Enhancing resistance to Sclerotinia minor in peanut by expressing a barley oxalate oxidase gene

Abstract

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.

Figure 1.

Comparison of two oxalate oxidase activity assays using transgenic and control peanut plants. Transgenic plants were regenerated from embryogenic peanut cultures bombarded with an oxalate oxidase gene under the control of the cauliflower mosaic virus 35S promoter and selected in the presence of hygromycin. Transformants were identified by screening for transgene expression using a leaf disc assay for oxalate oxidase activity. Quantitative measure of enzyme activity was determined for eight transformants and three untransformed controls using two different detection methods. A, The colorimetric detection protocol used 100 μL of the enzyme reaction, and results were measured spectrophotometrically at A₅₅₀. B, The Amplex Red detection kit used 20 μL of the enzyme reaction, and results were measured with a fluorescence plate reader with an excitation filter of 530 nm and emission filter of 590 nm. (The same reaction was assayed by both methods for each of the eight leaf discs per plant.) Untransformed controls were Perry, Wilson, and NC-7. P9, P30, and P39 were regenerated from bombardment of Perry embryogenic cultures. W1, W3, W73, and W100 were regenerated from bombardment of Wilson embryogenic cultures. N6 is derived from NC-7 embryos, although some caution in making this assignment was described in “Materials and Methods.” “Blank” indicates a reaction performed without plant material (buffer alone). Means and se are presented for eight replicates for each plant.

Figure 2.

Southern-blot analysis of peanut genomic DNA with the oxalate oxidase probe. BstXI-digested DNA was probed with a ³²P-labeled fragment amplified by PCR from the barley oxalate oxidase cDNA. Size markers were HindIII-digested lambda DNA (sizes to the left of the blot are in base pairs). The first three lanes show hybridization of the probe to the PCR product used as the probe (470 bp), to undigested pOxOx plasmid, and to BstXI-digested pOxOx plasmid. Untransformed controls are cultivars Perry, Wilson, and NC-7. P9, P30, and P39 were Perry transformants. W3, W73, and W100 were Wilson transformants, and N6 is believed to be a NC-7 transformant.

Figure 3.

Northern-blot analysis of peanut leaflet RNA with the oxalate oxidase probe. A, Total RNA from transformants and nontransformed controls was probed with the same 470-bp probe as described for the Southern blot in Figure 2. Expression in transgenic peanuts is observed by the presence of a 700-nucleotide mRNA band. B, Ethidium bromide (EtBr) staining was performed to demonstrate sample loading.

Figure 4.

Resistance of transgenic plants to application of oxalic acid. Different concentrations of oxalic acid were applied to detached leaflets from transformants and nontransformed controls. Oxalic acid in A was applied at 0, 2, 4, 6, 8, and 10 mm. Oxalic acid in B to D was applied at 0, 20, 50, 100, and 200 mm. A, Nontransformed Perry and three transformants (P9, P30, and P39) at low oxalic acid concentrations. B, Same samples as in A except for high range of oxalic acid concentrations. C, Nontransformed Wilson and three transformants (W3, W73, and W100). D, Nontransformed NC-7 and transformed N6. Means and se are shown for four replicates.

Figure 5.

Resistance to oxalic acid in control and transformed peanut. A, Bar graph showing lesion size (in mm²) for transformed lines and nontransformed controls following application of 200 mm oxalic acid to peanut lines. B, Photograph of selected peanut lines showing lesion size and appearance on detached leaflets after application of increasing amounts of oxalic acid. Means and se are shown for four replicates.

Figure 6.

Resistance to S. minor in oxalate oxidase-expressing peanut lines compared to controls. A, Lesion size (in mm²) on detached leaflets in response to inoculation with S. minor. B, Comparison of selected peanut lines in response to inoculation with S. minor showing lesion size and appearance on detached leaflets. Means and se are shown for 16 replicates.