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

Abstract

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.

Figure 1.

Expression of wheat OXO transgene in different sunflower lines. OXO activity (A) and steady-state level of OXO mRNA (B) in leaf tissues from 6-week-old transgenic lines (610255, 539154, and 539149) and untransformed control (SMF3). DW, Dry weight; rRNA, ribosomal RNAs that were stained with ethidium bromide in agrose gel. Error bars = sd (n = 6).

Figure 2.

Phenotype of HR-like lesions and accumulation of autofluorescent compounds in OXO transgenic leaves. A, Phenotype of the lesions on OXO transgenic leaves (610255) was compared with that of untransformed control leaves (SMF3) at the 6-week-old stage. B, UV-stimulated autoflurescence of OXO transgenic leaves was detected in the early development of HR-like lesions in OXO leaves from 6-week-old plants. White scale bar = 0.05 mm.

Figure 3.

Changes of OXO activity, H₂O₂, and SA during the first 8-week growth period of OXO transgenic (610255, 539149, and 539154) and untransformed control (SMF3) plants. A, OXO activity. Values are the means of three samples; error bars = sd of the mean (n = 3). B, Endogenous levels of total SA. Values are the means of five samples; error bars = sd of the mean (n = 5). C, Accumulation of H₂O₂ in untransformed (SMF3) and transgenic (610255) leaves of 6-week-old plants. The purple color indicates the presence of H₂O₂. FW, Fresh weight; DW, dry weight.

Figure 4.

Differential expression profiling of genes in the OXO transgenic (line 610255) and untransformed leaves and detection of cDNA-fragments associated with four genes. The leaves were harvested from 6-week-old plants. 1, OXO transgene (fragment ID: h0a0-302.2); 2, PR5-1 (fragment ID: h0a0-231.3); 3, Defensin (fragment ID: d0l0-113.9); 4, SCO (fragment ID: n0s0-162.7). A, Schematic diagrams of the four full-length genes showing the relative positions of detected fragments (shaded area). The lengths of the boxes are not proportional to the gene lengths. B, Detection of the cDNA fragments of the four differentially modulated genes in OXO transgenic (red trace) compared with untransformed (blue trace) leaf tissues. Vertical red lines show the specific fragments of the genes. C, Confirmation of the cDNA fragments associated with the four genes after competitive PCR with (green trace) or without (red trace) an unlabeled oligonucleotide specific to the transgene (for details, see “Materials and Methods”).

Figure 5.

Expression of PR5-1, Defensin, and SCO in S. sclerotiorum-infected SMF3 and OXO transgenic sunflower (line 610255) leaves of 6-week-old plants. The leaves of SMF3 and line 610255 plants were also used to generate the mRNA profiling data shown in Figure 4. DAI, Days after inoculation; I, infected plants; U, uninfected plants; 18S, 18S ribosomal RNA.

Figure 6.

Accumulation of OXO, PR5-1, Defensin, and SCO transcripts in OXO transgenic sunflower leaves. A, Developmental regulation of the defense gene expression. Lane a, SMF3; lane b, 610255; w, week; rRNA, ribosomal RNAs that were stained with ethidium bromide in agrose gel. B, Expression of OXO, PR5-1, defensin, and SCO genes in the leaves of transgenic lines 610255, 539149, and 539154 and in the untransformed SMF3 at the 6-week-old stage.

Figure 7.

Effects of exogenous JA, SA, and H₂O₂ on the steady-state levels of PR5-1, Defensin, and SCO mRNAs in the leaves of 6-week-old untransformed sunflower plants. The numbers on the top of the figure indicate the time (hours) after spraying the chemicals. C, Untreated control; 18S, 18S ribosomal RNA.

Figure 8.

Inhibition of S. sclerotiorum infection in OXO transgenic sunflower leaf and stem tissues. A, S. sclerotiorum leaf rot assay. The length and width of the lesions in leaf segments were measured 24 h after inoculation. Leave lesion diameter represents the average of length and width of the lesion on the leaf segment. Data shown are the average of eight replicates ± sd (n = 8). B, S. sclerotiorum stalk rot assay. Plants were inoculated at the 6-week-old stage. Lesion size is presented as the vertical length of lesions on the stem. Data shown are the average of nine replicates ± sd (n = 9). SMF3, Untransformed control; line 610255, OXO transgenic line.