Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus × euramericana (`Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic `Ogy'. The enzyme activity in the transformed `Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic `Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic `Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from `Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

Oxalate decarboxylase from Collybia velutipes. Molecular cloning and its overexpression to confer resistance to fungal infection in transgenic tobacco and tomato

Oxalic acid is present as nutritional stress in many crop plants like Amaranth and Lathyrus. Oxalic acid has also been found to be involved in the attacking mechanism of several phytopathogenic fungi. A full-length cDNA for oxalate decarboxylase, an oxalate-catabolizing enzyme, was isolated by using 5'-rapid amplification of cDNA ends-polymerase chain reaction of a partial cDNA as cloned earlier from our laboratory (Mehta, A., and Datta, A. (1991) J. Biol. Chem. 266, 23548-23553). By screening a genomic library from Collybia velutipes with this cDNA as a probe, a genomic clone has been isolated. Sequence analyses and comparison of the genomic sequence with the cDNA sequence revealed that the cDNA is interrupted with 17 small introns. The cDNA has been successfully expressed in cytosol and vacuole of transgenic tobacco and tomato plants. The transgenic plants show normal phenotype, and the transferred trait is stably inherited to the next generation. The recombinant enzyme is partially glycosylated and shows oxalate decarboxylase activity in vitro as well as in vivo. Transgenic tobacco and tomato plants expressing oxalate decarboxylase show remarkable resistance to phytopathogenic fungus Sclerotinia sclerotiorum that utilizes oxalic acid during infestation. The result presented in the paper represents a novel approach to develop transgenic plants resistant to fungal infection.     

Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower

Oxalate oxidase (OXO) converts oxalic acid (OA) and O(2) to CO(2) and hydrogen peroxide (H(2)O(2)), and acts as a source of H(2)O(2) in certain plant-pathogen interactions. To determine if the H(2)O(2) produced by OXO can function as a messenger for activation of defense genes and if OXO can confer resistance against an OA-producing pathogen, we analyzed transgenic sunflower (Helianthus annuus cv SMF3) plants constitutively expressing a wheat (Triticum aestivum) OXO gene. The transgenic leaf tissues could degrade exogenous OA and generate H(2)O(2). Hypersensitive response-like lesion mimicry was observed in the transgenic leaves expressing a high level of OXO, and lesion development was closely associated with elevated levels of H(2)O(2), salicylic acid, and defense gene expression. Activation of defense genes was also observed in the transgenic leaves that had a low level of OXO expression and had no visible lesions, indicating that defense gene activation may not be dependent on hypersensitive response-like cell death. To further understand the pathways that were associated with defense activation, we used GeneCalling, an RNA-profiling technology, to analyze the alteration of gene expression in the transgenic plants. Among the differentially expressed genes, full-length cDNAs encoding homologs of a PR5, a sunflower carbohydrate oxidase, and a defensin were isolated. RNA-blot analysis confirmed that expression of these three genes was significantly induced in the OXO transgenic sunflower leaves. Furthermore, treatment of untransformed sunflower leaves with jasmonic acid, salicylic acid, or H(2)O(2) increased the steady-state levels of these mRNAs. Notably, the transgenic sunflower plants exhibited enhanced resistance against the OA-generating fungus Sclerotinia sclerotiorum.     

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71–82.73% reduction of lesion length in a detached stem assay (T₃ and T₄ generations) and 76.67–82.0% reduction of lesion area in a detached leaf assay (T₄ generation). The transgenic plants also showed increased tolerance to the externally applied OA (60 mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T₃ and T₄ transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H₂O₂) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1 h and 4 h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H₂O₂ at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H₂O₂ levels, and eliciting defense responses mediated by multiple signaling pathways.

     

Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on oxalate oxidase and SIRE technology

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Oxalate oxidase: a novel reporter gene for monocot and dicot transformations

A wheat germin gene, with oxalate oxidase (OxO) activity, can be used as a sensitive reporter gene in both monocot and dicot transformations. Detection of H₂O₂ generated from OxO oxidation of oxalate provides simple, rapid detection of gene expression. Inexpensive substrates are required for both assays. OxO activity, could be detected histochemically in minutes, without chlorophyll clearing procedures. This assay was used to optimize transformation procedures and to track stable transgene expression in breeding populations over many generations. A simple spectrophotometric quantitative enzyme activity assay was used to select lines with various levels of transgene expression and to monitor transgene silencing phenomena. The quantitative OxO assay can also be used as an internal DNA delivery standard with a second reporter gene used in gene expression studies. The simplicity of the assay is ideal for screening large populations to identify primary transgenics, for monitoring transgene segregation in large populations in field studies and for assessing stability of transgene expression over numerous generations.     

Germin-like oxalate oxidase, a H2O2-producing enzyme, accumulates in barley attacked by the powdery mildew fungus

Germin-like oxalate oxidase is an oligomeric enzyme which generates H₂O₂. This paper reports increased activity of this enzyme in association with the response of barley to the powdery mildew fungus, Erysiphe graminis f.sp. hordei . The increase is detected in a colorimetric assay as well as on activity blots using extracts of both resistant and susceptible leaves. The increase is generally apparent from 24 h after inoculation. From 48 h after inoculation, there is an approximately 10-fold higher oxalate oxidase activity in the samples from inoculated plants compared with the controls. The oxalate oxidase activity increase appears 1–3 days earlier than PR-1 protein accumulation. SDS—PAGE analysis suggests that this pathogen-response oxalate oxidase is different from a commercially available barley oxalate oxidase, and from a constitutive barley root oxalate oxidase.     

Assessing in vitro efficacy of certain fungicides to control Sclerotinia sclerotiorum in peanut

The fungal pathogen Sclerotinia sclerotiorum is responsible for Sclerotinia blight in several crops around the world, including peanut. This study was conducted under laboratory conditions to determine the effects of four registered fungicides, Propulse?, Fontelis^®, Omega^® and Endura^® on mycelial growth and pigmentation, as well as sclerotia and oxalic acid production on a growth medium modified with a fungicide and on the pathogenicity of S. sclerotiorum on leaflets detached from Valencia peanut. Propulse, Omega and Fontelis inhibited mycelial growth of S. sclerotiorum, while, mycelial growth on a modified support with Endura was similar to the control treatment. All fungicides, except Endura, inhibited the production of oxalic acid. Pigmentation of the mycelium was observed in both the control and endura treatments. Sclerotia production was observed only in the control treatment. With the exception of Endura, all fungicides were effective in controlling the development of lesions on Valencia peanut leaflets.     

PDF1.2基因在转草酸氧化酶基因甘蓝型油菜中的表达

采用RT-PCR方法检测茉莉酸（JA）／乙烯（ET）依赖性信号途径中关键基因PDF1。2在转草酸氧化酶基因（OXO）油菜株系与未转化对照中的表达差异。结果表明,在转基因油菜不同株系中PDF1．2都有不同程度的上调表达,预示着转OXO油菜对菌核病的抗性增强可能与激活JA／ET依赖性信号途径有关。     

Oxalic Acid Production and Mycelial Biomass Yield of Sclerotinia minor for the Formulation Enhancement of a Granular Turf Bioherbicide

The fungus Sclerotinia minor is presently under development in this laboratory as a granular bioherbicide for broadleaf weed species. With a view to enhancing the virulence of the fungus, the effect of increasing endogenous oxalic acid concentration through modification of the growth conditions was investigated. S. minor was grown in 125 ml of eight different liquid culture media in shake flasks incubated at 20°C for 7 days. The final pH, mycelial dry weight, and oxalic acid content of the spent growth media were determined and the virulence of S. minor grown on each solid culture medium was screened on detached dandelion leaves. A 330% increase in oxalic acid was obtained plus 56 mM of sodium succinate to Modified Richard's solution (MRS) as compared to MRS alone. A concomitant increase in virulence of 218% was expressed as increased lesion diameter.     

Comparisons of Ectomycorrhizal Colonization of Transgenic American Chestnut with Those of the Wild Type,a Conventionally Bred Hybrid,and Related Fagaceae Species

American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.     

pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate‐minus mutants of Sclerotinia sclerotiorum

The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV‐induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate‐minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate‐minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate‐minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate‐minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.     

草酸氧化酶基因转化烟草的研究

为研究草酸氧化酶基因(OxO)对植物抗病的作用,将含有CaMV 35s启动子的植物表达载体以根癌农杆菌(Agrobacterium tumefaciens)介导的叶盘方法,转化了烟草97131。具有卡那霉素抗性的再生植株经PCR检测,得到了与阳性对照一致的470bp的片段,进一步对PCR产物测序表明OxO基因已整合进烟草基因组中。在对草酸的耐受性实验中,转基因烟草对草酸的抗性明显高于未转化烟草。