins at the plasma membrane was further determined by plasmolysis of leaf trichome cells. To elucidate AtAGP17/18/19 function(s), these AGPs were expressed without the green fluorescent protein (GFP) tag under the control of 35S cauliflower mosaic virus promoter. In contrast to AtAGP17/AtAGP19 overexpressors which showed phenotypes identical to wild-type plants, AtAGP18 overexpressors displayed several phenotypes distinct from wild-type plants. Specifically, these overexpressors had smaller rosettes and shorter stems and roots, produced more branches and had less viable seeds. Moreover, these AtAGP18 overexpressors exhibited similar phenotypes to tomato LeAGP-1 overexpressors, suggesting these two AGP genes may have similar function(s) in Arabidopsis and tomato.

Involvement of a cucumber MAPK gene ( CsNMAPK) in positive regulation of ROS scavengence and osmotic adjustment under salt stress

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as biotic and abiotic stress response. To unravel the roles of MAPK in cucumber, its expression in transgenic cucumber plants was reduced by an antisense approach. For this purpose, a 1113 bp cDNA fragment of cucumber MAPK gene ( CsNMAPK) was expressed in antisense orientation driven by the 35S promoter of cauliflower mosaic virus. The pBI-CsNMAPK plasmid DNA was introduced into cucumber embryo by the pollen-tube pathway method. All seeds were sown in the soil and screened for transformants with kanamycin, polymerase chain reaction (PCR), Northern blot and real-time quantitative reverse transcriptative PCR (RT-PCR) analysis. Two independent transgenic plants were obtained and analyzed. The results showed that the transgenic cucumber plants exhibited retarded growth. The transgenic plant height was shorter and the leaves were smaller. Transgenic cucumber plants suppression CsNMAPK were more sensitive to salt stress than the wild-type (WT) plants. The fresh weight of shoot of the transgenic plants decreased more than WT after 50 mM NaCl treatment for 7 days. The malondialdehyde (MDA) content was higher, while the superoxide dismutase (SOD) activity and proline accumulation were lower in the transgenic plants than the WT plants after NaCl stress treatment. The evidence indicated that CsNMAPK was involved in positive regulation of reactive oxygen species (ROS) scavengence and osmotic adjustment under salt stress.

The Nod Factor–Independent Symbiotic Signaling Pathway: Development of Agrobacterium rhizogenes–Mediated Transformation for the Legume Aeschynomene indica

The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes-mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.

LPA19, a Psb27 Homolog in Arabidopsis thaliana, Facilitates D1 Protein Precursor Processing during PSII Biogenesis

The biogenesis and assembly of photosystem II (PSII) are mainly regulated by the nuclear-encoded factors. To further identify the novel components involved in PSII biogenesis, we isolated and characterized a high chlorophyll fluorescence low psii accumulation19 (lpa19) mutant, which is defective in PSII biogenesis. LPA19 encodes a Psb27 homolog (At1g05385). Interestingly, another Psb27 homolog (At1g03600) in Arabidopsis was revealed to be required for the efficient repair of photodamaged PSII. These results suggest that the Psb27 homologs play distinct functions in PSII biogenesis and repair in Arabidopsis. Chloroplast protein labeling assays showed that the C-terminal processing of D1 in the lpa19 mutant was impaired. Protein overlay assays provided evidence that LPA19 interacts with D1, and coimmunoprecipitation analysis demonstrated that LPA19 interacts with mature D1 (mD1) and precursor D1 (pD1). Moreover, LPA19 protein was shown to specifically interact with the soluble C terminus present in the precursor and mature D1 through yeast two-hybrid analyses. Thus, these studies suggest that LPA19 is involved in facilitating the D1 precursor protein processing in Arabidopsis.

Insertion and Characterization of the cry1Ia1 Gene in the Potato Cultivar Spunta for Resistance to Potato Tuber Moth

Potato tuber moth (Phthorimaea operculella) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The cry1Ia1 gene (from Bacillus thuringiensis) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (Solanum tuberosum) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the cry1Ia1 gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the cry1Ia1 gene in ‘SpuntaG2’ was compared with the known sequence of the cry1Ia1 gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid “backbone” genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the cry1Ia1 gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzyme-linked immunoabsorbent assays (ELISA) tests indicate that the cry1Ia1 gene was expressed at a mean concentration of 2.24 μg·g−1 fresh weight in leaf tissue and 0.12 μg·g−1 fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’.

Comparison of expression systems for the production of human interferon-α2b

Abstract The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 µg/L of culture, while the amount of nuclear expression in the plant was 4.46 µg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.

Reliability of PCR based screening for identification and quantification of GMOs

Handling with genetically modified organisms (GMOs) is regulated namely in EC. Laboratories often use polymerase chain reaction (PCR) based screening methods to monitor the presence of GM particles in food commodities as a cost effective approach. The reliability was tested of such screening using 35S CaMV promoter as the target sequences. Soya grown from non-GM cultivar as declared by a seed company was investigated after the harvest, transport to the silo, and before processing. The results based on PCR and real-time PCR analysis clearly showed that, the contamination with debris of other species, dust during transport, storage, and other kind of handling led to contamination with detectable amounts of Cauliflower mosaic virus (CaMV). Impurities are allowed by EC regulations but may, as we have shown, interfere with the analytical procedures based on PCR. The identification of 35S CaMV promoter and NOS terminator in food with uncertain history and no approved specific events may indicate unknown GMOs and perhaps emergency situation.

Efficient in vitro plant regeneration from shoot apices and gene transfer by particle bombardment in Jatropha curcas

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5-6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm-3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.

Pattern Recognition Receptors Require N-Glycosylation to Mediate Plant Immunity

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR(N143Q) lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR(N143Q) in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.