Tissue-specific expression of a defence-related peroxidase in transgenic wheat potentiates cell death in pathogen-attacked leaf epidermis

Abstract

Gene technology can offer creative solutions to problems of agronomical relevance, which may not be solved by conventional breeding methods. One of the major problems of wheat cultivation is disease caused by a number of fungal pathogens including the wheat powdery mildew fungus Blumeria graminis f.sp. tritici (Bgt). Transgenic wheat plants that constitutively express the coding sequence of the defence-related wheat peroxidase TaPrx103 (previously TaPERO) in shoot epidermis under the control of the wheat GstA1 promoter were generated and found to exhibit enhanced resistance to Bgt (Altpeter et al., Plant. Mol. Biol. 57, 271-283). Here, I report on physiological and molecular analyses of these plants in order to assess the mode of action of the peroxidase encoded by the TaGstA1:TaPrx103 transgene. Epidermal cells of transgenic lines with enhanced resistance were found to respond to Bgt attack more frequently with hypersensitive cell death and the generation of hydrogen peroxide. By contrast, resistance of epidermal cell walls to degradation by fungal enzymes appeared to be similar in transgenic and wild-type plants. Moreover, the analysis of the abundance of approximately 10,000 wheat transcripts revealed no significant effect of the GstA1i:TaPrx103 transgene on host gene expression in non-inoculated leaves and only a marginal effect in Bgt-challenged leaves, compared with wild-type plants treated in the same manner. The results indicate that the TaPrx103 protein is involved in generating reactive oxygen species specifically in pathogen-attacked cells, which may lead to localized cell death and resistance. I therefore suggest that the transgenic plants presented here can be regarded as substantially equivalent to non-transgenic wheat.

Figure 1

Analysis by Western blotting of TaPrx103 protein accumulation in T₁ seedlings of transgenic lines carrying GstA1i::TaPrx103. Acid‐soluble (pH 2.8) proteins in intercellular washing fluid (IWF) were separated by SDS‐PAGE and blotted onto nitrocellulose. TaPrx103 protein was detected by an antiserum directed against the homologous protein HvPrx08 of barley. The transgene product has an expected mass of approximately 31 kDa. Coomassie‐stained IWF run in parallel served as loading controls (C). Shown is the major protein band corresponding to the germin‐like protein HvGER2 (Vallelian‐Bindschedler et al., 1998).

Figure 2

Resistance phenotype of adult transgenic T₂ plants compared with wild‐type wheat. Pictures were taken approximately 4 weeks after the outbreak of a spontaneous Bgt epidemic in the greenhouse. Numbers in lower left corners of panels indicate transgenic lines.

Figure 3

Segregation analysis of GstA1i:TaPrx103 expression in T₂ transgenic lines. (A) A spotted RNA array was used to measure the abundance of TaPrx103 mRNA in the non‐inoculated leaf immediately below the flag leaf of adult individual T₂ plants (transgenic lines indicated to the right side of the array). BW, non‐inoculated leaf immediately below the flag leaf of five individual wild‐type plants; BW pools, primary leaves of five seedlings (either non‐inoculated or Bgt‐challenged) were pooled for each RNA extraction. The lower panel shows hybridization signals of a 26S rRNA probe that served for signal normalization. (B) Resistance data of two sublines of transgenic line 2193 that either carry the transgene (2193b) or the null‐allele (2193d). Susceptibility (%) relative to wild‐type plants is shown. Statistical significance of difference from the control was tested by chi‐squared analysis, based on two independent experiments with different batches of plants. (C) Bulk analysis of transgene presence in lines 2193b and 2193d by genomic PCR.

Figure 4

Co‐segregation analysis of transgene expression with resistance to Bgt. Expression of TaPrx103 in non‐inoculated leaves immediately below the flag leaf of adult transgenic plants was related to Bgt resistance of flag leaves from the same individuals. Two groups with highest or lowest TaPrx103 mRNA abundance were formed, each consisting of 48 plants.. For this analysis, plants from all transgenic lines shown in Fig. 3 were pooled. Cl0 to Cl3, disease scoring classes 0 (resistant) to 3 (fully susceptible).

Figure 5

Whole‐cell autofluorescence and H₂O₂ burst revealed by DAB staining of Bgt‐attacked epidermal cells of transgenic plants. Whole‐cell autofluorescence (A) and DAB staining (B) was recorded at 36 and 20 h post‐inoculation, respectively. Arrows point at fungal appressoria. Scale bars = 25 µm.

Figure 6

Epidermal cell walls of transgenic plants do not exhibit enhanced resistance to fungal cell‐wall‐degrading enzymes. (A) The cell‐wall degradation of immobilized epidermis was measured after incubation beneath a drop of a cell‐wall‐degrading enzyme cocktail. (B) Microscopic image (100× magnification) of partially degraded epidermal cross‐cell walls after 120 min of incubation. Black arrows, still intact walls; red arrows, digested walls. Scale bar = 50 µm. (C) Different rates of cross‐cell walls from old versus young, elongating leaves. Data are shown as mean ± SE from four parallel plants. (D) Comparison of cell‐wall digestibility of wild‐type plants versus homozygous transgenic line #2013. Data are shown as mean ± SE from five parallel plants. A repeated independent experiment using plants from a different sowing date produced a similar result.

Figure 7

GstA1:TaPrx103 expression in wheat epidermis does not affect host gene expression in leaves. Hierarchical clustering of 558 pathogen‐regulated host genes in wild‐type and transgenic plants. Signal intensities were log‐transformed and median centred per gene, prior to hierarchical clustering by using Euclidian distance and average linkage settings.

Figure 8

Model of the pathogen‐specific effect of the GstA1i:TaPrx103 transgene. Together with the pathogen‐induced TaGLP4 proteins (orthologous to extracellular SODs of barley), the recombinant TaPrx103 protein might constitute a shunt of reactive oxygen species in pathogen‐attacked epidermal cells, resulting in the accumulation of H₂O₂ that leads to cell death.