Effect of yeast CTA1 gene expression on response of tobacco plants to tobacco mosaic virus infection

Abstract

The response of tobacco (Nicotiana tabacum L. cv Xanthi-nc) plants with elevated catalase activity was studied after infection by tobacco mosaic virus (TMV). These plants contain the yeast (Saccharomyces cerevisiae) peroxisomal catalase gene CTA1 under the control of the cauliflower mosaic virus 35S promoter. The transgenic lines exhibited 2- to 4-fold higher total in vitro catalase activity than untransformed control plants under normal growth conditions. Cellular localization of the CTA1 protein was established using immunocytochemical analysis. Gold particles were detected mainly inside peroxisomes, whereas no significant labeling was detected in other cellular compartments or in the intercellular space. The physiological state of the transgenic plants was evaluated in respect to growth rate, general appearance, carbohydrate content, and dry weight. No significant differences were recorded in comparison with non-transgenic tobacco plants. The 3,3'-diaminobenzidine-stain method was applied to visualize hydrogen peroxide (H(2)O(2)) in the TMV infected tissue. Presence of H(2)O(2) could be detected around necrotic lesions caused by TMV infection in non-transgenic plants but to a much lesser extent in the CTA1 transgenic plants. In addition, the size of necrotic lesions was significantly bigger in the infected leaves of the transgenic plants. Changes in the distribution of H(2)O(2) and in lesion formation were not reflected by changes in salicylic acid production. In contrast to the local response, the systemic response in upper noninoculated leaves of both CTA1 transgenic and control plants was similar. This suggests that increased cellular catalase activity influences local but not systemic response to TMV infection.

Figure 1

Influence of SA on yeast catalase activity in vitro. Catalase activity was measured in crude extracts isolated from isogenic yeast strains carrying mutations in either the CTT1 (A, black symbols) or CTA1 (B, white symbols) gene. Twenty microliters of extract (50–70 μg of protein) was added to a reaction mixture containing 0.05% (v/v) H₂O₂ and SA at appropriate concentration. Each point represents a mean of three assays performed with independently prepared extracts. Error bars represent the sd.

Figure 2

Expression of the yeast CTA1 gene in three selected transgenic lines and in an untransformed line (WT). The presence of CTA1 mRNA was detected by northern blotting (A, 10 μg of total RNA was loaded per lane), the presence of the CTA1 protein was detected by western blotting (B, 20 μg of protein was loaded per lane), and the catalase activity was measured in crude extracts from leaf tissue (C). Results shown in C are means of three measurements performed with independently prepared extracts. Error bars represent the sd.

Figure 3

Assessment of the physiological state of the CTA1/4 line: dry weight (mg) per mg of fresh weight (A) and carbohydrate content (B). Leaf weight was measured for 10 leaves taken from a single plant. The ratio was calculated using obtained arithmetic means. Carbohydrate content was measured in three plants (□, Glc; ▥, Fru; , Suc; ▪, starch). The results are arithmetic means with error bars representing the sd.

Figure 4

The influence of SA on in vitro catalase activity in untransformed plants (white symbols) and the CTA1/4 line (black symbols). Catalase activities were measured in crude extracts obtained from fully developed leaves in the presence of various SA concentrations. Each point is the mean of three independent experiments. Error bars represent the sd.

Figure 5

The subcellular localization of the CTA1 protein. A and B, CTA1/4 line; C, non-transgenic tobacco; and D, the number of gold particles in different cellular compartments. Positive immunolocalization (black dots) was counted on several independently obtained sections. Arrows indicate positions of gold labeling. Cellular compartments were abbreviated as follows: c, cytoplasm; ch, chloroplasts; cw, cell wall; is, intercellular space; m, mitochondria; p, peroxisomes; and v, vacuole.

Figure 6

TMV lesion phenotype on CTA1 and control tobacco plants. CTA1-expressing (CTA1/4) and untransformed tobacco cv Xanthi-nc were inoculated with 1.5 μg of TMV strain U1, and sections of leaves were photographed 2, 4, and 7 d after inoculation.

Figure 7

Visualization of H₂O₂ by DAB staining in leaf tissue after inoculation with TMV. A, Untransformed control plant 32 h postinfection (hpi); B, CTA1/4 32 hpi; C, untransformed control plant 36 hpi; and D, CTA1/4 36 hpi.

Figure 8

Local expression of genes coding for acidic and basic PR proteins. RNA was isolated at 4 d postinoculation from TMV- or mock-inoculated fully developed leaves. Each lane contained 10 μg of total RNA. After hybridization with a probe specific to an acidic PR isoform, the probe was removed and the same membrane was rehybridized with a probe for the corresponding basic isoform. The amount of RNA on the blot was visualized by rehybridizaton with a probe for rRNA. The experiment was done three times.

Figure 9

SA and SAG levels in the TMV- or mock-inoculated leaves 48 hpi (A) and 96 hpi (B). Results are the mean of two independent experiments. Inoculations were performed on three plants during each experiment. Error bars represent the sd. ▪, SA; □, SAG.

Figure 10

Expression of acidic PR-1 and PR-2 genes in the CTA1/4 and untransformed control plants during the systemic response to TMV infection. Seven days after the initial infection, upper uninoculated leaves were either mock inoculated (A) or inoculated with TMV (B). Ten micrograms of total RNA was loaded per lane. The blot was first probed with the PR-1 probe, then stripped, and reprobed with the PR-2 probe. Finally, the blot was probed with a probe for rRNA. The experiment was done three times.