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

Simmonds, John; Cass, Leslie; Routly, Elizabeth; Hubbard, Keith; Donaldson, Pauline A.; Bancroft, Bonnie; Davidson, Andrea L.; Hubbard, Sheryl; Simmonds, Daina H.

doi:10.1023/b:molb.0000012877.45556.09

Cited by 15 publications

(7 citation statements)

References 40 publications

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“…It is inexpensive and easy to assay, allowing screening of many progeny plants for transgene segregation patterns during generation of sufficient peanut seeds for field studies. The utility of oxalate oxidase as a reporter gene and its advantages over luciferase, b-glucuronidase, and green fluorescent protein have been recently described for other dicots (soybean) and monocots (maize) alike (Simmonds et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene

Livingstone

Hampton²,

Phipps³

et al. 2005

Plant Physiology

148

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Sclerotinia minor Jagger is the causal agent of Sclerotinia blight, a highly destructive disease of peanut (Arachis hypogaea). Based on evidence that oxalic acid is involved in the pathogenicity of many Sclerotinia species, our objectives were to recover transgenic peanut plants expressing an oxalic acid-degrading oxalate oxidase and to evaluate them for increased resistance to S. minor. Transformed plants were regenerated from embryogenic cultures of three Virginia peanut cultivars (Wilson, Perry, and NC-7). A colorimetric enzyme assay was used to screen for oxalate oxidase activity in leaf tissue. Candidate plants with a range of expression levels were chosen for further analysis. Integration of the transgene was confirmed by Southern-blot analysis, and gene expression was demonstrated in transformants by northern-blot analysis. A sensitive fluorescent enzyme assay was used to quantify expression levels for comparison to the colorimetric protocol. A detached leaflet assay tested whether transgene expression could limit lesion size resulting from direct application of oxalic acid. Lesion size was significantly reduced in transgenic plants compared to nontransformed controls (65%-89% reduction at high oxalic acid concentrations). A second bioassay examined lesion size after inoculation of leaflets with S. minor mycelia. Lesion size was reduced by 75% to 97% in transformed plants, providing evidence that oxalate oxidase can confer enhanced resistance to Sclerotinia blight in peanut.Sclerotinia blight of peanut, caused by the necrotrophic fungus Sclerotinia minor, is one of the most devastating diseases of peanut (Arachis hypogaea) in Virginia, northeastern North Carolina, Oklahoma, and Texas. The fungicide fluazinam provides some protection (Smith et al., 1992), but its benefit to peanut producers is offset by the expense of the multiple applications necessary to achieve modest control. Yield losses due to Sclerotinia blight can be significant. For example, prior to the availability of fluazinam, losses to Sclerotinia blight averaged 10% in Virginia, with pod loss exceeding 50% in severely affected fields (Porter and Melouk, 1997).Oxalic acid is considered a pathogenicity factor in Sclerotinia sclerotiorum and many other fungal pathogens (Maxwell and Lumsden, 1970;Godoy et al., 1990 (Rao and Tewari, 1987), and S. minor (Hollowell et al., 2001). Direct application of oxalic acid to stem or leaf tissue causes marked tissue injury and wilting, similar to plant responses to fungal infection by S. rolfsii (Bateman and Beer, 1965) and S. sclerotiorum (Noyes and Hancock, 1981). The most compelling evidence for the involvement of oxalic acid in disease initiation was the demonstration that mutant isolates of S. sclerotiorum, deficient in oxalic acid production, were not pathogenic on bean (Phaseolus vulgaris), but revertants became pathogenic once they regained the ability to produce oxalic acid (Godoy et al., 1990). Screening for resistance to oxalic acid has also been studied as an indirect test of physiological resist...

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Section: Discussionmentioning

confidence: 99%

Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene

Livingstone

Hampton²,

Phipps³

et al. 2005

Plant Physiology

148

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“…The soybean (Glycine max) genotype used in this study was the X5 (AAFC breeding line X2650-7-2-3) and the X5-derived transgenic line, OxO-OE (short for B71(5A1)-20B-7), carrying the wheat germin gene gf2.8 (Lane et al, 1991;Donaldson et al, 2001) that had been transformed using the biolistic particle delivery system (Bio-Rad) (Simmonds, 2003). Transgenic line OxO-OE was chosen for this study as it is a stable transgenic line that expresses OxO at a high level (Simmonds et al, 2004). All plants were grown at 26/24°C with a 15 h photoperiod, watered every day and fertilized with a 20-20-20 (N-P-K) nutrient solution.…”

Section: Plantsmentioning

confidence: 99%

“…Unless specified, each sample was replicated using three separate plants. The assay solution was prepared, prior to use, by adding 0.6 mg mL À1 4-chloro-1-naphthol to a solution of 25 mM succinic acid, 3.5 mM EDTA, 2.5 mM OA, pH 4 and dissolved by heating at 55°C as previously described (Simmonds et al, 2004). Control solutions lacking OA were used to detect activity that was not specific for oxalate oxidase.…”

Section: Oxalate Oxidase Histological Assaymentioning

confidence: 99%

“…Resistance to S. sclerotiorum has been attained in soybean transformed with the wheat germin gene, gf2.8 (Lane et al, 1991) that codes for oxalate oxidase (OxO), a catalyst for OA degradation to CO 2 and H 2 O 2 (Donaldson et al, 2001;Cober et al, 2003;Simmonds et al, 2004). In order to gain a better understanding of the mechanism of resistance provided by the transgenic soybean, the host-pathogen interaction was investigated using a transgenic line with high OxO activity (OxO-OE) (Simmonds et al, 2004) and its susceptible isogenic parent (WT). The inoculum used in this study was flowers infected with ascospores because this is the primary mode of infection in soybean (Mclean, 1958;Grau, 1988).…”

Section: Introductionmentioning

confidence: 99%

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Histopathology of Sclerotinia sclerotiorum infection and oxalic acid function in susceptible and resistant soybean

et al. 2016

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The success of the necrotrophic fungus Sclerotinia sclerotiorum is largely dependent on its major virulence factor, oxalic acid (OA). Virulence is lost in transgenic plants that express OA degrading enzymes, e.g. oxalate oxidase (OxO). The histopathology of S. sclerotiorum infection and OA accumulation was examined in a transgenic soybean line over-expressing OxO (OxO-OE) and its isogenic parent (WT). In situ flower inoculation showed that the OxO-OE plants were highly resistant to the pathogen while the WT parents were susceptible. This difference in resistance was not apparent in the floral tissues, as aggressive hyphal activity was similar on both hosts, showing that high OxO activity and low OA accumulation in OxO-OE was not a deterrent. However, the process of fungal infection on excised leaf tissue differed on the two hosts. Primary lesions developed and showed similar severe ultrastructural damage on both hosts but rapid lesion expansion (colonization) proceeded only on the WT, concomitant with OA accumulation. Oxalic acid rose in OxO-OE 1 day post-inoculation and did not change over the following 3 days, showing that colonization can be blocked by maintaining low levels of OA. However, OxO degradation of OA did not deter initial host penetration and primary lesion formation. This shows that OA, the major virulence factor of S. sclerotiorum, is critical for host colonization but may not be required during primary lesion formation, suggesting that other factors are contributing to the establishment of the primary lesion.

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“…Tables 1 and 2 summarize the characteristics of all the SMGs. Plant genes that can serve as reporters but do not confer a selective advantage to transgenic cells (Simmonds et al 2004;Kim et al 2010) will not be reviewed here.…”

Section: Introductionmentioning

confidence: 99%

Selectable marker genes from plants: reliability and potential

Rosellini

2011

In Vitro Cell.Dev.Biol.-Plant

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Selectable marker genes (SMGs) are still useful to efficiently obtain transgenic plants, although marker-free techniques are available, but with limitations. The presence of SMGs, especially bacterial antibiotic resistance genes, in transgenic crops is criticized. Fortunately, several genes isolated from plants are available that can serve as SMGs.Here, I review the plant genes reported to have been used as SMGs. Some are wild-type genes that, when overexpressed, confer a selective advantage during in vitro plant regeneration, whereas some are mutated genes encoding enzymes resistant to inhibitory chemicals. Most of the genes have not yet been tested in a significant number of species. The effect of SMGs expression on the phenotype has often been superficially examined and should be better characterized. The sequence conservation of some SMGs could allow derivation of a SMGs from any plant species, if an intragenic or cisgenic approach to genetic engineering is preferred. I conclude that several promising SMGs have been isolated from plants, allowing avoidance of bacterial genes for transformation, transgene stacking, and intragenic or cisgenic engineering approaches. Nonetheless, further testing in more plant species would be useful to fully assess phenotypic neutrality, efficiency, and versatility. Patent rights restrict the immediate use of most plant SMGs for commercial applications, but freely available marker systems do exist.

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

Cited by 15 publications

References 40 publications

Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene

Enhancing Resistance to Sclerotinia minor in Peanut by Expressing a Barley Oxalate Oxidase Gene

Histopathology of Sclerotinia sclerotiorum infection and oxalic acid function in susceptible and resistant soybean

Selectable marker genes from plants: reliability and potential

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